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Sevoflurane inhibits malignant progression of colorectal cancer via hsa_circ_0000231-mediated miR-622

Sevoflurane inhibits malignant progression of colorectal cancer via hsa_circ_0000231-mediated... Background: Sevoflurane (Sev), a commonly used volatile anesthetic, has been reported to inhibit the process of colorectal cancer (CRC). Circular RNAs (circRNAs) are revealed to participate in the pathogenesis of CRC. This study aims to reveal the mechanism of hsa_circ_0000231 in Sev-mediated CRC progression. Methods: The expression of hsa_circ_0000231 and microRNA-622 (miR-622) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein level was determined by western blot analysis. Cell proliferation was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ), cell colony formation and DNA content quantitation assays. Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate and propidium iodide double staining and caspase 3 activity assays. Cell migration and invasion were investigated by wound-healing and transwell invasion assays, respectively. The putative relationship between hsa_circ_0000231 and miR-622 was predicted by circular RNA Interactome online database, and identified by dual-luciferase reporter and RNA immuno - precipitation assays. The impacts of hsa_circ_0000231 on Sev-mediated tumor formation in vivo were presented by in vivo assay. Results: Hsa_circ_0000231 expression was upregulated, while miR-622 was downregulated in CRC tissues and cells compared with control groups. Sev treatment decreased hsa_circ_0000231 expression, but increased miR-622 expres- sion in CRC cells. Sev treatment suppressed cell proliferation, migration and invasion, and induced cell apoptosis. Hsa_ circ_0000231 overexpression restored Sev-mediated CRC progression in vitro. Additionally, hsa_circ_0000231 acted as a sponge of miR-622, and miR-622 inhibitors reversed the impacts of hsa_circ_0000231 silencing on CRC process. Furthermore, Sev treatment inhibited tumor growth by regulating hsa_circ_0000231 in vivo. Conclusion: Hsa_circ_0000231 attenuated Sev-aroused repression impacts on CRC development by sponging miR- 622. This findings may provide an appropriate anesthetic protocol for CRC sufferers undergoing surgery. Keywords: Sev, hsa_circ_0000231, miR-622, CRC Background Colorectal cancer (CRC) is a general pernicious can- cer worldwide with high mortality [1]. CRC ranks the third in incidence and the second in death rate among various cancers for the combination of men and women *Correspondence: wjpwjpwjpwang@163.com [2]. The data studied in 2017 presented nearly half of Department of Anaesthesiology, The Chengyang People’s Hospital, 1.8 million CRC patients died [3]. At present, despite No.76 Zhengyang Road, Chengyang District, Qingdao 266109, Shandong Province, China brilliant achievements have been achieved in disclos- Full list of author information is available at the end of the article ing the pathogenesis of CRC, the high mortality of CRC © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 2 of 14 sufferers is still intently correlated with recurrence and experiments. Additionally, whether hsa_circ_0000231 metastasis [4, 5]. Major therapeutic manners of CRC bound to miR-622 was demonstrated. are operating together with chemotherapy, radiother- apy or targeted treatment [6]. Numerous studies have Methods verified that Sevoflurane (Sev), a frequently utilized Specimen collection and the ethics committee inhaled anesthetic, plays significant part in cancer pro - Forty-seven pairs of human CRC tissues and matched cess. For instances, Liu and his colleagues explained healthy colorectal tissues were collected up from CRC Sev repressed cell growth via increasing microRNA-203 sufferers in the Chengyang People’s Hospital, and the (miR-203) expression in breast cancer [7]. Kang et  al. written informed consent was signed by the CRC cases also reported Sev had repressive impacts on cell prolif- before surgery. Obtained tissues were stored at −  80  °C. eration and invasion through mediating mitogen-acti- The Ethics Committee of the Chengyang People’s Hospi - vated protein kinase-related pathway in ovarian cancer tal approved this study. [8]. In this paper, the molecular mechanism of CRC development mediated by Sev was revealed. Cell culture and exposure to Sev Circular RNA (circRNA) is a novel noncoding Human CRC cell lines (HCT116 and SW620) and nor- RNA, characterised by high stability, high expression mal human colonic epithelial cell line NCM460 were and good conservatism [9]. An increasing number purchased from Procell (Wuhan, China). HCT116 and of research efforts have reported that circRNAs are NCM460 cells were grown in Roswell Park Memorial involved in the evolution of various cancers, such as Institute-1640 (RPMI-1640; Procell, Wuhan, China), and lung carcinoma [10], glioma [11], bladder cancer [12] SW620 cells were cultivated in Leibovitz’s L15 media and CRC [13]. Previous research also presented that (L15; Procell, Wuhan, China) at 37 °C in humid condition circRNAs were related to Sev-mediated cancer pro- with 5% CO . Media were supplemented with 10% fetal cess. For example, Li and his colleagues indicated Sev bovine serum (FBS; Procell, Wuhan, China) and 1% peni- repressed cell proliferation and metastasis by decreas- cillin/streptomycin (Procell, Wuhan, China). ing circ_0002755 and circ_0012129 expression in For Sev treatment, HCT116 and SW620 cells were glioma [14]. He et  al. revealed that Sev restrained cell placed in a sealed container with humid atmosphere of proliferation and induced cell apoptosis via controlling 37  °C. Sev (Seebio Biotech, Shanghai) was mixed with circ-3-hydroxy-3-methylglutaryl-CoA synthase 1 (circ- 95% air and 5% CO using a volatilization tank, and gas HMGCS1) in colon cancer [15]. In this study, we found monitor was used to adjust concentrations of Sev to 1.7%, that circ_0000231 (hsa_circ_0000231) was increased 3.4% and 5.1%, respectively. Then, the cells were treated in CRC specimen and cell lines, but decreased in with the various concentrations of Sev for 30 min. Sev-treated CRC cells. Thus, we hypothesized that hsa_circ_0000231 might participate in modulating Sev- Plasmid construction and oligonucleotide synthesis mediated CRC progression. The small RNAs against hsa_circ_0000231 (si-hsa_ MiRNA is a small RNA with about 20 nucleotides, circ_0000231#1, si-hsa_circ_0000231#2 and si-hsa_ commonly altering mRNA levels via binding to their circ_0000231#3), miR-622 mimics (miR-622), miR-622 non-coding regions [16]. Multiple data exhibited miR- inhibitors (anti-miR-622) and control groups (si-NC, NAs regulated cancer progression through interacting NC and anti-NC) were synthesized by GenePharma with circRNAs, including CRC. For example, enforced (Shanghai, China). The overexpression plasmids of hsa_ circ_100395 expression hindered cell proliferation and circ_0000231 (hsa_circ_0000231) and control group metastasis via sponging miR-1228 in lung cancer [10]. (circ-NC) were built by Geneseed (Guangzhou, China). Circ_001783 silencing repressed cell proliferation and Plasmids or oligonucleotides were transfected into cells invasion through binding to miR-200c-3p in breast can- with TurboFect Reagent (Thermo Fisher, Waltham, MA, cer [17]. In CRC, circ_0026344 modulated cell metas- USA). The synthesized sequences of oligonucleotides tasis, growth and apoptosis via sponging miR-183, were si-hsa_circ_0000231#1 5′-ACT GAA CAG ATA AGG miR-21 or miR-31 [18, 19]. In this paper, we found that GTT TAA-3′, si-hsa_circ_0000231#2 5′-CTG AAC AGA hsa_circ_0000231 possessed the binding sequence of TAA GGG TTT AAA-3′, si-hsa_circ_0000231#3 5′-CAC miR-622, which has been reported to act as a repressor in TGA ACA GAT AAG GGT TTA-3′, miR-622 5′-ACA GUC CRC progression [20].UGC UGA GGU UGG AGC-3′, anti-miR-622 5′-GCU Herein, the impacts of hsa_circ_0000231 silencing on CCA ACC UCA GCA GAC UGU-3′, si-NC 5′-CCT CTA cell proliferation, metastasis and apoptosis were con-CCT GTC GCT GAG CTG TAA T-3′, NC 5′-UUU GUA firmed. Whether hsa_circ_0000231 was involved in CUA CAC AAA AGU ACUG-3′ and anti-NC 5′-CAG UAC Sev-mediated CRC progression was disclosed by rescue UUU UGU GUA GUA CAAA-3′. W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 3 of 14 Quantitative real‑time polymerase chain reaction Cell colony formation assay (qRT‑PCR) HCT116 and SW620 cells diluted in RPMI-1640 (Pro- A miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was cell, Wuhan, China) or L15 media (Procell, Wuhan, firstly employed to isolate RNA. After that, cDNA was China) were grown in 6-well plates. After various treat- synthesized with a FastKing RT Kit (Tiangen, Beijing, ments, cells were continued to be cultured for about China) or Qiagen reverse transcription kit (Valencia, 14 days. During cell culture, media were renewed every CA, USA). Then, SuperReal PreMix Color (Tiangen, 3  days. Then, cell supernatant was removed, and para - Beijing, China) was utilized to detect the content of formaldehyde (Sigma, St. Louis, MO, USA) and crystal circRNA/miRNA/mRNA. Obtained data were assessed violet (Sigma, St. Louis, MO, USA) were dripped into −∆∆Ct with the 2 method with U6 or glyceraldehyde plates, respectively. Cell colony-forming ability was 3-phosphate dehydrogenase (GAPDH) as a reference. determined by analyzing the number of colonies. A col- The sequences of forward and reverse primers were ony was considered when cell numbers over 50. hsa_circ_0000231 5′-ACT TAG CAG CAG CTC CAC -3′ and 5′-CCA CTT CTG TCA GCC ATT -3′; miR-622 DNA content quantitation assay 5′ - AC A C T C C AG C T G G G A C AG T C T G C T G AG G T - 3′ Cells were collected, and eluted with phosphate buffer and 5′-TGG TGT CGT GGA GTCG-3′; GAPDH 5′-GGT solution (PBS; Procell, Wuhan, China). Afterwards, the CAC CAG GGC TGC TTT -3′ and 5′-GGA AGA TGG TGA cells were fixed with 70% ethanol (Millipore, Bradford, TGG GAT T-3′; U6 5′-CTC GCT TCG GCA GCACA-3′ MA, USA). RNase A (Solarbio, Beijing, China) was and 5′-AAC GCT TCA CGA ATT TGC GT-3′. incubated with the cells at 37  °C in water. Cells were stained with propidium iodide (PI; Solarbio, Beijing, China) in dark. Finally, samples were analyzed with a RNase R treatment assay flow cytometry (Thermo Fisher, Waltham, MA, USA). Cultured HCT116 and SW620 cells were harvested and RNA was isolated according to the method as described above. Then, obtained RNA was incubated Annexin V‑fluorescein isothiocyanate (Annexin V‑FITC) −1 with RNase R (RNase R+) (3  U  μg RNA; Geneseed, and PI double staining assay Guangzhou, China) and without RNase R (RNase Cell apoptosis was assessed with an Annexin V-FITC/ R-) at 37  °C, respectively. About 