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Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells

Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells Hindawi Journal of Oncology Volume 2022, Article ID 7526731, 10 pages https://doi.org/10.1155/2022/7526731 Research Article Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells 1 2 2 2 Ran Zhao, Xin Zhou, Wencan Zhang, and Le Li Department of Burns and Plastic Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021 Shandong, China Department of Orthopedic Surgery, Qilu Hospital of Shandong University, Jinan, 250021 Shandong, China Correspondence should be addressed to Le Li; qiluhosp_orthoplee@163.com Received 13 June 2022; Revised 2 September 2022; Accepted 15 September 2022; Published 30 September 2022 Academic Editor: Zhiqian Zhang Copyright © 2022 Ran Zhao 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. Background. Previous studies had shown that lncRNA HULC exhibited different effects in human cancers. However, the role of HULC was not reported in osteosarcoma. Hence, we designed this research to explore the function of HULC in osteosarcoma. Methods. Firstly, HULC expression was measured in osteosarcoma tissues and cells via the RT-qPCR assay. The protein expression was detected through western blot. Then, CCK-8 and Transwell assays were conducted to measure cell proliferation, migration, and invasion. Results. The expression of HULC was obviously higher in osteosarcoma tissues and cells compared with normal control. Moreover, cell proliferation, migration, and invasion were inhibited by HULC knockdown in osteosarcoma cells. HULC overexpression markedly increased osteosarcoma cell proliferation and tumor size in vivo. Furthermore, HULC increased the activity of AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K. Overexpressing HULC enhanced cell migration and invasion of SAOS-2 cells and MG63 cells, while LY294002 reversed the effects. Conclusion. HULC enhanced the progression of osteosarcoma through targeting PTEN. scripted into proteins, and 98% can be transcripted into 1. Introduction noncoding RNAs [7]. The function of these noncoding Osteosarcoma is caused by bone cell abnormal differentiation RNAs is still poorly understand. The long noncoding RNA and proliferation. Consistent with medicine information, the is a type of noncoding RNA that is longer than 200 nt [8]. incidence of osteogenic sarcoma is around 0.2–3/100000 per lncRNAs have been shown to be involved in a large number annum [1]. It is the foremost common primary bone tumor of cellular processes, such as cell proliferation, transcrip- in youngsters and adolescents with a high degree of malig- tional and posttranscriptional modification, epigenetic mod- nancy. Osteosarcoma typically shows a high tendency to path- ification, and invasion [9]. The large number of studies ologic process spread [2]. Osteosarcoma is commonly treated shows that lncRNA could play an important role in many with a mixture of therapies that may embody surgery, therapy, types of cancer [10]. For example, Chen et al. showed that and radiotherapy [3]. However, in recent years, the survival lncRNA HULC was overexpressed in epithelial ovarian car- rate of patients with osteosarcoma amid distant metastasis cinoma and target ATG7 [11]. Zheng et al. showed that has not been considerably improved, and also the impact of lncRNA HULC was highly expressed in HeLa cells and pro- therapy has not been considerably improved, and also the motes cell migration and invasion [12]. treatment of osteosarcoma remains controversial [4, 5]. Thus, lncRNA HULC was first reported by Panzitt et al. in new therapeutic targets ought to be found to produce clinical 2007, which was overexpressed lncRNA in human hepatocel- treatment choices to enhance the survival rate [6]. lular carcinoma [13]. HULC gene is located on chromosome High-throughput transcriptome sequencing analysis has 6p24.3 with approximately 500 nucleotides in length and shown that only 2% of the human genome can be tran- contains two exons [14]. Since then, the function of HULC 2 Journal of Oncology ⁎ 4 Non-tumor Tumor (a) (b) Figure 1: lncRNA HULC expression was increased in osteosarcoma tissue and cells. The expressions of HULC in osteosarcoma tissues (a) and cell lines (b) were detected by RT-qPCR. P <0:05. 