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miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3

miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3 Hindawi Journal of Oncology Volume 2022, Article ID 8697676, 8 pages https://doi.org/10.1155/2022/8697676 Research Article miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3 1 2 3,4 2 5 3,6 Xinzhi Yang, Dong Chen, Jiliang Hu, Qingsuo Zhao, Xing Fu, and Wen Lv Department of Neurosurgery, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong, China Department of Neurosurgery, Shenzhen Yantian District s People s Hospital, Shenzhen, Guangdong, China Department of Neurosurgery, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Guangdong Engineering Technological Research Center for Nervous Anatomy and Related Clinical Applications, Zhongshan, China Department of Neurosurgery, Changsha Central Hospital of University of South China, Changsha, Hunan, China Department of Neurosurgery, …e First A‡liated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, China Correspondence should be addressed to Wen Lv; lv.wen1@szhospital.com Received 19 May 2022; Accepted 28 June 2022; Published 2 August 2022 Academic Editor: Dong-Hua Yang Copyright © 2022 Xinzhi Yang et al. €is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. €is research aims to investigate the expression of miR-133a-5p in glioma tissues and its impact on glioma cell proliferation. Methods. Fluorescence-quantitative PCR was used to detect the expression of miR-133a-5p in 25 cases of glioma and adjuncent tissues. CCK-8 and colony formation analyses were used to evaluate the impact of transfection with miR-133a-5p inhibitors or mimics on glioma cell growth and colony formation. €e IGFBP3 (insulin-like growth factor-binding protein-3) and miR-133a-5p binding sites were predicted using Starbase, and the miR-133a-5p binding capacity with 3’UTR of IGFBP3 gene was determined using a luciferase gene reporter system. Following transfection with miR-133a-5p mimics or inhibitors, the IGFBP3 protein expression in glioma cells was determined by western blotting. €e colony formation assay was applied to evaluate the in›uence of IGFBP3 overexpression on the miR-133a-5p in glioma cell proliferation. For assessment of the IGFBP3 expression in glioma tissues and prognosis, TCGA database was employed. Results. €e expression of miR-133a-5p was considerably reduced in glioma tissue compared to adjuncent control tissue. In addition, miR-133a-5p expression decreased with increasing glioma malignancy. Glioma cell growth and colony formation were reduced after miR-133a-5p mimics were transfected, while transfection of miR-133a-5p inhibitors had a reverse impact. €e expression of IGFBP3 was aœected by miR-133a-5p by binding to its 3’UTR region. Additional study demonstrated that the overall survival (OS) of subjects with increased IGFBP3 expression was considerably lower compared to patients with decreased IGFBP3 expression. €e IGFBP3 overexpression eœectively counteracts the glioma cell proliferation-inhibiting impact of miR-133a-5p. Conclusion. miR-133a-5p acts as a glioma tumor suppressor gene. It reduces glioma cell proliferation by modulating IGFBP3 and could be a target for glioma therapy. advances in surgery, radiotherapy, and chemotherapy in 1. Introduction recent decades, the prognosis of glioma remains poor, with a Glioma arises from glial cells’ surrounding neurons and is mean survival time of only 14.6 month [3, 4]. Various the most prevalent tumor of the central nervous system fundamental and clinical research studies have revealed that (CNS) found in clinical settings. Among all gliomas, glio- glioma is a polygenic illness and that its onset and pro- blastoma has an extremely high recurrence rate and is re- gression are controlled by numerous genes [5, 6]. €erefore, sponsible for roughly 80% of all aggressive brain tumors. understanding the interaction among relevant factors at the Glioblastoma is one of the malignant tumors with the level of gene regulation and looking for additional molecular poorest prognostic outcome [1, 2]. Despite signi¤cant candidate genes have become important for glioma therapy. 2 Journal of Oncology ** 2.5 1.5 2.0 1.0 1.5 1.0 0.5 0.5 0.0 0.0 Low grade High grade NHAS U251 T98MG U87 Adjacent tissues Glioma tissues (a) (b) (c) Figure 1: miR-133a-5p is expressed at low level in glioma tissues and cell lines. (a) €e quantitative-›uorescence PCR assay identi¤ed the expression levels of miR-133a-5p in glioma and para-carcinoma tissues. (b) miR-133a-5p expression in the high and low-grade glioma ∗ ∗∗ tissues. (c) €e expression levels of miR-133a-5p in glioma cell lines ( P < 0.05 and P < 0.01). MicroRNA (miRNA) is a kind of non-coding single- 2. Materials and Methods stranded RNA (ncRNA) that can attach to the 3′UTR 2.1. Research Materials. Twenty-¤ve participants that un- domain of target genes and restrict gene expression at the derwent glioma excision at our hospital between June 2020 post-transcriptional level, ultimately leading to mRNA and January 2021 were included in this study. €e glioma breakdown and translational reduction [7, 8]. Prior re- and para-carcinoma specimens (∼5 cm away from tumor search has shown that miRNA plays a signi¤cant role in tissue edge) were obtained and preserved in the freezer at controlling cell diœerentiation, proliferation, apoptosis, −80 C. €ere was no preoperative chemotherapy, radio- and tumor growth and development [9]. miRNAs are therapy, immunotherapy, targeted therapy, or other relevant implicated in all tumor-related activities, including