30  min later, RNeasy PI apoptosis detection kit (Solarbio, Beijing, China). In MinElute Cleaning Kit (Qiagen, Valencia, CA, USA) brief, harvested cells were suspended in Binding buffer was employed to purify RNA. Hsa_circ_0000231 con- (Solarbio). Cell supernatant was discarded by centrifug- tent was determined by qRT-PCR with linear GAPDH ing at 300g for 12  min, followed by the incubation with mRNA (linear mRNA) as a reference. Annexin V-FITC (Solarbio, Beijing, China) and PI (Solar- bio, Beijing, China) in dark. Finally, cells were diluted in PBS (Procell, Wuhan, China) and assessed with flow 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium cytometer (Thermo Fisher, Waltham, MA, USA). bromide (MTT) assay Cultivated HCT116 and SW620 cells were diluted in RPMI-1640 (Procell, Wuhan, China) and L15 media Caspase‑3 activity assay (Procell, Wuhan, China), respectively, and grown in A caspase-3 activity detection kit (Beyotime, Shanghai, 96-well plates. Sixteen hours later, cells were treated China) was employed to detect caspase-3 activity. Briefly, with Sev (1.7%, 3.4% or 5.1%) (Sigma, St. Louis, MO, cell supernatant was discarded and cells were collected USA), si-hsa_circ_0000231#1, si-hsa_circ_0000231#2, up by centrifuging. Then, lysis buffer (Beyotime, Shang - hsa_circ_0000231 or anti-miR-622 based on the defined hai, China) was utilized to lyse cells. Supernatant was purposes with 0% Sev (Control), si-NC, circ-NC or harvested by centrifugation. Detection buffer (Beyotime, anti-NC as a reference. At 48  h after treatment, MTT Shanghai, China) and acetyl-Asp-Glu-Val-Asp p-nitroan- solution (Beyotime, Shanghai, China) was incubated ilide were mixed with the samples, and the output of with cells for 4  h. Then, dimethyl sulfoxide (Sigma, wavelength at 405 nm was determined with a microplate St. Louis, MO, USA) was added into plates to dissolve reader (Thermo Fisher, Waltham, MA, USA). Finally, cas - formazan. Cell viability was determined after samples pase-3 activity was determined by assessing the output. were analyzed with a microplate reader (Thermo Fisher, Waltham, MA, USA) with a wavelength at 490 nm. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 4 of 14 Wound‑healing assay 24  h. Proteins were digested with proteinase K (Mil- Cell migration was demonstrated in this part. In short, lipore, Bradford, MA, USA), and the contents of hsa_ cells were grown in 6-well plates after diverse treat- circ_0000231 and miR-622 were detected by qRT-PCR. ments. Cell wounds were created when the confluence of cells reached approximately 100%. Cells were cul- In vivo assay tured in RPMI-1640 (Procell, Wuhan, China) or L15 The Vital River Laboratories (Beijing, China) provided media (Procell, Wuhan, China) without FBS (Procell, male BALB/c nude mice (5  weeks of age), and the mice Wuhan, China). After 24  h, the migratory capacity of were fed in a sterile environment. All mice were divided cells was determined by analyzing the width of wounds into four groups (n = 5, respectively). SW620 cells were under microscope (Nikon, Tokyo, Japan) at a 40× cultivated for 24  h after treatment of 5.1% Sev, and the magnification. cells were transfected with hsa_circ_0000231 or circ-NC. After 48 h, the cells were digested and diluted in 200 μL Transwell invasion assay PBS (Procell, Wuhan, China), which were hypodermi- The invasive ability of CRC cells was revealed with tran - cally inoculated right anterior temporal region of mice. swell chambers with Matrigel (Corning, Madison, New Tumor volume was measured every one day. Five days York, USA). In brief, HCT116 and SW620 cells were later, nude mice were sacrificed by intraperitoneal injec - −1 cultivated in the upper chambers supplemented with tion of xylazine (10  mg  kg ; Seebio Biotech, Shanghai, FBS-free RPMI-1640 (Procell, Wuhan, China) and L15 China) and the neoplasms were harvested. The weight media (Procell, Wuhan, China), respectively. RPMI-1640 of every tumor was measured. A part of each tumor was (Procell, Wuhan, China) and L15 media (Procell, Wuhan, kept for further assessment in RNA or protein level. The China) containing 15% FBS (Procell, Wuhan, China) Animal Care and Use Committee of the Chengyang Peo- were added into the lower chambers. At 24  h after cul- ple’s Hospital approved this research. ture, supernatant was removed, and cells were orderly incubated with paraformaldehyde (Sigma, St. Louis, MO, Western blot analysis USA) and crystal violet (Sigma, St. Louis, MO, USA). Tissues were lysed using RIPA buffer (Beyotime, Shang - Results were determined by counting cell numbers in the hai, China), and lysates were loaded onto 12% bis–tris- lower chambers under microscope (Nikon) with a 100× acrylamide gels (Thermo Fisher, Waltham, MA, USA). magnification. The separated protein bands were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bradford, Dual‑luciferase reporter assay MA, USA), and then immersed in 5% nonfat dry milk Circular RNA Interactome online database (https:// circi (Solarbio, Beijing, China). After that, the membranes n t e r a c t o m e . n i a . n i h . g o v / a p i / v 2 / m i r n a s e a r c h ? c i r c u l a r _ were incubated with anti-BCL2-associated x protein r na_ quer y= hsa_ c ir c_ 00002 31& mir na_ quer y= hsa- miR - (anti-Bax) (1:5000; Abcam, Cambridge, UK), anti-matrix 622& submit= miRNA+ Target+ Search) was firstly uti - metalloprotein 2 (anti-MMP2) (1:5000; Abcam, Cam- lized to predict the binding sites of hsa_circ_0000231 bridge, UK), anti-MMP9 (1:3000; Abcam, Cambridge, in miR-622. Then, the wild-type (wt) and mutant (mut) UK), anti-caspase 3 (anti-t-caspase 3) (1:5000; Abcam, plasmids of hsa_circ_0000231 were constructed by Cambridge, UK), anti-cleaved-caspase 3 (anti-C-caspase Geneseed Co., Ltd. (Guangzhou, China) and named as 3) (1:8000; Abcam, Cambridge, UK), anti-proliferating hsa_circ_0000231-wt and hsa_circ_0000231-mut, respec- cell nuclear antigen (anti-PCNA) (1:3000; Abcam, Cam- tively. Built plasmids were co-transfected into HCT116 bridge, UK), anti-MYC proto-oncogene, bHLH tran- and SW620 cells with miR-622 or NC with TurboFect scription factor (anti-c-Myc; 1:1000; Abcam, Cambridge, Reagent (Thermo Fisher, Waltham, MA, USA) based on UK), anti-KRAS proto-oncogene, GTPase (anti-K-RAS; the instruction of manufacturer. Forty-eight hours later, 1:1000; Abcam, Cambridge, UK), anti-B-Raf proto- luciferase activities were detected by a Dual-Lucy Assay oncogene, serine/threonine kinase (anti-BRAF; 1:2000; Kit (Solarbio, Beijing, China). Renilla luciferase activity Abcam, Cambridge, UK) and anti-GAPDH (1:15,000; was used as a reference. Abcam, Cambridge, UK), respectively. Then, the mem - branes were incubated with secondary antibody (1:8000; RNA immunoprecipitation (RIP) assay Abcam, Cambridge, UK). Finally, RapidStep ECL Reagent Cells were lysed with RIP lysis buffer (Millipore, Brad - (Millipore, Bradford, MA, USA) was used to visualize the ford, MA, USA). Then, the lysates were incubated with protein bands. GAPDH was chosen as a control. magnetic beads coated with the antibodies against argo- naute-2 (Anti-Ago2; Abcam, Cambridge, UK) or immu- noglobulin G (Anti-IgG; Abcam, Cambridge, UK) for W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 5 of 14 Statistical analysis high hsa_circ_0000231 expression had a low survival rate Data derived from three independent duplicate tests as compared to those with low hsa_circ_0000231 expres- were assessed by GraphPad Prism (GraphPad Software, sion (Fig.  1B), which suggested hsa_circ_0000231 might La Jolla, CA, USA), and expressed as means ± stand- act as an oncogene in CRC process. Subsequently, RNase ard deviations (SD). Significant differences in Spear - R treatment assay showed that hsa_circ_0000231 expres- man correlation analysis and overall survival curve were sion had no obvious change after RNase R treatment, compared with Spearman’s correlation test and log-rank but linear mRNA expression was dramatically decreased test, respectively. Additionally, significant differences (Fig.  1C), suggesting hsa_circ_0000231 was more stable were compared with two-tailed Student’s t-tests or Wil- than linear mRNA. Furthermore, the expression level of coxon rank-sum test between the two groups and with hsa_circ_0000231 was detected in HCT116 and SW620 one-way analysis of variance (ANOVA) with Tukey’s test cells, and it was found that hsa_circ_0000231 expression or Kruskal–Wallis test between the multiple groups. p was greatly upregulated in HCT116 and SW620 cells rel- value < 0.05 was deemed as statistical significance. ative to NCM460 cells (Fig. 1D). The above data demon - strated hsa_circ_0000231 might positively regulate CRC Results progression. Hsa_circ_0000231 expression was upregulated in the tissues of CRC patients with poor prognosis Sev repressed cell proliferation, migration and invasion, Hsa_circ_0000231 expression was firstly determined whereas induced cell apoptosis in CRC cells in CRC tissues, and results showed  that its expression The study then explored the impacts of Sev on CRC was dramatically increased in CRC tissues compared progression. HCT116 and SW620 cells were initially with paracancerous normal colorectal tissues (Fig.  1A). treated with Sev at various concentrations (1.7%, 3.4% Kaplan–Meier methods presented CRC patients with and 5.1%), and cell proliferative and metastatic abilities Fig. 1 Hsa_circ_0000231 was overexpressed in CRC tissues and cells with poor prognosis of CRC patients. A, D Hsa_circ_0000231 expression was detected by qRT-PCR in 47 pairs of CRC and matched healthy colorectal tissues as well as NCM460, HCT116 and SW620 cells. B The overall survival curve of CRC sufferers with high or low hsa_circ_0000231 expression was assessed by Kaplan–Meier methods. C RNase R treatment assay was employed to demonstrate hsa_circ_0000231 was more stable than linear mRNA. *p < 0.05 ( Wilcoxon rank-sum test, two-tailed Student’s t-tests and ANOVA with Tukey’s test) Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 6 of 14 Fig. 2 Sev treatment repressed cell proliferation and induced cell apoptosis in CRC. A, B The impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on cell viability and cell colony-forming ability were presented by MTT and cell colony formation assays, respectively. C The effects of Sev (0%, 1.7%, 3.4% and 5.1%) on cell cycle were determined by DNA content quantitation assay in HCT116 and SW620 cells. D The effects of Sev (0%, 1.7%, 3.4% and 5.1%) on cell apoptosis were determined by Annexin V-FITC and PI double staining assay in HCT116 and SW620 cells. E Caspase-3 activity was determined by caspase-3 activity assay in HCT116 and SW620 cells treated with Sev