1.5 MG63 1.5 SAOS-2 1.0 1.0 0.5 0.5 0.0 0.0 sh-NC sh-HULC (a) (b) MG63 SAOS-2 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 24 48 72 96 24 48 72 96 Time (h) Time (h) sh-NC sh-NC sh-HULC sh-HULC (c) (d) Figure 2: Downregulation of HULC impeded the proliferation of osteosarcoma cells. (a, b) The relative expression of HULC in cells was detected in SAOS-2 (a) and MG63 cells (b) by RT-qPCR. (c, d) Cell viability in SAOS-2 (c) and MG63 cells (d) was measured by CCK-8 assay. P <0:05. OD value (450 nm) Relative expression of HULC Relative expression of HULC Relative expression of HULC OD value (450 nm) Relative expression of HULC hFOB 1.19 sh-NC SAOS-2 U-2 OS sh-HULC MG63 Journal of Oncology 3 sh-NC sh-HULC (a) sh-NC sh-HULC SAOS-2 (b) sh-NC sh-HULC MG63 (c) Figure 3: Continued. Number of migrated cells Number of invasive cells Number of migrated cells sh-NC sh-NC sh-NC sh-HULC sh-HULC sh-HULC 4 Journal of Oncology sh-NC sh-HULC MG63 (d) Figure 3: Decreased HULC impeded the malignant behaviors of osteosarcoma cells. (a, b) Cell migration (a) and invasion (b) assays in SAOS-2 cells were detected by Transwell assay. (c, d) Cell migration (c) and invasion (d) assays in MG63 cells were measured by Transwell assay. P <0:05. has been demonstrated in multiple cancer types [15, 16]. separated by 12% SDS-polyacrylamide gel electrophoresis HULC could promote cancer cell survival, proliferation, (SDS-PAGE) and then were transferred on polyvinylidene and invasion. These studies indicated that HULC played an fluoride membranes (Millipore, Billerica, MA, USA). The important role in the development of cancer [16, 17]. HULC membranes were treated with 5% fat-free milk for 2 h and was overexpressed in human osteosarcoma cell which has then were incubated with the antibodies of AKT (PTG, been shown in the previous studies. However, the mecha- USA, 1 : 1000), p-AKT (PTG, USA, 1 : 10000), PI3K (CST, nism of HULC involved in the progression of osteosarcoma USA, 1 : 1000), p-PI3K (CST, USA, 1 : 1000), mTOR (PTG, is unknown, and the interaction of HULC and PTEN has USA, 1 : 20000), p-mTOR (PTG, USA, 1 : 10000), PTEN not been proved in osteosarcoma. (ABclonal, China, 1 : 1000), or β-actin (CST, USA, 1 : 1000). Therefore, we investigated the interaction of HULC and After washing and incubating with the second antibodies, PTEN in promoting osteosarcoma cell proliferation and the abundance of the proteins was analyzed by enhanced migration. chemiluminescence (ECL). 2.4. shRNA Transfection. In short, the specific oligonucleo- 2. Materials and Methods tides targeting HULC were synthesized by Genepharmacy 2.1. Cell Lines and Culture. The human osteosarcoma cell ′ Technology (China): sense: 5 -GATCCGCCACATGAAC lines, including Saos-2, U-2OS, and MG63, were selected GCCCAGAGATTTTCAAGAGAAATCTCTGGGCGTTC as subjects for subsequent analysis. All cells were purchased ′ ′ ATGTGGTTTTTTG-3 and antisense: 5 -AATTCAAAA from the American Type Culture Collection (ATCC) AACCACATGAACGCCCAGAGATTTCTCTTGAAAAT (Manassas, VA, USA). CTCTGGGCGTTCATGTGGCG-3 . After that, the plas- mids and control were transfected into Saos-2 and MG63 2.2. RNA Extraction and Quantitative Real-Time PCR (qRT- cells. RT-qPCR was performed to confirm the knockdown PCR). Total RNA was extracted by TRIzol reagent (Invitro- efficiency of HULC. gen Inc., Carlsbad, CA, USA). Subsequently, the RNA was reversely transcribed to the cDNA. The M-MLV reverse 2.5. Cell Viability Assay. The cell viability was detected using transcriptase (Promega, Madison, WI, USA) was used for 4 Cell Counting Kit-8 (CCK-8). Briefly, 1×10 cells were reverse