im- treatments. All participants signed an informed consent munological response and angiogenesis. €ey can enhance form. All specimens were obtained and operated following or suppress cancer growth by blocking the production of the ethical standards of clinical trials. speci¤c molecules in signaling networks [7, 10]. For in- stance, miRNA-451 can modulate the NF-B signaling cascade by activating IKKβ, reducing glioma cell prolif- 2.2. Cell Culture and Transfection. €e Shanghai Cell Bank eration both in vivo and in vitro [11]. miR-23b-5p increases (Chinese Academy of Sciences) provided normal astrocyte sensitivity of glioma to temozolomide treatment by neg- (NHAS) cells and human glioma cell lines T98MG, U251, atively regulating TLR4 expression [12]. €ese ¤ndings and U87. €ese cells were maintained in a DMEM con- suggest that miRNAs play important roles in the incidence, taining 10% FBS. €e culture conditions were 5% of CO drug resistance, and progression of glioma. concentration, incubation temperature of 37 C, relative Recent research has indicated that miR-133a-5p is a humidity of 95%, and incubation in darkness. €ey were tumor suppressor gene that is poorly expressed in a range subcultured when the cells grew to an appropriate cell of tumor tissues and plays an antitumor role via modu- density (∼80% con›uence). €e cells at logarithmic growth lating downstream target genes in gastric cancer, bladder were implanted into 6-well cell plates (1 × 10 cells per well). cancer, and non-small-cell lung cancer. For example, €e transfection was performed when the cell fusion reached miR-133a-5p is modestly expressed in gastric cancer cell about 60% as per the instruction of the Lipofectamine 2000 lines and tissues. €e analysis of its molecular mechanism transfection kit. has revealed that miR-133a-5p suppresses metastasis and cell growth while promoting apoptosis by targeting TCF4 [13]. Furthermore, in prostate cancer cells, miR-133a-5p 2.3. Fluorescence-Quantitative PCR Detection. €e TRIzol reduces cellular invasiveness and proliferation via tar- method was utilized for RNA extraction from para-carci- geting the androgen receptor (AR) [14]. Other molecules, noma and glioma tissues. €e glioma cell lines and con- such as circRNA circP4HB increases the metastasis and centration and purity of the RNA were determined using a invasiveness of non-small-cell lung cancer (NSCLC) by spectrophotometer. €rough a reverse transcription kit, the sponging miR-133a-5p, can also control the regulating mRNA was reversedly-transcribed into cDNA. miR-133a-5p eœect of miR-133a-5p to exert biological functions in and internal reference U6 primers were added after the tumor cells [15]. reverse transcription and then ampli¤ed in ABI ›uores- However, the expression and regulatory eœects of miR- cence-quantitative PCR instrument. Each sample was re- 133a-5p in glioma tissue is unknown. €is study sought to peated three times to quantify the relative expression level −ΔΔC determine the expression of miR-133a-5p in glioma cells and using the 2 T method. tissues. Moreover, the impact of miR-133a-5p over- expression or inhibition on glioma cell proliferation was investigated. €e regulatory link between IGFBP and miR- 2.4. CCK-8 for Cell Viability Detection. After transfection, 133a-5p was validated. €is study will provide a theoretical the CCK-8 kit was utilized to detect the proliferative ability foundation for clinical treatment of glioma. of glioma cells. Glioma cells in the log growth phase were Relative miR-133a-5p expression Relative miR-133a-5p expression Relative miR-133a-5p expression Journal of Oncology 3 1.5 ** 1.5 ** 1.0 ** 1.0 0.5 0.5 0.0 0.0 Control miR-133a-5p Control miR-133a-5p mimics mimics inhibitor inhibitor Culture time (day) Control mimics miR-133a-5p mimics (a) (b) (c) 1.5 150 ** Control miR-133a-5p mimics mimics 1.0 0.5 0.0 1234 Control miR-133a-5p mimics mimics Culture time (day) Control inhibitor miR-133a-5p inhibitor (d) (e) ** Control miR-133a-5p inhibitor inhibitor Control miR-133a-5p inhibitor inhibitor (f) Figure 2: Effect of miR-133a-5p on glioma cell proliferative ability. (a, b) Fluorescence-quantitative PCR detected the miR-133a-5p expression level in U87 and T98MG cells following transfection with miR-133a-5p mimics or inhibitors. (c, d) In U87 and T98MG cells, the MTT assay was used to examine the transfection of miR-133a-5p mimics or inhibitors that affected cellular proliferation. (e, f) A colony formation test was utilized to examine the impact of transfection of miR-133a-5p mimics or inhibitors on colony formation ability in U87 ∗ ∗∗ and T98MG cells ( P< 0.05 and P< 0.01). paused and enumerated following transfection. Cells were cell staining was performed using 1% crystal violet solution. kept in a 5% CO incubator and inoculated into a 96-well Cell pictures were collected. -e cell mass with ≥50 cells was plate at 37 C with 100 μL (1 × 10 cells) per well. At four taken as the number of colonies, and the colony formation periods of time (24, 48, 72, and 96 h) following transfection, rate was analyzed. -e procedure was repeated three times. 