at various doses (0%, 1.7%, 3.4% and 5.1%). *p < 0.05 (ANOVA with Tukey’s test) as well as apoptotic rate were determined. MTT and concentration-dependently promoted the apopto- cell colony formation assays displayed that Sev expo- sis and caspase-3 activity of HCT116 and SW620 cells sure inhibited cell viability and cell colony-forming abil- (Fig.  2D, E). Additionally, Sev treatment inhibited cell ity in a dose-dependent manner (Fig.  2A, B). The cell migration and invasion of HCT116 and SW620 cells cycle of HCT116 and SW620 cells was also arrested in dose-dependent fashion (Fig.  3A, B). Sev treatment at G0/G1 phase after Sev treatment in a concentra- upregulated Bax protein expression and the value of tion-dependent manner (Fig.  2C). These findings sug - C-caspase 3/t-caspase 3, and downregulated the protein gested that Sev treatment repressed the proliferation of expression of MMP2 as well as MMP9 in a concentra- HCT116 and SW620 cells. On the contrary, Sev exposure tion-dependent manner (Fig. 3C). In support, the protein W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 7 of 14 Fig. 3 The migration and invasion of HCT116 and SW620 cells were repressed by Sev. A The influences of Sev (0%, 1.7%, 3.4% and 5.1%) on the migration of HCT116 and SW620 cells were revealed by wound-healing assay. B Transwell invasion assay was employed to demonstrate the impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on the invasion of HCT116 and SW620 cells. C The impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. D Hsa_ circ_0000231 expression was determined by qRT-PCR in HCT116 and SW620 cells treated with 0%, 1.7%, 3.4% and 5.1% of Sev. *p < 0.05 (ANOVA with Tukey’s test) expression of oncogenes including c-Myc, K-RAS and was notably downregulated after transfection of si- BRAF was dose-dependently reduced by Sev (Additional hsa_circ_0000231#1, si-hsa_circ_0000231#2 or si-hsa_ file  1: Figure S1A, B). The above data demonstrated Sev circ_0000231#3 in HCT116 and SW620 cells (Fig.  4A). could repress CRC process. Given the negative correla- si-hsa_circ_0000231#1 and si-hsa_circ_0000231#2 tion between hsa_circ_0000231 expression and survival were employed in subsequent study owing to their rate of CRC sufferers as well as the repressive impacts higher efficiency. Subsequently, our results presented of Sev on CRC progression, whether Sev regulated hsa_ hsa_circ_0000231 silencing suppressed cell viability circ_0000231 expression was further analyzed. As shown and colony-forming ability in HCT116 and SW620 cells in Fig.  3D, hsa_circ_0000231 expression was apparently (Fig.  4B, C). Hsa_circ_0000231 knockdown also induced decreased after Sev treatment in a dose-dependent man- cell arrested at G0/G1 phase and promoted cell apop- ner, suggesting that Sev might regulate CRC progression tosis (Fig.  4D, E). Meanwhile, the caspase-3 activity was by repressing hsa_circ_0000231. also promoted after hsa_circ_0000231 silencing (Fig. 4F), which further demonstrated that hsa_circ_0000231 Hsa_circ_0000231 silencing repressed cell proliferation, downregulation could induce cell apoptosis. The migra - migration and invasion, but induced cell apoptosis tory and invasive abilities of HCT116 and SW620 cells in HCT116 and SW620 cells were restrained after hsa_circ_0000231 absence (Fig. 4G, Considering the high hsa_circ_0000231 expression in H). Hsa_circ_0000231 silencing increased Bax protein HCT116 and SW620 cells, the small interfering RNAs expression and the value of C-caspase 3/t-caspase 3, and against hsa_circ_0000231 were built and their knock- decreased the protein expression levels of MMP2 as well down efficiency was determined. The data from qRT- as MMP9 (Fig.  4I). Thus, these evidences demonstrated PCR analysis displayed that hsa_circ_0000231 expression Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 8 of 14 Fig. 4 Hsa_circ_0000231 knockdown repressed CRC process. A The knockdown efficiency of hsa_circ_0000231#1, si-hsa_circ_0000231#2 and si-hsa_circ_0000231#3 was determined by qRT-PCR in HCT116 and SW620 cells. B, C The impacts of hsa_circ_0000231 silencing on the viability and colony-forming ability were revealed by MTT and cell colony formation assays, respectively. D The effect of hsa_circ_0000231 absence on cell cycle was demonstrated by DNA content quantitation assay. E The effect of hsa_circ_0000231 absence on cell apoptosis was demonstrated by Annexin V-FITC and PI double staining assay. F The influence of hsa_circ_0000231 silencing on caspase-3 activity was unveiled by caspase-3 activity assay. G, H Wound-healing and transwell invasion assays were performed to demonstrate the impacts of hsa_circ_0000231 downregulation on the migration and invasion of HCT116 and SW620 cells, respectively. I The impacts of hsa_circ_0000231 silencing on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey ’s test) that hsa_circ_0000231 positively regulated CRC reversed the repressive impact of Sev treatment on hsa_ progression. circ_0000231 expression in HCT116 and SW620 cells (Fig. 5A). Sev-mediated inhibitory impacts on cell viabil- Hsa_circ_0000231 attenuated Sev‑mediated effects on CRC ity and cell colony-forming ability were also restored after process hsa_circ_0000231 upregulation (Fig.  5B, C). Addition- Given the repressive role of hsa_circ_0000231 silencing ally, Sev treatment induced cell arrest at G0/G1 phase, on CRC progression, whether it participated in Sev-medi- whereas ectopic hsa_circ_0000231 expression impaired ated CRC process was subsequently explored; the effects this impact (Fig.  5D). Sev-induced cell apoptosis was between Sev treatment and hsa_circ_0000231 overex- also partly abolished after hsa_circ_0000231 overexpres- pression on CRC progression were analyzed. Results sion (Fig. 5E). Caspase-3 activity assay also presented the firstly exhibited that hsa_circ_0000231 overexpression promotion impact of Sev treatment on caspase-3 activity W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 9 of 14 Fig. 5 Hsa_circ_0000231 reversed Sev-mediated influences on CRC development. A The impacts between Sev treatment and hsa_circ_0000231 overexpression on hsa_circ_0000231 expression were detected by qRT-PCR in HCT116 and SW620 cells. B, C The impacts between Sev treatment and hsa_circ_0000231 overexpression on the viability and colony-forming ability of HCT116 and SW620 cells were determined by MTT and cell colony formation assays, respectively. D DNA content quantitation assay was performed to reveal the effects between Sev treatment and hsa_circ_0000231 overexpression on cell cycle. E Annexin V-FITC and PI double staining assay was conducted to show the effects between Sev treatment and hsa_circ_0000231 overexpression on the apoptosis of HCT116 and SW620 cells. F Caspase-3 activity assay was used to demonstrate the impacts between Sev treatment and ectopic hsa_circ_0000231 expression on caspase-3 activity. G, H The impacts between Sev treatment and hsa_circ_0000231 overexpression on the migration and invasion of HCT116 and SW620 cells were revealed by wound-healing and transwell invasion assays, respectively. I The effects between Sev treatment and hsa_circ_0000231 overexpression on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey’s test) was hindered after transfection of hsa_circ_0000231 and invasion, and induced cell apoptosis by regulating (Fig. 5F). Furthermore, Sev exposure inhibited cell migra- hsa_circ_0000231. tion and invasion, whereas these impacts were restrained by enforced hsa_circ_0000231 expression (Fig.  5G, H). Hsa_circ_0000231 acted as a sponge of miR‑622 Sev-mediated effects on the protein expression of Bax, In order to reveal the regulatory mechanism of hsa_ MMP2 and MMP9 as well as the value of C-caspase circ_0000231 in CRC process, the miRNA with the 3/t-caspase 3, were attenuated after hsa_circ_0000231 ability to bind to hsa_circ_0000231 was sought. Cir- overexpression (Fig.  5I). Taken together, the above data cular RNA Interactome online database presented suggested that Sev repressed cell proliferation, migration hsa_circ_0000231 contained the binding sequence of miR-622 (Fig.  6A). To prove the binding relationship Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 10 of 14 Fig. 6 Hsa_circ_0000231 directly interacted with miR-622. A Circular RNA Interactome online database was performed to predict the putative relationship between hsa_circ_0000231 and miR-622. B, C Dual-luciferase reporter and RIP assays were carried out to prove that hsa_circ_0000231 directly bound to miR-622. D The impacts between Sev treatment and hsa_circ_0000231 overexpression on miR-622 expression were demonstrated by qRT-PCR in HCT116 and SW620 cells. E, G MiR-622 expression was detected by qRT-PCR in 47 pairs of CRC and paracancerous normal colorectal tissues as well as NCM460, HCT116 and SW620 cells. F Spearman correlation analysis was conducted to disclose the linear relationship between hsa_circ_0000231 and miR-622 expression in CRC tissues. H The influences of various concentrations of Sev (0%, 1.7%, 3.4% and 5.1%) on miR-622 expression were determined by qRT-PCR in HCT116 and SW620 cells. *p < 0.05 (two-tailed Student’s t-tests, Wilcoxon rank-sum test, ANOVA with Tukey’s test and Spearman’s correlation test) between hsa_circ_0000231 and miR-622, dual- expression and miR-622 expression was observed luciferase reporter and RIP assays were employed. (Fig.  6F). Furthermore, the impact of Sev exposure Results exhibited the relative luciferase activity on miR-622 expression was unveiled in HCT116 and was dramatically repressed after co-transfection of SW620 cells, and we found that miR-622 expression was hsa_circ_0000231-wt and miR-622 in HCT116 and dose-dependently increased by Sev (Fig. 6H). Thus, our SW620 cells, whereas that had no apparent change in evidences demonstrated hsa_circ_0000231 was directly hsa_circ_0000231-mut and miR-622 group (Fig.  6B). associated with miR-622, and Sev could upregulate RIP assay also presented that both hsa_circ_0000231 miR-622 expression by regulating hsa_circ_0000231. and miR-622 were notably enriched by Anti-Ago2 as compared to Anti-IgG in HCT116 and SW620 cells Hsa_circ_0000231 regulated cell proliferation, apoptosis, (Fig.  6C). Subsequently, qRT-PCR data revealed miR- migration and invasion via binding to miR‑622 622 expression was dramatically upregulated after Sev Given the bound relationship between hsa_circ_0000231 treatment, but this impact was reversed by enforced and miR-622, whether hsa_circ_0000231 regulated CRC hsa_circ_0000231 expression (Fig. 6D). Our results also progression by interacting with miR-622 was investi- showed that miR-622 expression was apparently down- gated. Our