transcription reaction. SYBR-Green Real-Time Mas- seeded into 96-well plates and cultured for 1, 2, 3, and 4 d. ter Mix (Toyobo, Tokyo, Japan) was applied for qRT-PCR Subsequently, osteosarcoma cells were incubated with −ΔΔCt reaction. Finally, the data were calculated with 2 ° CCK-8 at 37 C for 1 h. The absorbency at 450 nm of the cells method. was measured with microplate reader Thermo Plate (Rayto ′ ′ HULC P1: 5 -AACCTCCAGAACTGTGAT-3 and Life and Analytical Sciences Co., Ltd., Germany). ′ ′ HULC P2: 5 -CATAATTCAGGGAGAAAG-3 2.6. Cell Migration and Invasion Assays. For migration assay, ′ ′ β-Actin P1: 5 -CTTCCTTCCTGGGCATGGAG-3 and 2×10 cells and 100 μl serum-free medium were injected ′ ′ β-actin P2: 5 -GGAACGCTTCACGAATTTGC-3 into upper chamber. For Transwell invasion assay, Matrigel-coated chamber was used for invasion assay, and 3 2.3. Western Blot. The cells were treated with RAPA buffer (Beyotime, Jiangsu, China) on ice for 30 min. BCA kit ×10 cells were plated in 100 μl serum-free medium in the (Pierce, Rockford, IL, USA) was applied for the measurement upper. In both assays, 500 μl of the medium and 20% FBS of protein concentration. Subsequently, total proteins were were added to the lower chamber. Subsequently, the cells Number of invasive cells sh-NC sh-HULC Journal of Oncology 5 SAOS-2 MG63 5 5 4 4 3 3 2 2 1 1 0 0 (a) (b) SAOS-2 MG63 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 24 48 72 96 24 48 72 96 Time (h) Time (h) pcDNA3.1 pcDNA3.1 pcDNA3.1-HULC pcDNA3.1-HULC (c) (d) 2.0 1.5 1.0 0.5 0.0 pcDNA3.1-HULC pcDNA3.1 (e) (f) Figure 4: Continued. OD value (450 nm) Relative expression of HULC pcDNA3.1 pcDNA3.1-HULC OD value (450 nm) Relative expression of HULC pcDNA3.1 Tumor weight (g) pcDNA3.1-HULC pcDNA3.1 pcDNA3.1-HULC 6 Journal of Oncology 9 14192429 34 39 44 Days pcDNA3.1 pcDNA3.1-HULC (g) Figure 4: Overexpression of HULC increased the proliferation of osteosarcoma cells. (a, b) HULC expression in cells was detected by RT- qPCR in SAOS-2 (a) and MG63 cells (b). (c, d) CCK-8 assay was applied to measure the cell proliferation of SAOS-2 (c) and MG63 cells (d). (e) Mouse tumorigenesis assay was conducted. (f, g) The tumor weight (f) and volume (g) were detected. P <0:05. were cultured at 37 C and 5% CO for 24 h. After that, the knockdown notably inhibited the migration of osteosarcoma cells were fixed with 100% methanol for 30 min, and then, cell lines, including SAOS-2 (Figure 3(a)) and MG63 the cells in upper chamber were removed. Finally, the cells (Figure 3(c)). HULC knockdown markedly inhibited the were stained with 0.5% crystal violet (Sigma, St. Louis, MO, invasion of osteosarcoma cell lines, including SAOS-2 USA) for 20 min for cell count. (Figure 3(b)) and MG63 (Figure 3(d)). 2.7. Xenograft Tumor Formation. Male BALB/c nude mice (6 3.3. Overexpression of HULC Increased the Proliferation of weeks) were obtained from HFK Biosciences and main- Osteosarcoma Cells. Overexpression experiment was per- tained under pathogen-free conditions. The experiment formed to detect the effect of HULC in osteosarcoma cell was approved by the Shandong Provincial Hospital Affili- proliferation. In SAOS-2 (Figure 4(a)) and MG63 cells ated to Shandong First Medical University. For tumor prop- (Figure 4(b)), we successfully overexpressed HULC using agation analysis, the mice accepted the subcutaneous overexpression vectors. Overexpression of HULC signifi- injection including 2×10 HULC overexpression cells. After cantly increased cell proliferation of SAOS-2 (Figure 4(c)) 5 week, the weight of the tumors was measured. and MG63 cells (Figure 4(d)). Then, we injected the cultured cells into nude mice for tumorigenesis. As shown in 2.8. Statistical Analysis. SPSS 20.0 (SPSS Inc., Chicago, IL, Figure 4(e), HULC overexpression markedly raised tumor USA) was applied for data analyses. The Kaplan-Meier size. As observed by statistical data, HULC overexpression method and log-rank test were applied for the visualization markedly raised tumor weight (Figure 4(f)) and volume of survival curves. Moreover, one-way analyses of variance (Figure 4(g)). and two-tailed Student’s t-tests were applied for difference analysis. P <0:05 was considered statistically significant. 