10 μl CCK-8 solution was poured into each well 2 h before detection, and a microplate reader was used to quantify the 2.6. Dual-Luciferase Reporter Gene. Starbase online predic- absorbance rate. Each sample was repeated in triplicate. tion software was applied to identify the probable down- stream miR-133a-5p target genes. It was found that miR- 2.5. Colony Formation Assay. After transfection, 0.25% 133a-5p had a binding affinity with the 3′-UTR region of IGFBP, resulting in a dual-luciferase reporter gene vector trypsin was applied to digest the glioma cells in the log growth phase, dispersed into a single-cell suspension, and formation. Glioma cells were harvested and plated at a density centrifuged at 1000 rpm at room temperature for 5 min. -e of 5 ×10 cells per well in a 24-well plate. -e no-load vector supernatant was discarded, and the remaining cells were and reporter vector were prepared with miR-133a-5p mimics enumerated after resuspension. A total of 500 cells were and control, respectively, to form a transfection mixture. After plated into a six-well plate, the media were replaced every adding the transfection mixture to the cells, they were kept in two to three days, and cell development was constantly a 37 C incubator with 5% CO . -e cells were cultured and monitored. After around 2 weeks, the medium was removed, kept in a complete medium for 48 h after being cultured for 4% paraformaldehyde was introduced for 15 min, and the 12 h. -e cells were lysed per the Promega dual-luciferase OD Value (450nm) Relative miR-133a-5p expression Relative miR-133a-5p expression Colony number OD Value (450nm) Colony number 4 Journal of Oncology Control miR-133a-5p inhibitor inhibitor IGFBP3 GAPDH 1.5 ** ** 1.0 0.5 IGFBP3Wt: miR-133a-5p: IGFBP3Mut: 0.0 IGFBP3 Wt IGFBP3 Mut Control miR-133a-5p inhibitor inhibitor Control mimics miR-133a5p mimics (a) (b) (c) Control miR-133a-5p mimics mimics IGFBP3 GAPDH 1.5 ** 1.0 0.5 0.0 Control miR-133a-5p mimics mimics (d) Figure 3: -e IGFBP3 expression in glioma cells is regulated by miR-133a-5p. (a) miR-133a-5p had a binding site with IGFBP3. (b) -e dual-luciferase reporting assay measured miR-133a-5p and IGFBP3 binding activity. (c) -e western blot measured the transfection of miR- ∗∗ 133a-5p mimics or inhibitors on IGFBP3 expression in U87 and T98MG cells ( P< 0.01). reporter gene detection kit protocol, and luciferase activity imaging, the absorbance values of each band were quantified was determined by adding a fluorescent substrate. using a density scanner. 2.7. Western Blot Analysis. -e expression of IGFBP protein 2.8. StatisticalData Analysis. -e SPSS software (v.19.0) was in glioma cells transfected with miR-133a-5p inhibitors or used for data analysis. -e statistical data were described as mimics was analyzed using a western blot technique. Equal mean± SD. For independent samples, a t-test was used to amounts of cells from each transfection group were taken, look at measured values. -e ANOVA was applied to search and the SDS lysis method was applied to extract the total for differences between each group. Statistical significance protein. After protein denaturation, SDS-PACE electro- was defined as a P value of less than 0.05. phoresis was performed, and the membrane was transferred, and then it was blocked overnight with 3% BSA. -e primary antibody (1 : 800 dilution) was applied and kept at 4 C 3. Results overnight before washing the membrane. -e HRP-labelled secondary antibody was used and incubated at room tem- 3.1. miR-133a-5p Was Expressed at Low Level in Glioma perature for 1 h before washing the membrane and per- Tissues and Cell Lines. First, fluorescence-quantitative PCR forming ECL chromogenic exposure. For the film after determined the expression of miR-133a-5p in glioma and Relative protein expression luciferase activity Relative protein expression Journal of Oncology 5 ** Glioma tissues Adjacent tissues GBM LGG (num(T)=163; num(N)=207) (num(T)=518; num(N)=207) (a) (b) Overall Survival Disease Free Survival 1.0 1.0 Logrank p=0 Logrank p=2.3e-15 HR(high)=3.1 HR(high)=2.7 p(HR)=2.5e-14 p(HR)=3.3e-16 0.8 0.8 n(high)=338 n(high)=338 n(low)=338 n(low)=338 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 50 100 150 200 0 50 100 150 Months Months Low IGFBP3 TPM Low IGFBP3 TPM High IGFBP3 TPM High IGFBP3 TPM (c) (d) Figure 4: IGFBP3 was overexpressed in gliomas and could be exploited as a prognostic biomarker. (a) IGFBP3 expression levels in gliomas, and para-cancerous tissues determined by quantitative-fluorescence PCR. (b) -e TCGA database was used to compare IGFBP3 expression level between GBM and LGG. (c, d) TCGA database was used to examine the association between IGFBP3 expression level, disease-free ∗∗ survival (DFS), and overall survival (OS) ( P< 0.01). para-carcinoma tissues. -e analysis indicated that glioma mimics, and fluorescence-quantitative PCR detection tissue had less miR-133a-5p than para-carcinoma tissue revealed that miR-133a-5p mimic transfection substantially (Figure 1(a)). -e glioma tissues were then subdivided into enhanced the miR-133a-5p expression level in cells as high-grade and low-grade glioma tissues. -e findings compared to the control (Figure 2(a)). In addition, T98MG demonstrated that the miR-133a-5p expression reduced as cells were transfected with miR-133a-5p inhibitor and the the malignant degree of the glioma increased (Figure 1(b)). control. Compared to the control, transfection of miR-133a- Furthermore, compared to normal astrocyte (NHAS) cells, 5p inhibitor significantly reduced miR-133a-5p expression in the cells (Figure 2(b)). Compared to the control, the miR- the expression level of miR-133a-5p was lowered in glioma cell lines, particularly in U87 cells (Figure 1(c)). -ese results 133a-5p mimic transfection decreased cell growth consider- suggested that the expression of miR-133a-5p reduced in ably in U87 cells, as revealed by MTT assay findings glioma cells and tissues. (Figure 2(c)). -e control mimics, transfected with miR-133a- 5p inhibitor, greatly increased cell proliferation in