data firstly showed that hsa_circ_0000231 regulated in CRC tissues and HCT116 and SW620 silencing apparently upregulated miR-622 expression, cells when compared with normal colorectal tissues whereas miR-622 inhibitors attenuated this impact and NCM460 cells, respectively (Fig.  6E, G). Addition- (Fig.  7A). Subsequently, hsa_circ_0000231 knockdown ally, a negative correlation between hsa_circ_0000231 repressed cell viability and cell colony-forming ability, W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 11 of 14 Fig. 7 Hsa_circ_0000231 mediated CRC development by binding to miR-622. HCT116 and SW620 cells were transfected with si-NC, si-hsa_ circ_0000231#1, si-hsa_circ_0000231#1 + anti-NC and si-hsa_circ_0000231#1 + anti-miR-622, respectively. A MiR-622 expression was detected by qRT-PCR in HCT116 and SW620 cells. B, C The viability and colony-forming ability of HCT116 and SW620 cells were detected by MTT and colony-forming assays, respectively. D, E Cell cycle and apoptosis were detected by DNA content quantitation assay and Annexin V-FITC and PI double staining assay, respectively, in HCT116 and SW620 cells. F Caspase-3 activity was detected by caspase-3 activity assay in HCT116 and SW620 cells. G, H The migration and invasion of HCT116 and SW620 cells were detected by wound-healing and transwell invasion assays, respectively. I Western blot analysis was performed to reveal the influences between hsa_circ_0000231 silencing and miR-622 downregulation on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey’s test) which were reversed after downregulation of miR-622 were also repressed by si-hsa_circ_0000231#1; however, (Fig.  7B, C). Data also presented that hsa_circ_0000231 these influences were restored after miR-622 down - silencing induced cell arrest at G0/G1 phase and cell regulation (Fig.  7G, H). The effects of hsa_circ_0000231 apoptosis, but these effects were restored after transfec - downregulation on the protein expression of Bax, MMP2 tion of anti-miR-622 (Fig. 7D, E). In order to further ana- and MMP9 as well as the value of C-caspase 3/t-caspase lyze the impacts between hsa_circ_0000231 and miR-622 3 were attenuated after transfection of anti-miR-622 on cell apoptosis, caspase-3 activity assay was performed. (Fig.  7I). Collectively, the above data demonstrated that Results exhibited that hsa_circ_0000231 downregula- hsa_circ_0000231 could modulate CRC progression by tion promoted caspase-3 activity, while miR-622 inhibi- interacting with miR-622. tors restrained this impact (Fig.  7F). Additionally, the migration and invasion of HCT116 and SW620 cells Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 12 of 14 Fig. 8 Sev treatment repressed tumor formation by repressing hsa_circ_0000231 in vivo. A, B The impacts between Sev and hsa_circ_0000231 overexpression on tumor volume and weight were revealed. C The effects between Sev treatment and enforced hsa_circ_0000231 expression on the levels of hsa_circ_0000231 and miR-622 were determined by qRT-PCR. D Western blot analysis was employed to reveal the influences between Sev treatment and ectopic hsa_circ_0000231 expression on the expression of PCNA protein and the value of C-caspase 3/t-caspase 3. *p < 0.05 (ANOVA with Tukey’s test) Hsa_circ_0000231 overexpression restrained Sev‑mediated hsa_circ_0000231. These data demonstrated  that Sev impacts on tumor formation in vivo repressed tumor growth by controlling hsa_circ_0000231 To further affirm the promotion influences of hsa_ expression in vivo. circ_0000231 on Sev-mediated CRC process, in  vivo assay was conducted. Results showed Sev treatment Discussion reduced tumor volume and weight, whereas these effects Emerging evidences suggest that anesthetics can influ - were reversed by hsa_circ_0000231 overexpression ence cancer evolution [21]. Sev was found to serve as a (Fig.  8A, B). Additionally, we found hsa_circ_0000231 cancer suppressor in CRC through various mechanisms. expression was obviously downregulated, while miR- As reported by previous researchers, Sev restrained cell 622 expression was dramatically upregulated after Sev metastasis of CRC via modulating extracellular signal- treatment in the excised tissues; however, these impacts regulated kinase/matrix metalloproteinase-9 signal path were impaired by enforced hsa_circ_0000231 expression through increasing miR-203 [22]. Additionally, Sev sup- (Fig.  8C). Furthermore, the protein expression of prolif- pressed cell metastasis via regulating miR-34a/ADAM eration-related maker PCNA was decreased after Sev metallopeptidase domain 10 (ADAM10) pathway in CRC treatment, which was reversed after hsa_circ_0000231 [23]. In this paper, we found that Sev repressed CRC overexpression (Fig. 8D). The value of C-caspase 3/t-cas - development. Different from the above results, Sev hin - pase 3 was increased by Sev, but this result was reversed dered CRC progression by regulating hsa_circ_0000231/ after upregulation of hsa_circ_0000231 (Fig.  8D), sug- miR-622 axis. In this research, to reveal the mecha- gesting Sev could induce cell apoptosis by regulating nism of Sev in mediating CRC process, the reasonable W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 13 of 14 concentration of Sev was firstly determined. Thus, the cancer [32] and renal cell carcinoma [33]. In CRC pro- impacts of various concentrations of Sev (0%, 1.7%, 3.4% cess, miR-622 could repress angiogenesis [34] and radio- and 5.1%) on CRC progression were explored. Results sensitivity [35]. In this study, a decreased expression of showed that Sev repressed cell viability, colony-forming miR-622 was observed in CRC samples and cells, and ability and metastasis, and upregulated apoptosis rate as miR-622 silencing promoted cell migration and invasion, well as induced cell arrest in a concentration-dependent which were supported by the current evidences [20, 36]. fashion. Also, Sev dose-dependently reduced the expres- Besides, miR-622 inhibitors facilitated cellular prolif- sion of c-Myc, K-RAS and BRAF. Based on these results, eration and repressed cellular apoptosis. The impacts of CRC cells were treated with 5.1% Sev in subsequent miR-622 inhibitors on hsa_circ_0000231 absence-medi- research. ated CRC process also suggested that hsa_circ_0000231 Previous research revealed that circRNAs acted modulated CRC development through sponging as tumor suppressors or promoters in CRC progres- miR-622. sion. For example, circ_001680 [24], circ_101555 Additionally, our results supported that miR-622 [25] and circ_0053277 [26] contributed to CRC pro- expression was apparently upregulated after Sev cess, but circ_0009361 [27], circ_0020397 [28] and treatment, which was reversed by enforced hsa_ circ_0007534 [29] hindered CRC development via circ_0000231 expression. This result also suggested that regulating cell proliferation, migration or apoptosis. Sev could increase miR-622 expression by regulating In this paper, it was found that hsa_circ_0000231 con- hsa_circ_0000231. tent was increased in HCT116 and SW620 cells and the Collectively, ectopic hsa_circ_0000231 expression specimens of CRC patients, and its absence hindered restrained Sev-mediated CRC progression. Besides, CRC development by repressing cellular abilities in hsa_circ_0000231 bound to miR-622. MiR-622 inhibi- proliferation and metastasis as well as enhancing the tors were further proved to hinder hsa_circ_0000231 capacity in apoptosis, which were approved by the find - absence-mediated repressive impacts on CRC process. ings of Liu et  al. [30]. Beyond that, an inverse correla- Summarily, hsa_circ_0000231 restrained Sev-triggered tion between hsa_circ_0000231 level and the survival repressive influences on CRC progression via binding time of CRC sufferers was shown in this paper. Given to miR-622. This finding not only provides a theoreti - the repressive impacts both Sev and the silencing of cal foundation for further studying the application of hsa_circ_0000231 on CRC progression, Sev-mediated Sev in CRC surgery, but also lays a basis for researching effect on hsa_circ_0000231 expression was explored circRNA-directed CRC therapy. further. Data exhibited that hsa_circ_0000231 was dose-dependently downregulated by Sev. Thus, we sug - Supplementary Information gested that hsa_circ_0000231 might be involved in Sev- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40709- 021- 00145-6. mediated CRC progression. To elaborate on that, the overexpression plasmid of hsa_circ_0000231 was trans- Additional file 1: Figure S1. The effects of Sev (0%, 1.7%, 3.4% and 5.1%) fected into Sev-stimulated HCT116 and SW620 cells on the protein expression of c-Myc, K-RAS and BRAF were detected by with control group, and same indices were detected. western blot in both HCT116 (A) and SW620 cells (B). *p < 0.05 (ANOVA As expected, results exhibited that hsa_circ_0000231 with Tukey’s test). overexpression attenuated Sev-mediated impacts on CRC process. Meanwhile, in  vivo assay also showed Acknowledgements enforced hsa_circ_0000231 expression restrained Sev- None. aroused inhibitive influences on tumor formation. The Authors’ contributions inhibition impact of Sev on PCNA protein expres- JW performed the experiments and drafted the manuscript, SL and GZ con- sion and upregulation influence of that on the value of ducted the study. HH collected and analyzed the data. All authors read and approved the final manuscript. C-caspase 3/t-caspase 3 were also abolished after hsa_ circ_0000231 overexpression in  vivo. These evidences Funding suggested that Sev could repress CRC development via None. modulating hsa_circ_0000231. Availability of data and materials CircRNAs commonly interact with miRNAs in can- Please contact the correspondence author for the data request. cer progression [31]. Thus, the miRNA bound to hsa_ circ_0000231 was assessed. Our results presented that Declarations hsa_circ_0000231 directly interacted with miR-622. As Ethics approval and consent participate reported in previous research efforts, miR-622 acted as Written informed consent was obtained from patients with approval by the an anti-oncogene in cancer progression, such as gastric Institutional Review Board in The Chengyang People’s Hospital. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 14 of 14 Competing interests 17. Liu Z, Zhou Y, Liang G, Ling Y, Tan W, Tan L, et al. 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Sevoflurane inhibits malignant progression of colorectal cancer via hsa_circ_0000231-mediated miR-622

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Copyright © The Author(s) 2021
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2241-5793