3.4. HULC Impeded PTEN to Activate AKT-PI3K-mTOR Pathway in Osteosarcoma Cells. In order to investigate the 3. Results interaction between HULC and PTEN, we overexpressed HULC, and western blot was applied to detect the expression 3.1. lncRNA HULC Was Increased in Osteosarcoma Tissue. of PTEN and other proteins (Figure 5(a)). We found that First, the expression of lncRNA HULC abundance was overexpression of HULC could result in decreased expres- examined in osteosarcoma tissue and normal tissue using sion of PTEN protein (Figure 5(b)). Moreover, excessive RT-qPCR. The expression of HULC in tumor tissues was HULC increased the phosphorylation of AKT higher than normal tissues (Figure 1(a)). Similarly, the (Figure 5(c)), mTOR (Figure 5(d)), and PI3K (Figure 5(e)), expression of HULC was also increased in tumor cell lines while overexpressed PTEN fully abrogated the HULC’s Saos-2, MG63, and U-2OS in contrast to normal cell (hFOB action. This suggested that HULC interacts in vivo with sig- 1.19) (Figure 1(c)). naling pathways that promote cancer formation. However, 3.2. HULC Knockdown Inhibited the Malignant Behaviors of the overexpression of PTEN cancels the role of HULC. Osteosarcoma Cells. Then, the effect of downregulation Together, PTEN determined the carcinogenic function of HULC on osteosarcoma cells was investigated. First, HULC HULC in liver cancer cells. was knockdown in osteosarcoma cell lines successfully (Figures 2(a) and 2(b)). In CCK-8 assay, we found that 3.5. Overexpressing HULC Enhanced the Malignant HULC knockdown significantly reduced cell proliferation Behaviors of Osteosarcoma Cells. To explore whether the in osteosarcoma cell lines (Figures 2(a) and 2(d)). We used effect of HULC on osteosarcoma cells depends on PI3K/ Transwell assay to detect cell migration and invasion. HULC Akt/mTOR signaling pathway, we added LY294002, an Tumor volume (mm ) Journal of Oncology 7 0.8 0.6 𝛽 -Actin 0.4 PTEN 0.2 AKT p-AKT 0.0 PI3K p-PI3K mTOR p-mTOR (a) (b) 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 (c) (d) 1.5 1.0 0.5 0.0 (e) Figure 5: HULC increases activity AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. (a) Protein expression was detected by western blot. (b) Overexpression of HULC could result in PTEN downregulation. (c–e) HULC advanced the phosphorylation of AKT (c), PI3K (d), and mTOR (e). P <0:05. Protein expression of p-AKT/AKT pcDNA3.1 pcDNA3.1 Protein expression of pcDNA3.1-HULC p-mTOR/mTOR pcDNA3.1-HULC HULC + PTEN HULC + PTEN pcDNA3.1 Protein expression of Protein expression of PTEN pcDNA3.1-HULC p-PI3K/PI3K HULC + PTEN pcDNA3.1 pcDNA3.1 pcDNA3.1-HULC pcDNA3.1-HULC HULC + PTEN HULC + PTEN 8 Journal of Oncology 1.5 𝛽 -Actin ⁎ 1.0 AKT p-AKT 0.5 PI3K p-PI3K 0.0 mTOR p-mTOR HULC HULC + LY294002 (a) III III 150 150 100 100 50 50 0 0 I II III III III (b) I II III 80 ⁎ I II III III III (c) Figure 6: Overexpressing HULC enhanced the malignant behaviors of osteosarcoma cells. I = pcDNA3:1, II = pcDNA3:1 − HULC, and III = pcDNA3:1 − HULC + LY294002. (a, b) Cell migration (a) and invasion (b) assay in SAOS-2 cells was detected by Transwell assay. (c, d) Cell migration (c) and invasion (d) assays in MG63 cells were measured by Transwell assay. P <0:05. inhibitor of PI3K signaling pathway. LY294002 inhibited the 4. Discussion phosphorylation of AKT, mTOR, and PI3K (Figure 6(a)). Overexpressing HULC enhanced cell migration and inva- Of all bone cancers, osteosarcoma is the second leading sion of SAOS-2 cells (Figure 6(b)) and MG63 cells cause of death in children [2]. Osteosarcoma is a fibrogenic (Figure 6(c)), while LY294002 reversed the effects of overex- malignant bone tumor that can directly or indirectly form pressing HULC on the cell migration