T98MG 3.2. ,e Effect of miR-133a-5p on Glioma Cell Proliferation. cells (Figure 2(d)). Furthermore, the colony formation ex- -e initial experimental results suggested that the expression periment indicated that miR-133a-5p mimic transfection of miR-133a-5p was lower in U87 cells than in T98MG cells. considerably lowered the competence of U87 cells to form U87 cells were transfected with miR-133a-5p and control colonies when compared to the control (Figure 2(e)). Percent survival Relative IGFBP3 expression Percent survival Relative IGFBP3 expression 6 Journal of Oncology IGFBP3 GAPDH 1.5 1.5 ** ** 1.0 1.0 0.5 0.5 0.0 0.0 miR-1332-5p mimics – + + miR-1332-5p mimics – + + Control mimics + – – + – – Control mimics Vector-control + + – Vector-control + + – Vector-IGFBP3 – – + Vector-IGFBP3 – – + (a) (b) Figure 5: IGFBP3 overexpression mitigates the inhibitory effect of miR-133a-5p on the proliferation of glioma cells. (a) Effect of overexpression of IGFBP3 on upregulation of IGFBP3 protein expression in miR-199a-5p cells. (b) IGFBP3 overexpression mitigates the ∗∗ ∗ inhibitory effect of miR-133a-5p on glioma cell proliferation ( P< 0.01 and P< 0.01). Transfection with miR-133a-5p inhibitor substantially ele- multiforme (GBM)) compared to para-cancerous tissues vated the ability of T98MG cells to form colonies when (Figure 4(b)). Significantly shorter overall survival (OS) and compared to the control (Figure 2(f)). disease-free survival (DFS) were seen in patients with high IGFBP3 expression compared to those with low IGFBP3 expression (Figures 4(c) and 4(d)). 3.3. miR-133a-5p Regulates the IGFBP3 (Insulin-Like Growth Factor-Binding Protein-3) Expression in Glioma Cells. -e TargetScan prediction software study revealed that miR- 3.5. IGFBP3 Overexpression Can Mitigate the miR-133a-5p 133a-5p had a binding site with IGFBP3 (Figure 3(a)). -e Inhibitory Effect on the Proliferation of Glioma Cells. -e luciferase activity of U87 cells in the miR-133a-5p mimic western blot analysis showed that the miR-133a-5p mimics and IGFBP3 Wt co-transfection group was substantially and the vector group could reduce IGFBP3 expression, lower than that in the control and IGFBP3 Wt co-trans- confirming IGFBP3 gene suppression by miR-133a-5p. fection group, according to the findings of the dual-lu- Following transfection with an overexpression vector ciferase report analysis. Compared with the control mimics (Vector-IGFBP3) that overexpressed IGFBP3, the inhibition and IGFBP3 Mut co-transfection group, the luciferase of IGFBP3 by overexpression of miR-133a-5p was dra- activity of U87 cells in the miR-133a-5p mimic and IGFBP3 matically reduced in the miR-133a-5p mimic group Mut co-transfection group did not change significantly (Figure 5(a)). Compared to the vector group and miR-133a- (Figure 3(b)). According to western blotting assay results, 5p mimics, both groups effectively suppressed cell colony IGFBP3 protein expression in U87 cells was decreased in formation. Similarly, the inhibitory effect of miR-133a-5p the miR-199a-5p mimic group compared to the control overexpression on cell colony formation was inhibited after group (Figure 3(c)). In comparison to the control group, transfection with an overexpression vector (Vector-IGFBP3) the IGFBP3 protein expression in T98MG cells was dra- that overexpressed IGFBP3 (Figure 5(b)). matically increased in the miR-199a-5p inhibitor group (Figure 3(d)). 4. Discussion 3.4. IGFBP3 Was Expressed in Gliomas and Could Be a Bio- Glioma is the most frequent recurrent malignant brain marker for Patient Prognosis. To evaluate the expression of tumor. Due to the unsatisfactory effect of surgery, radio- IGFBP3 in glioma, the expression of IGFBP3 in glioma and therapy, and chemotherapy and its poor prognosis, gliomas para-cancerous brain tissues was determined by fluores- seriously endanger human health [16, 17]. According to cence-quantitative PCR. -e findings confirmed that previous research, miRNAs contribute significantly to the IGFBP3 was overexpressed in glioma tissues compared to transduction of intracellular signaling pathways, initiation, para-cancerous tissues (Figure 4(a)). Analysis of published and progression of gliomas by modulating the expression studies from TCGA database on IGFBP3 expression in of target genes and are a special class of prospective in- glioma revealed that IGFBP3 was strongly elevated in glioma dicators in targeted therapies [18, 19]. miR-133a-5p was tissues (low-grade glioma (LGG) and glioblastoma expressed at low level in glioma tissues, and its level of Relative protein expression Colony number Journal of Oncology 7 expression decreased substantially as glioma malignancy Conflicts of Interest progressed, according to this study’s results. Cell function -e authors declare that they have no conflicts of interest. experiments demonstrated that miR-133a-5p over- expression greatly decreased the glioma cell proliferation and colony formation, whereas the inhibition of miR-133a- Acknowledgments 5p had the opposite effect. Molecular mechanistic studies have reported the binding capacity of miR-133a-5p with -is study was supported by Shenzhen Science and Tech- the 3′-UTR region of IGFBP3 gene, influencing its ex- nology Research and Development Fund (no. pression. IGFBP3 overexpression can drastically counteract JCYJ20210324114008023). the inhibitory activity of miR-133a-5p on glioma cell growth. -erefore, miR-133a-5p could be utilized as a References potential treatment target for glioma therapy. -e latest evidence has suggested that abnormal [1] J. G. Nicholson and H. A. Fine, “Diffuse glioma heterogeneity miRNA expression is closely related to the development and its therapeutic implications,” Cancer Discovery, vol. 11, and proliferation of tumor cells. For example, the over- pp. 575–590, 2021. expression of miR-191 can enhance cell proliferation both [2] R. E. Phillips, A. A. Soshnev, and C. D. 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miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3