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10.1186/s40709-021-00145-6
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Abstract

Background: Sevoflurane (Sev), a commonly used volatile anesthetic, has been reported to inhibit the process of colorectal cancer (CRC). Circular RNAs (circRNAs) are revealed to participate in the pathogenesis of CRC. This study aims to reveal the mechanism of hsa_circ_0000231 in Sev-mediated CRC progression. Methods: The expression of hsa_circ_0000231 and microRNA-622 (miR-622) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Protein level was determined by western blot analysis. Cell proliferation was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ), cell colony formation and DNA content quantitation assays. Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate and propidium iodide double staining and caspase 3 activity assays. Cell migration and invasion were investigated by wound-healing and transwell invasion assays, respectively. The putative relationship between hsa_circ_0000231 and miR-622 was predicted by circular RNA Interactome online database, and identified by dual-luciferase reporter and RNA immuno - precipitation assays. The impacts of hsa_circ_0000231 on Sev-mediated tumor formation in vivo were presented by in vivo assay. Results: Hsa_circ_0000231 expression was upregulated, while miR-622 was downregulated in CRC tissues and cells compared with control groups. Sev treatment decreased hsa_circ_0000231 expression, but increased miR-622 expres- sion in CRC cells. Sev treatment suppressed cell proliferation, migration and invasion, and induced cell apoptosis. Hsa_ circ_0000231 overexpression restored Sev-mediated CRC progression in vitro. Additionally, hsa_circ_0000231 acted as a sponge of miR-622, and miR-622 inhibitors reversed the impacts of hsa_circ_0000231 silencing on CRC process. Furthermore, Sev treatment inhibited tumor growth by regulating hsa_circ_0000231 in vivo. Conclusion: Hsa_circ_0000231 attenuated Sev-aroused repression impacts on CRC development by sponging miR- 622. This findings may provide an appropriate anesthetic protocol for CRC sufferers undergoing surgery. Keywords: Sev, hsa_circ_0000231, miR-622, CRC Background Colorectal cancer (CRC) is a general pernicious can- cer worldwide with high mortality [1]. CRC ranks the third in incidence and the second in death rate among various cancers for the combination of men and women *Correspondence: wjpwjpwjpwang@163.com [2]. The data studied in 2017 presented nearly half of Department of Anaesthesiology, The Chengyang People’s Hospital, 1.8 million CRC patients died [3]. At present, despite No.76 Zhengyang Road, Chengyang District, Qingdao 266109, Shandong Province, China brilliant achievements have been achieved in disclos- Full list of author information is available at the end of the article ing the pathogenesis of CRC, the high mortality of CRC © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 2 of 14 sufferers is still intently correlated with recurrence and experiments. Additionally, whether hsa_circ_0000231 metastasis [4, 5]. Major therapeutic manners of CRC bound to miR-622 was demonstrated. are operating together with chemotherapy, radiother- apy or targeted treatment [6]. Numerous studies have Methods verified that Sevoflurane (Sev), a frequently utilized Specimen collection and the ethics committee inhaled anesthetic, plays significant part in cancer pro - Forty-seven pairs of human CRC tissues and matched cess. For instances, Liu and his colleagues explained healthy colorectal tissues were collected up from CRC Sev repressed cell growth via increasing microRNA-203 sufferers in the Chengyang People’s Hospital, and the (miR-203) expression in breast cancer [7]. Kang et  al. written informed consent was signed by the CRC cases also reported Sev had repressive impacts on cell prolif- before surgery. Obtained tissues were stored at −  80  °C. eration and invasion through mediating mitogen-acti- The Ethics Committee of the Chengyang People’s Hospi - vated protein kinase-related pathway in ovarian cancer tal approved this study. [8]. In this paper, the molecular mechanism of CRC development mediated by Sev was revealed. Cell culture and exposure to Sev Circular RNA (circRNA) is a novel noncoding Human CRC cell lines (HCT116 and SW620) and nor- RNA, characterised by high stability, high expression mal human colonic epithelial cell line NCM460 were and good conservatism [9]. An increasing number purchased from Procell (Wuhan, China). HCT116 and of research efforts have reported that circRNAs are NCM460 cells were grown in Roswell Park Memorial involved in the evolution of various cancers, such as Institute-1640 (RPMI-1640; Procell, Wuhan, China), and lung carcinoma [10], glioma [11], bladder cancer [12] SW620 cells were cultivated in Leibovitz’s L15 media and CRC [13]. Previous research also presented that (L15; Procell, Wuhan, China) at 37 °C in humid condition circRNAs were related to Sev-mediated cancer pro- with 5% CO . Media were supplemented with 10% fetal cess. For example, Li and his colleagues indicated Sev bovine serum (FBS; Procell, Wuhan, China) and 1% peni- repressed cell proliferation and metastasis by decreas- cillin/streptomycin (Procell, Wuhan, China). ing circ_0002755 and circ_0012129 expression in For Sev treatment, HCT116 and SW620 cells were glioma [14]. He et  al. revealed that Sev restrained cell placed in a sealed container with humid atmosphere of proliferation and induced cell apoptosis via controlling 37  °C. Sev (Seebio Biotech, Shanghai) was mixed with circ-3-hydroxy-3-methylglutaryl-CoA synthase 1 (circ- 95% air and 5% CO using a volatilization tank, and gas HMGCS1) in colon cancer [15]. In this study, we found monitor was used to adjust concentrations of Sev to 1.7%, that circ_0000231 (hsa_circ_0000231) was increased 3.4% and 5.1%, respectively. Then, the cells were treated in CRC specimen and cell lines, but decreased in with the various concentrations of Sev for 30 min. Sev-treated CRC cells. Thus, we hypothesized that hsa_circ_0000231 might participate in modulating Sev- Plasmid construction and oligonucleotide synthesis mediated CRC progression. The small RNAs against hsa_circ_0000231 (si-hsa_ MiRNA is a small RNA with about 20 nucleotides, circ_0000231#1, si-hsa_circ_0000231#2 and si-hsa_ commonly altering mRNA levels via binding to their circ_0000231#3), miR-622 mimics (miR-622), miR-622 non-coding regions [16]. Multiple data exhibited miR- inhibitors (anti-miR-622) and control groups (si-NC, NAs regulated cancer progression through interacting NC and anti-NC) were synthesized by GenePharma with circRNAs, including CRC. For example, enforced (Shanghai, China). The overexpression plasmids of hsa_ circ_100395 expression hindered cell proliferation and circ_0000231 (hsa_circ_0000231) and control group metastasis via sponging miR-1228 in lung cancer [10]. (circ-NC) were built by Geneseed (Guangzhou, China). Circ_001783 silencing repressed cell proliferation and Plasmids or oligonucleotides were transfected into cells invasion through binding to miR-200c-3p in breast can- with TurboFect Reagent (Thermo Fisher, Waltham, MA, cer [17]. In CRC, circ_0026344 modulated cell metas- USA). The synthesized sequences of oligonucleotides tasis, growth and apoptosis via sponging miR-183, were si-hsa_circ_0000231#1 5′-ACT GAA CAG ATA AGG miR-21 or miR-31 [18, 19]. In this paper, we found that GTT TAA-3′, si-hsa_circ_0000231#2 5′-CTG AAC AGA hsa_circ_0000231 possessed the binding sequence of TAA GGG TTT AAA-3′, si-hsa_circ_0000231#3 5′-CAC miR-622, which has been reported to act as a repressor in TGA ACA GAT AAG GGT TTA-3′, miR-622 5′-ACA GUC CRC progression [20].UGC UGA GGU UGG AGC-3′, anti-miR-622 5′-GCU Herein, the impacts of hsa_circ_0000231 silencing on CCA ACC UCA GCA GAC UGU-3′, si-NC 5′-CCT CTA cell proliferation, metastasis and apoptosis were con-CCT GTC GCT GAG CTG TAA T-3′, NC 5′-UUU GUA firmed. Whether hsa_circ_0000231 was involved in CUA CAC AAA AGU ACUG-3′ and anti-NC 5′-CAG UAC Sev-mediated CRC progression was disclosed by rescue UUU UGU GUA GUA CAAA-3′. W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 3 of 14 Quantitative real‑time polymerase chain reaction Cell colony formation assay (qRT‑PCR) HCT116 and SW620 cells diluted in RPMI-1640 (Pro- A miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) was cell, Wuhan, China) or L15 media (Procell, Wuhan, firstly employed to isolate RNA. After that, cDNA was China) were grown in 6-well plates. After various treat- synthesized with a FastKing RT Kit (Tiangen, Beijing, ments, cells were continued to be cultured for about China) or Qiagen reverse transcription kit (Valencia, 14 days. During cell culture, media were renewed every CA, USA). Then, SuperReal PreMix Color (Tiangen, 3  days. Then, cell supernatant was removed, and para - Beijing, China) was utilized to detect the content of formaldehyde (Sigma, St. Louis, MO, USA) and crystal circRNA/miRNA/mRNA. Obtained data were assessed violet (Sigma, St. Louis, MO, USA) were dripped into −∆∆Ct with the 2 method with U6 or glyceraldehyde plates, respectively. Cell colony-forming ability was 3-phosphate dehydrogenase (GAPDH) as a reference. determined by analyzing the number of colonies. A col- The sequences of forward and reverse primers were ony was considered when cell numbers over 50. hsa_circ_0000231 5′-ACT TAG CAG CAG CTC CAC -3′ and 5′-CCA CTT CTG TCA GCC ATT -3′; miR-622 DNA content quantitation assay 5′ - AC A C T C C AG C T G G G A C AG T C T G C T G AG G T - 3′ Cells were collected, and eluted with phosphate buffer and 5′-TGG TGT CGT GGA GTCG-3′; GAPDH 5′-GGT solution (PBS; Procell, Wuhan, China). Afterwards, the CAC CAG GGC TGC TTT -3′ and 5′-GGA AGA TGG TGA cells were fixed with 70% ethanol (Millipore, Bradford, TGG GAT T-3′; U6 5′-CTC GCT TCG GCA GCACA-3′ MA, USA). RNase A (Solarbio, Beijing, China) was and 5′-AAC GCT TCA CGA ATT TGC GT-3′. incubated with the cells at 37  °C in water. Cells were stained with propidium iodide (PI; Solarbio, Beijing, China) in dark. Finally, samples were analyzed with a RNase R treatment assay flow cytometry (Thermo Fisher, Waltham, MA, USA). Cultured HCT116 and SW620 cells were harvested and RNA was isolated according to the method as described above. Then, obtained RNA was incubated Annexin V‑fluorescein isothiocyanate (Annexin V‑FITC) −1 with RNase R (RNase R+) (3  U  μg RNA; Geneseed, and PI double staining assay Guangzhou, China) and without RNase R (RNase Cell apoptosis was assessed with an Annexin V-FITC/ R-) at 37  °C, respectively. About 30  min later, RNeasy PI apoptosis detection kit (Solarbio, Beijing, China). In MinElute Cleaning Kit (Qiagen, Valencia, CA, USA) brief, harvested