and invasion. tumor bone-like tissue and bone tissue during development, MG63 SAOS-2 HULC HULC + LY294002 Protein expression levels Invasion Migration Invasion Migration Number of migrated cells p-AKT/AKT Number of migrated cells p-PI3K/PI3K p-mTOR/mTOR Number of invasive cells Number of invasive cells Journal of Oncology 9 which is a common primary tumor of bone [3]. Osteosar- gested that HULC interacts in vivo with signaling pathways coma has atypical clinical symptoms in the early stage of that promote cancer formation. However, the overexpres- onset, which is easy to be confused with other traumatic sion of PTEN cancels the role of HULC. In addition, the swelling and pain diseases. Due to the invasive growth of present study found that LY294002 inhibited the phosphor- osteosarcoma and early rapid proliferation, the cure rate ylation of AKT, mTOR, and PI3K. Overexpressing HULC and prognosis of osteosarcoma are poor. Recent studies have enhanced cell migration and invasion of SAOS-2 cells and shown that the occurrence, development, and biological MG63 cells, while LY294002 reversed the effects. A large characteristics of osteosarcoma are the result of polygenic number of literature reports pointed out that PI3K/Akt/ and multifactorial abnormalities dominated by oncogene mTOR signaling pathway is an important signaling pathway activation or tumor suppressor gene inactivation [18]. in cells, which is widely involved in the regulation of cell Therefore, to study the occurrence and development of oste- functions such as cell proliferation, apoptosis, and invasion osarcoma, we need to understand the changes of its gene [27]. The abnormal activation of PI3K/Akt/mTOR signaling level. For the treatment of osteosarcoma, there are mainly pathway has been confirmed in a variety of cancer cells. radiotherapy, chemotherapy, and combination therapy, but Miao et al. believed that PI3K/Akt/mTOR signaling pathway the prognosis is still not improved [5]. Despite numerous is the key to driving tumor cell proliferation and invasion, and emp1 can promote glioblastoma cell proliferation by studies, the pathogenic mechanisms of osteosarcoma are still not fully understood. activating PI3K/Akt/mTOR signaling pathway [28]. Previous reports have shown that lncRNA is involved in In conclusion, this study supported that the expression the formation and development of a variety of tumors [19], of lncRNA HULC was increased in osteosarcoma, which mainly because it is involved in important cellular processes enhanced the progression of osteosarcoma in vivo and such as regulating genome expression, transcription, and in vitro. translation [20]. lncRNA can participate in the pathological process of tumor cell proliferation, metastasis, and invasion Data Availability [21, 22]. As the lncRNA located on human chromosome 6p24.3, HULC is primarily located in the cytoplasm and The data that supports the findings of this study is available can play an important role in various physiopathological on reasonable request from the corresponding author. processes by binding to the ribosome. Previous reports have shown that HULC was involved in various processes of tumor formation and metastasis. Plasma HULC is consid- Conflicts of Interest ered to be a biomarker for detecting liver cancer [23]. In gas- The authors do not have conflicts of interest to declare. tric cancer, high expression of HULC promotes cell proliferation, inhibits apoptosis of cancer cells, and enhances tumor metastasis [24]. Xu et al. found that HULC regulates References PTPRO/NF-κB signaling pathway that promotes the devel- opment of lung squamous cell carcinoma [25]. The present [1] S. Wang, F. Ma, Y. Feng, T. Liu, and S. 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Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells

Journal of Oncology , Volume 2022 – Sep 30, 2022

Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells

Abstract