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Copyright © 2022 Xinzhi Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2022, Article ID 8697676, 8 pages https://doi.org/10.1155/2022/8697676 Research Article miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3 1 2 3,4 2 5 3,6 Xinzhi Yang, Dong Chen, Jiliang Hu, Qingsuo Zhao, Xing Fu, and Wen Lv Department of Neurosurgery, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong, China Department of Neurosurgery, Shenzhen Yantian District s People s Hospital, Shenzhen, Guangdong, China Department of Neurosurgery, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Guangdong Engineering Technological Research Center for Nervous Anatomy and Related Clinical Applications, Zhongshan, China Department of Neurosurgery, Changsha Central Hospital of University of South China, Changsha, Hunan, China Department of Neurosurgery, …e First A‡liated Hospital, Southern University of Science and Technology, Shenzhen, Guangdong, China Correspondence should be addressed to Wen Lv; lv.wen1@szhospital.com Received 19 May 2022; Accepted 28 June 2022; Published 2 August 2022 Academic Editor: Dong-Hua Yang Copyright © 2022 Xinzhi Yang et al. €is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. €is research aims to investigate the expression of miR-133a-5p in glioma tissues and its impact on glioma cell proliferation. Methods. Fluorescence-quantitative PCR was used to detect the expression of miR-133a-5p in 25 cases of glioma and adjuncent tissues. CCK-8 and colony formation analyses were used to evaluate the impact of transfection with miR-133a-5p inhibitors or mimics on glioma cell growth and colony formation. €e IGFBP3 (insulin-like growth factor-binding protein-3) and miR-133a-5p binding sites were predicted using Starbase, and the miR-133a-5p binding capacity with 3’UTR of IGFBP3 gene was determined using a luciferase gene reporter system. Following transfection with miR-133a-5p mimics or inhibitors, the IGFBP3 protein expression in glioma cells was determined by western blotting. €e colony formation assay was applied to evaluate the in›uence of IGFBP3 overexpression on the miR-133a-5p in glioma cell proliferation. For assessment of the IGFBP3 expression in glioma tissues and prognosis, TCGA database was employed. Results. €e expression of miR-133a-5p was considerably reduced in glioma tissue compared to adjuncent control tissue. In addition, miR-133a-5p expression decreased with increasing glioma malignancy. Glioma cell growth and colony formation were reduced after miR-133a-5p mimics were transfected, while transfection of miR-133a-5p inhibitors had a reverse impact. €e expression of IGFBP3 was aœected by miR-133a-5p by binding to its 3’UTR region. Additional study demonstrated that the overall survival (OS) of subjects with increased IGFBP3 expression was considerably lower compared to patients with decreased IGFBP3 expression. €e IGFBP3 overexpression eœectively counteracts the glioma cell proliferation-inhibiting impact of miR-133a-5p. Conclusion. miR-133a-5p acts as a glioma tumor suppressor gene. It reduces glioma cell proliferation by modulating IGFBP3 and could be a target for glioma therapy. advances in surgery, radiotherapy, and chemotherapy in 1. Introduction recent decades, the prognosis of glioma remains poor, with a Glioma arises from glial cells’ surrounding neurons and is mean survival time of only 14.6 month [3, 4]. Various the most prevalent tumor of the central nervous system fundamental and clinical research studies have revealed that (CNS) found in clinical settings. Among all gliomas, glio- glioma is a polygenic illness and that its onset and pro- blastoma has an extremely high recurrence rate and is re- gression are controlled by numerous genes [5, 6]. €erefore, sponsible for roughly 80% of all aggressive brain tumors. understanding the interaction among relevant factors at the Glioblastoma is one of the malignant tumors with the level of gene regulation and looking for additional molecular poorest prognostic outcome [1, 2]. Despite signi¤cant candidate genes have become important for glioma therapy. 2 Journal of Oncology ** 2.5 1.5 2.0 1.0 1.5 1.0 0.5 0.5 0.0 0.0 Low grade High grade NHAS U251 T98MG U87 Adjacent tissues Glioma tissues (a) (b) (c) Figure 1: miR-133a-5p is expressed at low level in glioma tissues and cell lines. (a) €e quantitative-›uorescence PCR assay identi¤ed the expression levels of miR-133a-5p in glioma and para-carcinoma tissues. (b) miR-133a-5p expression in the high and low-grade glioma ∗ ∗∗ tissues. (c) €e expression levels of miR-133a-5p in glioma cell lines ( P < 0.05 and P < 0.01). MicroRNA (miRNA) is a kind of non-coding single- 2. Materials and Methods stranded RNA (ncRNA) that can attach to the 3′UTR 2.1. Research Materials. Twenty-¤ve participants that un- domain of target genes and restrict gene expression at the derwent glioma excision at our hospital between June 2020 post-transcriptional level, ultimately leading to mRNA and January 2021 were included in this study. €e glioma breakdown and translational reduction [7, 8]. Prior re- and para-carcinoma specimens (∼5 cm away from tumor