cells were suspended in Binding buffer was employed to purify RNA. Hsa_circ_0000231 con- (Solarbio). Cell supernatant was discarded by centrifug- tent was determined by qRT-PCR with linear GAPDH ing at 300g for 12  min, followed by the incubation with mRNA (linear mRNA) as a reference. Annexin V-FITC (Solarbio, Beijing, China) and PI (Solar- bio, Beijing, China) in dark. Finally, cells were diluted in PBS (Procell, Wuhan, China) and assessed with flow 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium cytometer (Thermo Fisher, Waltham, MA, USA). bromide (MTT) assay Cultivated HCT116 and SW620 cells were diluted in RPMI-1640 (Procell, Wuhan, China) and L15 media Caspase‑3 activity assay (Procell, Wuhan, China), respectively, and grown in A caspase-3 activity detection kit (Beyotime, Shanghai, 96-well plates. Sixteen hours later, cells were treated China) was employed to detect caspase-3 activity. Briefly, with Sev (1.7%, 3.4% or 5.1%) (Sigma, St. Louis, MO, cell supernatant was discarded and cells were collected USA), si-hsa_circ_0000231#1, si-hsa_circ_0000231#2, up by centrifuging. Then, lysis buffer (Beyotime, Shang - hsa_circ_0000231 or anti-miR-622 based on the defined hai, China) was utilized to lyse cells. Supernatant was purposes with 0% Sev (Control), si-NC, circ-NC or harvested by centrifugation. Detection buffer (Beyotime, anti-NC as a reference. At 48  h after treatment, MTT Shanghai, China) and acetyl-Asp-Glu-Val-Asp p-nitroan- solution (Beyotime, Shanghai, China) was incubated ilide were mixed with the samples, and the output of with cells for 4  h. Then, dimethyl sulfoxide (Sigma, wavelength at 405 nm was determined with a microplate St. Louis, MO, USA) was added into plates to dissolve reader (Thermo Fisher, Waltham, MA, USA). Finally, cas - formazan. Cell viability was determined after samples pase-3 activity was determined by assessing the output. were analyzed with a microplate reader (Thermo Fisher, Waltham, MA, USA) with a wavelength at 490 nm. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 4 of 14 Wound‑healing assay 24  h. Proteins were digested with proteinase K (Mil- Cell migration was demonstrated in this part. In short, lipore, Bradford, MA, USA), and the contents of hsa_ cells were grown in 6-well plates after diverse treat- circ_0000231 and miR-622 were detected by qRT-PCR. ments. Cell wounds were created when the confluence of cells reached approximately 100%. Cells were cul- In vivo assay tured in RPMI-1640 (Procell, Wuhan, China) or L15 The Vital River Laboratories (Beijing, China) provided media (Procell, Wuhan, China) without FBS (Procell, male BALB/c nude mice (5  weeks of age), and the mice Wuhan, China). After 24  h, the migratory capacity of were fed in a sterile environment. All mice were divided cells was determined by analyzing the width of wounds into four groups (n = 5, respectively). SW620 cells were under microscope (Nikon, Tokyo, Japan) at a 40× cultivated for 24  h after treatment of 5.1% Sev, and the magnification. cells were transfected with hsa_circ_0000231 or circ-NC. After 48 h, the cells were digested and diluted in 200 μL Transwell invasion assay PBS (Procell, Wuhan, China), which were hypodermi- The invasive ability of CRC cells was revealed with tran - cally inoculated right anterior temporal region of mice. swell chambers with Matrigel (Corning, Madison, New Tumor volume was measured every one day. Five days York, USA). In brief, HCT116 and SW620 cells were later, nude mice were sacrificed by intraperitoneal injec - −1 cultivated in the upper chambers supplemented with tion of xylazine (10  mg  kg ; Seebio Biotech, Shanghai, FBS-free RPMI-1640 (Procell, Wuhan, China) and L15 China) and the neoplasms were harvested. The weight media (Procell, Wuhan, China), respectively. RPMI-1640 of every tumor was measured. A part of each tumor was (Procell, Wuhan, China) and L15 media (Procell, Wuhan, kept for further assessment in RNA or protein level. The China) containing 15% FBS (Procell, Wuhan, China) Animal Care and Use Committee of the Chengyang Peo- were added into the lower chambers. At 24  h after cul- ple’s Hospital approved this research. ture, supernatant was removed, and cells were orderly incubated with paraformaldehyde (Sigma, St. Louis, MO, Western blot analysis USA) and crystal violet (Sigma, St. Louis, MO, USA). Tissues were lysed using RIPA buffer (Beyotime, Shang - Results were determined by counting cell numbers in the hai, China), and lysates were loaded onto 12% bis–tris- lower chambers under microscope (Nikon) with a 100× acrylamide gels (Thermo Fisher, Waltham, MA, USA). magnification. The separated protein bands were electrotransferred onto polyvinylidene fluoride membranes (Millipore, Bradford, Dual‑luciferase reporter assay MA, USA), and then immersed in 5% nonfat dry milk Circular RNA Interactome online database (https:// circi (Solarbio, Beijing, China). After that, the membranes n t e r a c t o m e . n i a . n i h . g o v / a p i / v 2 / m i r n a s e a r c h ? c i r c u l a r _ were incubated with anti-BCL2-associated x protein r na_ quer y= hsa_ c ir c_ 00002 31& mir na_ quer y= hsa- miR - (anti-Bax) (1:5000; Abcam, Cambridge, UK), anti-matrix 622& submit= miRNA+ Target+ Search) was firstly uti - metalloprotein 2 (anti-MMP2) (1:5000; Abcam, Cam- lized to predict the binding sites of hsa_circ_0000231 bridge, UK), anti-MMP9 (1:3000; Abcam, Cambridge, in miR-622. Then, the wild-type (wt) and mutant (mut) UK), anti-caspase 3 (anti-t-caspase 3) (1:5000; Abcam, plasmids of hsa_circ_0000231 were constructed by Cambridge, UK), anti-cleaved-caspase 3 (anti-C-caspase Geneseed Co., Ltd. (Guangzhou, China) and named as 3) (1:8000; Abcam, Cambridge, UK), anti-proliferating hsa_circ_0000231-wt and hsa_circ_0000231-mut, respec- cell nuclear antigen (anti-PCNA) (1:3000; Abcam, Cam- tively. Built plasmids were co-transfected into HCT116 bridge, UK), anti-MYC proto-oncogene, bHLH tran- and SW620 cells with miR-622 or NC with TurboFect scription factor (anti-c-Myc; 1:1000; Abcam, Cambridge, Reagent (Thermo Fisher, Waltham, MA, USA) based on UK), anti-KRAS proto-oncogene, GTPase (anti-K-RAS; the instruction of manufacturer. Forty-eight hours later, 1:1000; Abcam, Cambridge, UK), anti-B-Raf proto- luciferase activities were detected by a Dual-Lucy Assay oncogene, serine/threonine kinase (anti-BRAF; 1:2000; Kit (Solarbio, Beijing, China). Renilla luciferase activity Abcam, Cambridge, UK) and anti-GAPDH (1:15,000; was used as a reference. Abcam, Cambridge, UK), respectively. Then, the mem - branes were incubated with secondary antibody (1:8000; RNA immunoprecipitation (RIP) assay Abcam, Cambridge, UK). Finally, RapidStep ECL Reagent Cells were lysed with RIP lysis buffer (Millipore, Brad - (Millipore, Bradford, MA, USA) was used to visualize the ford, MA, USA). Then, the lysates were incubated with protein bands. GAPDH was chosen as a control. magnetic beads coated with the antibodies against argo- naute-2 (Anti-Ago2; Abcam, Cambridge, UK) or immu- noglobulin G (Anti-IgG; Abcam, Cambridge, UK) for W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 5 of 14 Statistical analysis high hsa_circ_0000231 expression had a low survival rate Data derived from three independent duplicate tests as compared to those with low hsa_circ_0000231 expres- were assessed by GraphPad Prism (GraphPad Software, sion (Fig.  1B), which suggested hsa_circ_0000231 might La Jolla, CA, USA), and expressed as means ± stand- act as an oncogene in CRC process. Subsequently, RNase ard deviations (SD). Significant differences in Spear - R treatment assay showed that hsa_circ_0000231 expres- man correlation analysis and overall survival curve were sion had no obvious change after RNase R treatment, compared with Spearman’s correlation test and log-rank but linear mRNA expression was dramatically decreased test, respectively. Additionally, significant differences (Fig.  1C), suggesting hsa_circ_0000231 was more stable were compared with two-tailed Student’s t-tests or Wil- than linear mRNA. Furthermore, the expression level of coxon rank-sum test between the two groups and with hsa_circ_0000231 was detected in HCT116 and SW620 one-way analysis of variance (ANOVA) with Tukey’s test cells, and it was found that hsa_circ_0000231 expression or Kruskal–Wallis test between the multiple groups. p was greatly upregulated in HCT116 and SW620 cells rel- value < 0.05 was deemed as statistical significance. ative to NCM460 cells (Fig. 1D). The above data demon - strated hsa_circ_0000231 might positively regulate CRC Results progression. Hsa_circ_0000231 expression was upregulated in the tissues of CRC patients with poor prognosis Sev repressed cell proliferation, migration and invasion, Hsa_circ_0000231 expression was firstly determined whereas induced cell apoptosis in CRC cells in CRC tissues, and results showed  that its expression The study then explored the impacts of Sev on CRC was dramatically increased in CRC tissues compared progression. HCT116 and SW620 cells were initially with paracancerous normal colorectal tissues (Fig.  1A). treated with Sev at various concentrations (1.7%, 3.4% Kaplan–Meier methods presented CRC patients with and 5.1%), and cell proliferative and metastatic abilities Fig. 1 Hsa_circ_0000231 was overexpressed in CRC tissues and cells with poor prognosis of CRC patients. A, D Hsa_circ_0000231 expression was detected by qRT-PCR in 47 pairs of CRC and matched healthy colorectal tissues as well as NCM460, HCT116 and SW620 cells. B The overall survival curve of CRC sufferers with high or low hsa_circ_0000231 expression was assessed by Kaplan–Meier methods. C RNase R treatment assay was employed to demonstrate hsa_circ_0000231 was more stable than linear mRNA. *p < 0.05 ( Wilcoxon rank-sum test, two-tailed Student’s t-tests and ANOVA with Tukey’s test) Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 6 of 14 Fig. 2 Sev treatment repressed cell proliferation and induced cell apoptosis in CRC. A, B The impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on cell viability and cell colony-forming ability were presented by MTT and cell colony formation assays, respectively. C The effects of Sev (0%, 1.7%, 3.4% and 5.1%) on cell cycle were determined by DNA content quantitation assay in HCT116 and SW620 cells. D The effects of Sev (0%, 1.7%, 3.4% and 5.1%) on cell apoptosis were determined by Annexin V-FITC and PI double staining assay in HCT116 and SW620 cells. E Caspase-3 activity was determined by caspase-3 activity assay in HCT116 and SW620 cells treated with Sev at various doses (0%, 1.7%, 3.4% and 5.1%). *p < 0.05 (ANOVA with Tukey’s test) as well as apoptotic rate were determined. MTT