<i>Background</i>. Previous studies had shown that lncRNA HULC exhibited different effects in human cancers. However, the role of HULC was not reported in osteosarcoma. Hence, we designed this research to explore the function of HULC in osteosarcoma. <i>Methods</i>. Firstly, HULC expression was measured in osteosarcoma tissues and cells via the RT-qPCR assay. The protein expression was detected through western blot. Then, CCK-8 and Transwell assays were conducted to measure cell proliferation, migration, and invasion. <i>Results</i>. The expression of HULC was obviously higher in osteosarcoma tissues and cells compared with normal control. Moreover, cell proliferation, migration, and invasion were inhibited by HULC knockdown in osteosarcoma cells. HULC overexpression markedly increased osteosarcoma cell proliferation and tumor size in vivo. Furthermore, HULC increased the activity of AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K. Overexpressing HULC enhanced cell migration and invasion of SAOS-2 cells and MG63 cells, while LY294002 reversed the effects. <i>Conclusion</i>. HULC enhanced the progression of osteosarcoma through targeting PTEN.

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Publisher
Hindawi Publishing Corporation
ISSN
1687-8450
eISSN
1687-8469
DOI
10.1155/2022/7526731
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Abstract

Hindawi Journal of Oncology Volume 2022, Article ID 7526731, 10 pages https://doi.org/10.1155/2022/7526731 Research Article Effect of Long Noncoding RNA HULC on Proliferation, Migration, and Invasion of Osteosarcoma Cells 1 2 2 2 Ran Zhao, Xin Zhou, Wencan Zhang, and Le Li Department of Burns and Plastic Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021 Shandong, China Department of Orthopedic Surgery, Qilu Hospital of Shandong University, Jinan, 250021 Shandong, China Correspondence should be addressed to Le Li; qiluhosp_orthoplee@163.com Received 13 June 2022; Revised 2 September 2022; Accepted 15 September 2022; Published 30 September 2022 Academic Editor: Zhiqian Zhang Copyright © 2022 Ran Zhao 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. Background. Previous studies had shown that lncRNA HULC exhibited different effects in human cancers. However, the role of HULC was not reported in osteosarcoma. Hence, we designed this research to explore the function of HULC in osteosarcoma. Methods. Firstly, HULC expression was measured in osteosarcoma tissues and cells via the RT-qPCR assay. The protein expression was detected through western blot. Then, CCK-8 and Transwell assays were conducted to measure cell proliferation, migration, and invasion. Results. The expression of HULC was obviously higher in osteosarcoma tissues and cells compared with normal control. Moreover, cell proliferation, migration, and invasion were inhibited by HULC knockdown in osteosarcoma cells. HULC overexpression markedly increased osteosarcoma cell proliferation and tumor size in vivo. Furthermore, HULC increased the activity of AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K. Overexpressing HULC enhanced cell migration and invasion of SAOS-2 cells and MG63 cells, while LY294002 reversed the effects. Conclusion. HULC enhanced the progression of osteosarcoma through targeting PTEN. scripted into proteins, and 98% can be transcripted into 1. Introduction noncoding RNAs [7]. The function of these noncoding Osteosarcoma is caused by bone cell abnormal differentiation RNAs is still poorly understand. The long noncoding RNA and proliferation. Consistent with medicine information, the is a type of noncoding RNA that is longer than 200 nt [8]. incidence of osteogenic sarcoma is around 0.2–3/100000 per lncRNAs have been shown to be involved in a large number annum [1]. It is the foremost common primary bone tumor of cellular processes, such as cell proliferation, transcrip- in youngsters and adolescents with a high degree of malig- tional and posttranscriptional modification, epigenetic mod- nancy. Osteosarcoma