search has shown that miRNA plays a signi¤cant role in tissue edge) were obtained and preserved in the freezer at controlling cell diœerentiation, proliferation, apoptosis, −80 C. €ere was no preoperative chemotherapy, radio- and tumor growth and development [9]. miRNAs are therapy, immunotherapy, targeted therapy, or other relevant implicated in all tumor-related activities, including im- treatments. All participants signed an informed consent munological response and angiogenesis. €ey can enhance form. All specimens were obtained and operated following or suppress cancer growth by blocking the production of the ethical standards of clinical trials. speci¤c molecules in signaling networks [7, 10]. For in- stance, miRNA-451 can modulate the NF-B signaling cascade by activating IKKβ, reducing glioma cell prolif- 2.2. Cell Culture and Transfection. €e Shanghai Cell Bank eration both in vivo and in vitro [11]. miR-23b-5p increases (Chinese Academy of Sciences) provided normal astrocyte sensitivity of glioma to temozolomide treatment by neg- (NHAS) cells and human glioma cell lines T98MG, U251, atively regulating TLR4 expression [12]. €ese ¤ndings and U87. €ese cells were maintained in a DMEM con- suggest that miRNAs play important roles in the incidence, taining 10% FBS. €e culture conditions were 5% of CO drug resistance, and progression of glioma. concentration, incubation temperature of 37 C, relative Recent research has indicated that miR-133a-5p is a humidity of 95%, and incubation in darkness. €ey were tumor suppressor gene that is poorly expressed in a range subcultured when the cells grew to an appropriate cell of tumor tissues and plays an antitumor role via modu- density (∼80% con›uence). €e cells at logarithmic growth lating downstream target genes in gastric cancer, bladder were implanted into 6-well cell plates (1 × 10 cells per well). cancer, and non-small-cell lung cancer. For example, €e transfection was performed when the cell fusion reached miR-133a-5p is modestly expressed in gastric cancer cell about 60% as per the instruction of the Lipofectamine 2000 lines and tissues. €e analysis of its molecular mechanism transfection kit. has revealed that miR-133a-5p suppresses metastasis and cell growth while promoting apoptosis by targeting TCF4 [13]. Furthermore, in prostate cancer cells, miR-133a-5p 2.3. Fluorescence-Quantitative PCR Detection. €e TRIzol reduces cellular invasiveness and proliferation via tar- method was utilized for RNA extraction from para-carci- geting the androgen receptor (AR) [14]. Other molecules, noma and glioma tissues. €e glioma cell lines and con- such as circRNA circP4HB increases the metastasis and centration and purity of the RNA were determined using a invasiveness of non-small-cell lung cancer (NSCLC) by spectrophotometer. €rough a reverse transcription kit, the sponging miR-133a-5p, can also control the regulating mRNA was reversedly-transcribed into cDNA. miR-133a-5p eœect of miR-133a-5p to exert biological functions in and internal reference U6 primers were added after the tumor cells [15]. reverse transcription and then ampli¤ed in ABI ›uores- However, the expression and regulatory eœects of miR- cence-quantitative PCR instrument. Each sample was re- 133a-5p in glioma tissue is unknown. €is study sought to peated three times to quantify the relative expression level −ΔΔC determine the expression of miR-133a-5p in glioma cells and using the 2 T method. tissues. Moreover, the impact of miR-133a-5p over- expression or inhibition on glioma cell proliferation was investigated. €e regulatory link between IGFBP and miR- 2.4. CCK-8 for Cell Viability Detection. After transfection, 133a-5p was validated. €is study will provide a theoretical the CCK-8 kit was utilized to detect the proliferative ability foundation for clinical treatment of glioma. of glioma cells. Glioma cells in the log growth phase were Relative miR-133a-5p expression Relative miR-133a-5p expression Relative miR-133a-5p expression Journal of Oncology 3 1.5 ** 1.5 ** 1.0 ** 1.0 0.5 0.5 0.0 0.0 Control miR-133a-5p Control miR-133a-5p mimics mimics inhibitor inhibitor Culture time (day) Control mimics miR-133a-5p mimics (a) (b) (c) 1.5 150 ** Control miR-133a-5p mimics mimics 1.0 0.5 0.0 1234 Control miR-133a-5p mimics mimics Culture time (day) Control inhibitor miR-133a-5p inhibitor (d) (e) ** Control miR-133a-5p inhibitor inhibitor Control miR-133a-5p inhibitor inhibitor (f) Figure 2: Effect of miR-133a-5p on glioma cell proliferative ability. (a, b) Fluorescence-quantitative PCR detected the miR-133a-5p expression level in U87 and T98MG cells following transfection with miR-133a-5p mimics or inhibitors. (c, d) In U87 and T98MG cells, the MTT assay was used to examine the transfection of miR-133a-5p mimics or inhibitors that affected cellular proliferation. (e, f) A colony formation test was utilized to examine the impact of transfection of miR-133a-5p mimics or inhibitors on colony formation ability in U87 ∗ ∗∗ and T98MG cells ( P< 0.05 and P< 0.01). paused and enumerated following transfection. Cells were cell staining was performed using 1% crystal violet solution. kept in a 5% CO incubator and inoculated into a 96-well Cell pictures were collected. -e cell mass with ≥50 cells was plate at 37 C with 100 μL (1 × 10 cells) per well. At four taken as the number of colonies, and the colony formation periods of time (24, 48, 72, and 96 h) following transfection, rate was analyzed. -e procedure was repeated three times. 