and concentration-dependently promoted the apopto- cell colony formation assays displayed that Sev expo- sis and caspase-3 activity of HCT116 and SW620 cells sure inhibited cell viability and cell colony-forming abil- (Fig.  2D, E). Additionally, Sev treatment inhibited cell ity in a dose-dependent manner (Fig.  2A, B). The cell migration and invasion of HCT116 and SW620 cells cycle of HCT116 and SW620 cells was also arrested in dose-dependent fashion (Fig.  3A, B). Sev treatment at G0/G1 phase after Sev treatment in a concentra- upregulated Bax protein expression and the value of tion-dependent manner (Fig.  2C). These findings sug - C-caspase 3/t-caspase 3, and downregulated the protein gested that Sev treatment repressed the proliferation of expression of MMP2 as well as MMP9 in a concentra- HCT116 and SW620 cells. On the contrary, Sev exposure tion-dependent manner (Fig. 3C). In support, the protein W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 7 of 14 Fig. 3 The migration and invasion of HCT116 and SW620 cells were repressed by Sev. A The influences of Sev (0%, 1.7%, 3.4% and 5.1%) on the migration of HCT116 and SW620 cells were revealed by wound-healing assay. B Transwell invasion assay was employed to demonstrate the impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on the invasion of HCT116 and SW620 cells. C The impacts of Sev (0%, 1.7%, 3.4% and 5.1%) on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. D Hsa_ circ_0000231 expression was determined by qRT-PCR in HCT116 and SW620 cells treated with 0%, 1.7%, 3.4% and 5.1% of Sev. *p < 0.05 (ANOVA with Tukey’s test) expression of oncogenes including c-Myc, K-RAS and was notably downregulated after transfection of si- BRAF was dose-dependently reduced by Sev (Additional hsa_circ_0000231#1, si-hsa_circ_0000231#2 or si-hsa_ file  1: Figure S1A, B). The above data demonstrated Sev circ_0000231#3 in HCT116 and SW620 cells (Fig.  4A). could repress CRC process. Given the negative correla- si-hsa_circ_0000231#1 and si-hsa_circ_0000231#2 tion between hsa_circ_0000231 expression and survival were employed in subsequent study owing to their rate of CRC sufferers as well as the repressive impacts higher efficiency. Subsequently, our results presented of Sev on CRC progression, whether Sev regulated hsa_ hsa_circ_0000231 silencing suppressed cell viability circ_0000231 expression was further analyzed. As shown and colony-forming ability in HCT116 and SW620 cells in Fig.  3D, hsa_circ_0000231 expression was apparently (Fig.  4B, C). Hsa_circ_0000231 knockdown also induced decreased after Sev treatment in a dose-dependent man- cell arrested at G0/G1 phase and promoted cell apop- ner, suggesting that Sev might regulate CRC progression tosis (Fig.  4D, E). Meanwhile, the caspase-3 activity was by repressing hsa_circ_0000231. also promoted after hsa_circ_0000231 silencing (Fig. 4F), which further demonstrated that hsa_circ_0000231 Hsa_circ_0000231 silencing repressed cell proliferation, downregulation could induce cell apoptosis. The migra - migration and invasion, but induced cell apoptosis tory and invasive abilities of HCT116 and SW620 cells in HCT116 and SW620 cells were restrained after hsa_circ_0000231 absence (Fig. 4G, Considering the high hsa_circ_0000231 expression in H). Hsa_circ_0000231 silencing increased Bax protein HCT116 and SW620 cells, the small interfering RNAs expression and the value of C-caspase 3/t-caspase 3, and against hsa_circ_0000231 were built and their knock- decreased the protein expression levels of MMP2 as well down efficiency was determined. The data from qRT- as MMP9 (Fig.  4I). Thus, these evidences demonstrated PCR analysis displayed that hsa_circ_0000231 expression Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 8 of 14 Fig. 4 Hsa_circ_0000231 knockdown repressed CRC process. A The knockdown efficiency of hsa_circ_0000231#1, si-hsa_circ_0000231#2 and si-hsa_circ_0000231#3 was determined by qRT-PCR in HCT116 and SW620 cells. B, C The impacts of hsa_circ_0000231 silencing on the viability and colony-forming ability were revealed by MTT and cell colony formation assays, respectively. D The effect of hsa_circ_0000231 absence on cell cycle was demonstrated by DNA content quantitation assay. E The effect of hsa_circ_0000231 absence on cell apoptosis was demonstrated by Annexin V-FITC and PI double staining assay. F The influence of hsa_circ_0000231 silencing on caspase-3 activity was unveiled by caspase-3 activity assay. G, H Wound-healing and transwell invasion assays were performed to demonstrate the impacts of hsa_circ_0000231 downregulation on the migration and invasion of HCT116 and SW620 cells, respectively. I The impacts of hsa_circ_0000231 silencing on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey ’s test) that hsa_circ_0000231 positively regulated CRC reversed the repressive impact of Sev treatment on hsa_ progression. circ_0000231 expression in HCT116 and SW620 cells (Fig. 5A). Sev-mediated inhibitory impacts on cell viabil- Hsa_circ_0000231 attenuated Sev‑mediated effects on CRC ity and cell colony-forming ability were also restored after process hsa_circ_0000231 upregulation (Fig.  5B, C). Addition- Given the repressive role of hsa_circ_0000231 silencing ally, Sev treatment induced cell arrest at G0/G1 phase, on CRC progression, whether it participated in Sev-medi- whereas ectopic hsa_circ_0000231 expression impaired ated CRC process was subsequently explored; the effects this impact (Fig.  5D). Sev-induced cell apoptosis was between Sev treatment and hsa_circ_0000231 overex- also partly abolished after hsa_circ_0000231 overexpres- pression on CRC progression were analyzed. Results sion (Fig. 5E). Caspase-3 activity assay also presented the firstly exhibited that hsa_circ_0000231 overexpression promotion impact of Sev treatment on caspase-3 activity W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 9 of 14 Fig. 5 Hsa_circ_0000231 reversed Sev-mediated influences on CRC development. A The impacts between Sev treatment and hsa_circ_0000231 overexpression on hsa_circ_0000231 expression were detected by qRT-PCR in HCT116 and SW620 cells. B, C The impacts between Sev treatment and hsa_circ_0000231 overexpression on the viability and colony-forming ability of HCT116 and SW620 cells were determined by MTT and cell colony formation assays, respectively. D DNA content quantitation assay was performed to reveal the effects between Sev treatment and hsa_circ_0000231 overexpression on cell cycle. E Annexin V-FITC and PI double staining assay was conducted to show the effects between Sev treatment and hsa_circ_0000231 overexpression on the apoptosis of HCT116 and SW620 cells. F Caspase-3 activity assay was used to demonstrate the impacts between Sev treatment and ectopic hsa_circ_0000231 expression on caspase-3 activity. G, H The impacts between Sev treatment and hsa_circ_0000231 overexpression on the migration and invasion of HCT116 and SW620 cells were revealed by wound-healing and transwell invasion assays, respectively. I The effects between Sev treatment and hsa_circ_0000231 overexpression on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 were determined by western blot analysis in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey’s test) was hindered after transfection of hsa_circ_0000231 and invasion, and induced cell apoptosis by regulating (Fig. 5F). Furthermore, Sev exposure inhibited cell migra- hsa_circ_0000231. tion and invasion, whereas these impacts were restrained by enforced hsa_circ_0000231 expression (Fig.  5G, H). Hsa_circ_0000231 acted as a sponge of miR‑622 Sev-mediated effects on the protein expression of Bax, In order to reveal the regulatory mechanism of hsa_ MMP2 and MMP9 as well as the value of C-caspase circ_0000231 in CRC process, the miRNA with the 3/t-caspase 3, were attenuated after hsa_circ_0000231 ability to bind to hsa_circ_0000231 was sought. Cir- overexpression (Fig.  5I). Taken together, the above data cular RNA Interactome online database presented suggested that Sev repressed cell proliferation, migration hsa_circ_0000231 contained the binding sequence of miR-622 (Fig.  6A). To prove the binding relationship Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 10 of 14 Fig. 6 Hsa_circ_0000231 directly interacted with miR-622. A Circular RNA Interactome online database was performed to predict the putative relationship between hsa_circ_0000231 and miR-622. B, C Dual-luciferase reporter and RIP assays were carried out to prove that hsa_circ_0000231 directly bound to miR-622. D The impacts between Sev treatment and hsa_circ_0000231 overexpression on miR-622 expression were demonstrated by qRT-PCR in HCT116 and SW620 cells. E, G MiR-622 expression was detected by qRT-PCR in 47 pairs of CRC and paracancerous normal colorectal tissues as well as NCM460, HCT116 and SW620 cells. F Spearman correlation analysis was conducted to disclose the linear relationship between hsa_circ_0000231 and miR-622 expression in CRC tissues. H The influences of various concentrations of Sev (0%, 1.7%, 3.4% and 5.1%) on miR-622 expression were determined by qRT-PCR in HCT116 and SW620 cells. *p < 0.05 (two-tailed Student’s t-tests, Wilcoxon rank-sum test, ANOVA with Tukey’s test and Spearman’s correlation test) between hsa_circ_0000231 and miR-622, dual- expression and miR-622 expression was observed luciferase reporter and RIP assays were employed. (Fig.  6F). Furthermore, the impact of Sev exposure Results exhibited the relative luciferase activity on miR-622 expression was unveiled in HCT116 and was dramatically repressed after co-transfection of SW620 cells, and we found that miR-622 expression was hsa_circ_0000231-wt and miR-622 in HCT116 and dose-dependently increased by Sev (Fig. 6H). Thus, our SW620 cells, whereas that had no apparent change in evidences demonstrated hsa_circ_0000231 was directly hsa_circ_0000231-mut and miR-622 group (Fig.  6B). associated with miR-622, and Sev could upregulate RIP assay also presented that both hsa_circ_0000231 miR-622 expression by regulating hsa_circ_0000231. and miR-622 were notably enriched by Anti-Ago2 as compared to Anti-IgG in HCT116 and SW620 cells Hsa_circ_0000231 regulated cell proliferation, apoptosis, (Fig.  6C). Subsequently, qRT-PCR data revealed miR- migration and invasion via binding to miR‑622 622 expression was dramatically upregulated after Sev Given the bound relationship between hsa_circ_0000231 treatment, but this impact was reversed by enforced and miR-622, whether hsa_circ_0000231 regulated CRC hsa_circ_0000231 expression (Fig. 6D). Our results also progression by interacting with miR-622 was investi- showed that miR-622 expression was apparently