10 μl CCK-8 solution was poured into each well 2 h before detection, and a microplate reader was used to quantify the 2.6. Dual-Luciferase Reporter Gene. Starbase online predic- absorbance rate. Each sample was repeated in triplicate. tion software was applied to identify the probable down- stream miR-133a-5p target genes. It was found that miR- 2.5. Colony Formation Assay. After transfection, 0.25% 133a-5p had a binding affinity with the 3′-UTR region of IGFBP, resulting in a dual-luciferase reporter gene vector trypsin was applied to digest the glioma cells in the log growth phase, dispersed into a single-cell suspension, and formation. Glioma cells were harvested and plated at a density centrifuged at 1000 rpm at room temperature for 5 min. -e of 5 ×10 cells per well in a 24-well plate. -e no-load vector supernatant was discarded, and the remaining cells were and reporter vector were prepared with miR-133a-5p mimics enumerated after resuspension. A total of 500 cells were and control, respectively, to form a transfection mixture. After plated into a six-well plate, the media were replaced every adding the transfection mixture to the cells, they were kept in two to three days, and cell development was constantly a 37 C incubator with 5% CO . -e cells were cultured and monitored. After around 2 weeks, the medium was removed, kept in a complete medium for 48 h after being cultured for 4% paraformaldehyde was introduced for 15 min, and the 12 h. -e cells were lysed per the Promega dual-luciferase OD Value (450nm) Relative miR-133a-5p expression Relative miR-133a-5p expression Colony number OD Value (450nm) Colony number 4 Journal of Oncology Control miR-133a-5p inhibitor inhibitor IGFBP3 GAPDH 1.5 ** ** 1.0 0.5 IGFBP3Wt: miR-133a-5p: IGFBP3Mut: 0.0 IGFBP3 Wt IGFBP3 Mut Control miR-133a-5p inhibitor inhibitor Control mimics miR-133a5p mimics (a) (b) (c) Control miR-133a-5p mimics mimics IGFBP3 GAPDH 1.5 ** 1.0 0.5 0.0 Control miR-133a-5p mimics mimics (d) Figure 3: -e IGFBP3 expression in glioma cells is regulated by miR-133a-5p. (a) miR-133a-5p had a binding site with IGFBP3. (b) -e dual-luciferase reporting assay measured miR-133a-5p and IGFBP3 binding activity. (c) -e western blot measured the transfection of miR- ∗∗ 133a-5p mimics or inhibitors on IGFBP3 expression in U87 and T98MG cells ( P< 0.01). reporter gene detection kit protocol, and luciferase activity imaging, the absorbance values of each band were quantified was determined by adding a fluorescent substrate. using a density scanner. 2.7. Western Blot Analysis. -e expression of IGFBP protein 2.8. StatisticalData Analysis. -e SPSS software (v.19.0) was in glioma cells transfected with miR-133a-5p inhibitors or used for data analysis. -e statistical data were described as mimics was analyzed using a western blot technique. Equal mean± SD. For independent samples, a t-test was used to amounts of cells from each transfection group were taken, look at measured values. -e ANOVA was applied to search and the SDS lysis method was applied to extract the total for differences between each group. Statistical significance protein. After protein denaturation, SDS-PACE electro- was defined as a P value of less than 0.05. phoresis was performed, and the membrane was transferred, and then it was blocked overnight with 3% BSA. -e primary antibody (1 : 800 dilution) was applied and kept at 4 C 3. Results overnight before washing the membrane. -e HRP-labelled secondary antibody was used and incubated at room tem- 3.1. miR-133a-5p Was Expressed at Low Level in Glioma perature for 1 h before washing the membrane and per- Tissues and Cell Lines. First, fluorescence-quantitative PCR forming ECL chromogenic exposure. For the film after determined the expression of miR-133a-5p in glioma and Relative protein expression luciferase activity Relative protein expression Journal of Oncology 5 ** Glioma tissues Adjacent tissues GBM LGG (num(T)=163; num(N)=207) (num(T)=518; num(N)=207) (a) (b) Overall Survival Disease Free Survival 1.0 1.0 Logrank p=0 Logrank p=2.3e-15 HR(high)=3.1 HR(high)=2.7 p(HR)=2.5e-14 p(HR)=3.3e-16 0.8 0.8 n(high)=338 n(high)=338 n(low)=338 n(low)=338 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 50 100 150 200 0 50 100 150 Months Months Low IGFBP3 TPM Low IGFBP3 TPM High IGFBP3 TPM High IGFBP3 TPM (c) (d) Figure 4: IGFBP3 was overexpressed in gliomas and could be exploited as a prognostic biomarker. (a) IGFBP3 expression levels in gliomas, and para-cancerous tissues determined by quantitative-fluorescence PCR. (b) -e TCGA database was used to compare IGFBP3 expression level between GBM and LGG. (c, d) TCGA database was used to examine the association between IGFBP3 expression level, disease-free ∗∗ survival (DFS), and overall survival (OS) ( P< 0.01). para-carcinoma tissues. -e analysis indicated that glioma mimics, and fluorescence-quantitative PCR detection tissue had less miR-133a-5p than para-carcinoma tissue revealed that miR-133a-5p mimic transfection substantially (Figure 1(a)). -e glioma tissues were then subdivided into enhanced the miR-133a-5p expression level in cells as high-grade and low-grade glioma tissues. -e findings compared to the control (Figure 2(a)). In addition, T98MG demonstrated that the miR-133a-5p expression reduced as cells were transfected with miR-133a-5p inhibitor and the the malignant degree of the glioma increased (Figure 1(b)). control. Compared to the control, transfection of miR-133a- Furthermore, compared to normal astrocyte (NHAS) cells, 5p inhibitor significantly reduced miR-133a-5p expression in the cells (Figure 2(b)). Compared to the control, the miR- the expression level of miR-133a-5p was lowered in