down- gated. Our data firstly showed that hsa_circ_0000231 regulated in CRC tissues and HCT116 and SW620 silencing apparently upregulated miR-622 expression, cells when compared with normal colorectal tissues whereas miR-622 inhibitors attenuated this impact and NCM460 cells, respectively (Fig.  6E, G). Addition- (Fig.  7A). Subsequently, hsa_circ_0000231 knockdown ally, a negative correlation between hsa_circ_0000231 repressed cell viability and cell colony-forming ability, W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 11 of 14 Fig. 7 Hsa_circ_0000231 mediated CRC development by binding to miR-622. HCT116 and SW620 cells were transfected with si-NC, si-hsa_ circ_0000231#1, si-hsa_circ_0000231#1 + anti-NC and si-hsa_circ_0000231#1 + anti-miR-622, respectively. A MiR-622 expression was detected by qRT-PCR in HCT116 and SW620 cells. B, C The viability and colony-forming ability of HCT116 and SW620 cells were detected by MTT and colony-forming assays, respectively. D, E Cell cycle and apoptosis were detected by DNA content quantitation assay and Annexin V-FITC and PI double staining assay, respectively, in HCT116 and SW620 cells. F Caspase-3 activity was detected by caspase-3 activity assay in HCT116 and SW620 cells. G, H The migration and invasion of HCT116 and SW620 cells were detected by wound-healing and transwell invasion assays, respectively. I Western blot analysis was performed to reveal the influences between hsa_circ_0000231 silencing and miR-622 downregulation on the protein expression of Bax, t-caspase 3, C-caspase 3, MMP2 and MMP9 in HCT116 and SW620 cells. *p < 0.05 (ANOVA with Tukey’s test) which were reversed after downregulation of miR-622 were also repressed by si-hsa_circ_0000231#1; however, (Fig.  7B, C). Data also presented that hsa_circ_0000231 these influences were restored after miR-622 down - silencing induced cell arrest at G0/G1 phase and cell regulation (Fig.  7G, H). The effects of hsa_circ_0000231 apoptosis, but these effects were restored after transfec - downregulation on the protein expression of Bax, MMP2 tion of anti-miR-622 (Fig. 7D, E). In order to further ana- and MMP9 as well as the value of C-caspase 3/t-caspase lyze the impacts between hsa_circ_0000231 and miR-622 3 were attenuated after transfection of anti-miR-622 on cell apoptosis, caspase-3 activity assay was performed. (Fig.  7I). Collectively, the above data demonstrated that Results exhibited that hsa_circ_0000231 downregula- hsa_circ_0000231 could modulate CRC progression by tion promoted caspase-3 activity, while miR-622 inhibi- interacting with miR-622. tors restrained this impact (Fig.  7F). Additionally, the migration and invasion of HCT116 and SW620 cells Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 12 of 14 Fig. 8 Sev treatment repressed tumor formation by repressing hsa_circ_0000231 in vivo. A, B The impacts between Sev and hsa_circ_0000231 overexpression on tumor volume and weight were revealed. C The effects between Sev treatment and enforced hsa_circ_0000231 expression on the levels of hsa_circ_0000231 and miR-622 were determined by qRT-PCR. D Western blot analysis was employed to reveal the influences between Sev treatment and ectopic hsa_circ_0000231 expression on the expression of PCNA protein and the value of C-caspase 3/t-caspase 3. *p < 0.05 (ANOVA with Tukey’s test) Hsa_circ_0000231 overexpression restrained Sev‑mediated hsa_circ_0000231. These data demonstrated  that Sev impacts on tumor formation in vivo repressed tumor growth by controlling hsa_circ_0000231 To further affirm the promotion influences of hsa_ expression in vivo. circ_0000231 on Sev-mediated CRC process, in  vivo assay was conducted. Results showed Sev treatment Discussion reduced tumor volume and weight, whereas these effects Emerging evidences suggest that anesthetics can influ - were reversed by hsa_circ_0000231 overexpression ence cancer evolution [21]. Sev was found to serve as a (Fig.  8A, B). Additionally, we found hsa_circ_0000231 cancer suppressor in CRC through various mechanisms. expression was obviously downregulated, while miR- As reported by previous researchers, Sev restrained cell 622 expression was dramatically upregulated after Sev metastasis of CRC via modulating extracellular signal- treatment in the excised tissues; however, these impacts regulated kinase/matrix metalloproteinase-9 signal path were impaired by enforced hsa_circ_0000231 expression through increasing miR-203 [22]. Additionally, Sev sup- (Fig.  8C). Furthermore, the protein expression of prolif- pressed cell metastasis via regulating miR-34a/ADAM eration-related maker PCNA was decreased after Sev metallopeptidase domain 10 (ADAM10) pathway in CRC treatment, which was reversed after hsa_circ_0000231 [23]. In this paper, we found that Sev repressed CRC overexpression (Fig. 8D). The value of C-caspase 3/t-cas - development. Different from the above results, Sev hin - pase 3 was increased by Sev, but this result was reversed dered CRC progression by regulating hsa_circ_0000231/ after upregulation of hsa_circ_0000231 (Fig.  8D), sug- miR-622 axis. In this research, to reveal the mecha- gesting Sev could induce cell apoptosis by regulating nism of Sev in mediating CRC process, the reasonable W ang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 13 of 14 concentration of Sev was firstly determined. Thus, the cancer [32] and renal cell carcinoma [33]. In CRC pro- impacts of various concentrations of Sev (0%, 1.7%, 3.4% cess, miR-622 could repress angiogenesis [34] and radio- and 5.1%) on CRC progression were explored. Results sensitivity [35]. In this study, a decreased expression of showed that Sev repressed cell viability, colony-forming miR-622 was observed in CRC samples and cells, and ability and metastasis, and upregulated apoptosis rate as miR-622 silencing promoted cell migration and invasion, well as induced cell arrest in a concentration-dependent which were supported by the current evidences [20, 36]. fashion. Also, Sev dose-dependently reduced the expres- Besides, miR-622 inhibitors facilitated cellular prolif- sion of c-Myc, K-RAS and BRAF. Based on these results, eration and repressed cellular apoptosis. The impacts of CRC cells were treated with 5.1% Sev in subsequent miR-622 inhibitors on hsa_circ_0000231 absence-medi- research. ated CRC process also suggested that hsa_circ_0000231 Previous research revealed that circRNAs acted modulated CRC development through sponging as tumor suppressors or promoters in CRC progres- miR-622. sion. For example, circ_001680 [24], circ_101555 Additionally, our results supported that miR-622 [25] and circ_0053277 [26] contributed to CRC pro- expression was apparently upregulated after Sev cess, but circ_0009361 [27], circ_0020397 [28] and treatment, which was reversed by enforced hsa_ circ_0007534 [29] hindered CRC development via circ_0000231 expression. This result also suggested that regulating cell proliferation, migration or apoptosis. Sev could increase miR-622 expression by regulating In this paper, it was found that hsa_circ_0000231 con- hsa_circ_0000231. tent was increased in HCT116 and SW620 cells and the Collectively, ectopic hsa_circ_0000231 expression specimens of CRC patients, and its absence hindered restrained Sev-mediated CRC progression. Besides, CRC development by repressing cellular abilities in hsa_circ_0000231 bound to miR-622. MiR-622 inhibi- proliferation and metastasis as well as enhancing the tors were further proved to hinder hsa_circ_0000231 capacity in apoptosis, which were approved by the find - absence-mediated repressive impacts on CRC process. ings of Liu et  al. [30]. Beyond that, an inverse correla- Summarily, hsa_circ_0000231 restrained Sev-triggered tion between hsa_circ_0000231 level and the survival repressive influences on CRC progression via binding time of CRC sufferers was shown in this paper. Given to miR-622. This finding not only provides a theoreti - the repressive impacts both Sev and the silencing of cal foundation for further studying the application of hsa_circ_0000231 on CRC progression, Sev-mediated Sev in CRC surgery, but also lays a basis for researching effect on hsa_circ_0000231 expression was explored circRNA-directed CRC therapy. further. Data exhibited that hsa_circ_0000231 was dose-dependently downregulated by Sev. Thus, we sug - Supplementary Information gested that hsa_circ_0000231 might be involved in Sev- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40709- 021- 00145-6. mediated CRC progression. To elaborate on that, the overexpression plasmid of hsa_circ_0000231 was trans- Additional file 1: Figure S1. The effects of Sev (0%, 1.7%, 3.4% and 5.1%) fected into Sev-stimulated HCT116 and SW620 cells on the protein expression of c-Myc, K-RAS and BRAF were detected by with control group, and same indices were detected. western blot in both HCT116 (A) and SW620 cells (B). *p < 0.05 (ANOVA As expected, results exhibited that hsa_circ_0000231 with Tukey’s test). overexpression attenuated Sev-mediated impacts on CRC process. Meanwhile, in  vivo assay also showed Acknowledgements enforced hsa_circ_0000231 expression restrained Sev- None. aroused inhibitive influences on tumor formation. The Authors’ contributions inhibition impact of Sev on PCNA protein expres- JW performed the experiments and drafted the manuscript, SL and GZ con- sion and upregulation influence of that on the value of ducted the study. HH collected and analyzed the data. All authors read and approved the final manuscript. C-caspase 3/t-caspase 3 were also abolished after hsa_ circ_0000231 overexpression in  vivo. These evidences Funding suggested that Sev could repress CRC development via None. modulating hsa_circ_0000231. Availability of data and materials CircRNAs commonly interact with miRNAs in can- Please contact the correspondence author for the data request. cer progression [31]. Thus, the miRNA bound to hsa_ circ_0000231 was assessed. Our results presented that Declarations hsa_circ_0000231 directly interacted with miR-622. As Ethics approval and consent participate reported in previous research efforts, miR-622 acted as Written informed consent was obtained from patients with approval by the an anti-oncogene in cancer progression, such as gastric Institutional Review Board in The Chengyang People’s Hospital. Wang et al. J of Biol Res-Thessaloniki (2021) 28:14 Page 14 of 14 Competing interests 17. Liu Z, Zhou Y, Liang G, Ling Y, Tan W, Tan L, et al. 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Journal

Journal of Biological Research-ThessalonikiSpringer Journals

Published: Jun 28, 2021

Keywords: Sev; hsa_circ_0000231; miR-622; CRC

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