glioma cell lines, particularly in U87 cells (Figure 1(c)). -ese results 133a-5p mimic transfection decreased cell growth consider- suggested that the expression of miR-133a-5p reduced in ably in U87 cells, as revealed by MTT assay findings glioma cells and tissues. (Figure 2(c)). -e control mimics, transfected with miR-133a- 5p inhibitor, greatly increased cell proliferation in T98MG 3.2. ,e Effect of miR-133a-5p on Glioma Cell Proliferation. cells (Figure 2(d)). Furthermore, the colony formation ex- -e initial experimental results suggested that the expression periment indicated that miR-133a-5p mimic transfection of miR-133a-5p was lower in U87 cells than in T98MG cells. considerably lowered the competence of U87 cells to form U87 cells were transfected with miR-133a-5p and control colonies when compared to the control (Figure 2(e)). Percent survival Relative IGFBP3 expression Percent survival Relative IGFBP3 expression 6 Journal of Oncology IGFBP3 GAPDH 1.5 1.5 ** ** 1.0 1.0 0.5 0.5 0.0 0.0 miR-1332-5p mimics – + + miR-1332-5p mimics – + + Control mimics + – – + – – Control mimics Vector-control + + – Vector-control + + – Vector-IGFBP3 – – + Vector-IGFBP3 – – + (a) (b) Figure 5: IGFBP3 overexpression mitigates the inhibitory effect of miR-133a-5p on the proliferation of glioma cells. (a) Effect of overexpression of IGFBP3 on upregulation of IGFBP3 protein expression in miR-199a-5p cells. (b) IGFBP3 overexpression mitigates the ∗∗ ∗ inhibitory effect of miR-133a-5p on glioma cell proliferation ( P< 0.01 and P< 0.01). Transfection with miR-133a-5p inhibitor substantially ele- multiforme (GBM)) compared to para-cancerous tissues vated the ability of T98MG cells to form colonies when (Figure 4(b)). Significantly shorter overall survival (OS) and compared to the control (Figure 2(f)). disease-free survival (DFS) were seen in patients with high IGFBP3 expression compared to those with low IGFBP3 expression (Figures 4(c) and 4(d)). 3.3. miR-133a-5p Regulates the IGFBP3 (Insulin-Like Growth Factor-Binding Protein-3) Expression in Glioma Cells. -e TargetScan prediction software study revealed that miR- 3.5. IGFBP3 Overexpression Can Mitigate the miR-133a-5p 133a-5p had a binding site with IGFBP3 (Figure 3(a)). -e Inhibitory Effect on the Proliferation of Glioma Cells. -e luciferase activity of U87 cells in the miR-133a-5p mimic western blot analysis showed that the miR-133a-5p mimics and IGFBP3 Wt co-transfection group was substantially and the vector group could reduce IGFBP3 expression, lower than that in the control and IGFBP3 Wt co-trans- confirming IGFBP3 gene suppression by miR-133a-5p. fection group, according to the findings of the dual-lu- Following transfection with an overexpression vector ciferase report analysis. Compared with the control mimics (Vector-IGFBP3) that overexpressed IGFBP3, the inhibition and IGFBP3 Mut co-transfection group, the luciferase of IGFBP3 by overexpression of miR-133a-5p was dra- activity of U87 cells in the miR-133a-5p mimic and IGFBP3 matically reduced in the miR-133a-5p mimic group Mut co-transfection group did not change significantly (Figure 5(a)). Compared to the vector group and miR-133a- (Figure 3(b)). According to western blotting assay results, 5p mimics, both groups effectively suppressed cell colony IGFBP3 protein expression in U87 cells was decreased in formation. Similarly, the inhibitory effect of miR-133a-5p the miR-199a-5p mimic group compared to the control overexpression on cell colony formation was inhibited after group (Figure 3(c)). In comparison to the control group, transfection with an overexpression vector (Vector-IGFBP3) the IGFBP3 protein expression in T98MG cells was dra- that overexpressed IGFBP3 (Figure 5(b)). matically increased in the miR-199a-5p inhibitor group (Figure 3(d)). 4. Discussion 3.4. IGFBP3 Was Expressed in Gliomas and Could Be a Bio- Glioma is the most frequent recurrent malignant brain marker for Patient Prognosis. To evaluate the expression of tumor. Due to the unsatisfactory effect of surgery, radio- IGFBP3 in glioma, the expression of IGFBP3 in glioma and therapy, and chemotherapy and its poor prognosis, gliomas para-cancerous brain tissues was determined by fluores- seriously endanger human health [16, 17]. According to cence-quantitative PCR. -e findings confirmed that previous research, miRNAs contribute significantly to the IGFBP3 was overexpressed in glioma tissues compared to transduction of intracellular signaling pathways, initiation, para-cancerous tissues (Figure 4(a)). Analysis of published and progression of gliomas by modulating the expression studies from TCGA database on IGFBP3 expression in of target genes and are a special class of prospective in- glioma revealed that IGFBP3 was strongly elevated in glioma dicators in targeted therapies [18, 19]. miR-133a-5p was tissues (low-grade glioma (LGG) and glioblastoma expressed at low level in glioma tissues, and its level of Relative protein expression Colony number Journal of Oncology 7 expression decreased substantially as glioma malignancy Conflicts of Interest progressed, according to this study’s results. Cell function -e authors declare that they have no conflicts of interest. experiments demonstrated that miR-133a-5p over- expression greatly decreased the glioma cell proliferation and colony formation, whereas the inhibition of miR-133a- Acknowledgments 5p had the opposite effect. 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Journal

Journal of OncologyHindawi Publishing Corporation

Published: Aug 2, 2022

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