typically shows a high tendency to path- ification, and invasion [9]. The large number of studies ologic process spread [2]. Osteosarcoma is commonly treated shows that lncRNA could play an important role in many with a mixture of therapies that may embody surgery, therapy, types of cancer [10]. For example, Chen et al. showed that and radiotherapy [3]. However, in recent years, the survival lncRNA HULC was overexpressed in epithelial ovarian car- rate of patients with osteosarcoma amid distant metastasis cinoma and target ATG7 [11]. Zheng et al. showed that has not been considerably improved, and also the impact of lncRNA HULC was highly expressed in HeLa cells and pro- therapy has not been considerably improved, and also the motes cell migration and invasion [12]. treatment of osteosarcoma remains controversial [4, 5]. Thus, lncRNA HULC was first reported by Panzitt et al. in new therapeutic targets ought to be found to produce clinical 2007, which was overexpressed lncRNA in human hepatocel- treatment choices to enhance the survival rate [6]. lular carcinoma [13]. HULC gene is located on chromosome High-throughput transcriptome sequencing analysis has 6p24.3 with approximately 500 nucleotides in length and shown that only 2% of the human genome can be tran- contains two exons [14]. Since then, the function of HULC 2 Journal of Oncology ⁎ 4 Non-tumor Tumor (a) (b) Figure 1: lncRNA HULC expression was increased in osteosarcoma tissue and cells. The expressions of HULC in osteosarcoma tissues (a) and cell lines (b) were detected by RT-qPCR. P <0:05. 1.5 MG63 1.5 SAOS-2 1.0 1.0 0.5 0.5 0.0 0.0 sh-NC sh-HULC (a) (b) MG63 SAOS-2 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 24 48 72 96 24 48 72 96 Time (h) Time (h) sh-NC sh-NC sh-HULC sh-HULC (c) (d) Figure 2: Downregulation of HULC impeded the proliferation of osteosarcoma cells. (a, b) The relative expression of HULC in cells was detected in SAOS-2 (a) and MG63 cells (b) by RT-qPCR. (c, d) Cell viability in SAOS-2 (c) and MG63 cells (d) was measured by CCK-8 assay. P <0:05. OD value (450 nm) Relative expression of HULC Relative expression of HULC Relative expression of HULC OD value (450 nm) Relative expression of HULC hFOB 1.19 sh-NC SAOS-2 U-2 OS sh-HULC MG63 Journal of Oncology 3 sh-NC sh-HULC (a) sh-NC sh-HULC SAOS-2 (b) sh-NC sh-HULC MG63 (c) Figure 3: Continued. Number of migrated cells Number of invasive cells Number of migrated cells sh-NC sh-NC sh-NC sh-HULC sh-HULC sh-HULC 4 Journal of Oncology sh-NC sh-HULC MG63 (d) Figure 3: Decreased HULC impeded the malignant behaviors of osteosarcoma cells. (a, b) Cell migration (a) and invasion (b) assays in SAOS-2 cells were detected by Transwell assay. (c, d) Cell migration (c) and invasion (d) assays in MG63 cells were measured by Transwell assay. P <0:05. has been demonstrated in multiple cancer types [15, 16]. separated by 12% SDS-polyacrylamide gel electrophoresis HULC could promote cancer cell survival, proliferation, (SDS-PAGE) and then were transferred on polyvinylidene and invasion. These studies indicated that HULC played an fluoride membranes (Millipore, Billerica, MA, USA). The important role in the development of cancer [16, 17]. HULC membranes were treated with 5% fat-free milk for 2 h and was overexpressed in human osteosarcoma cell which has then were incubated with the antibodies of AKT (PTG, been shown in the previous studies. However, the mecha- USA, 1 : 1000), p-AKT (PTG, USA, 1 : 10000), PI3K (CST, nism of HULC involved in the progression of osteosarcoma USA, 1 : 1000), p-PI3K (CST, USA, 1 : 1000), mTOR (PTG, is unknown, and the interaction of HULC and PTEN has USA, 1 : 20000), p-mTOR (PTG, USA, 1 : 10000), PTEN not been proved in osteosarcoma. (ABclonal, China, 1 : 1000), or β-actin (CST, USA, 1 : 1000). Therefore, we investigated the interaction of HULC and After washing and incubating with the second antibodies, PTEN in promoting osteosarcoma cell proliferation and the abundance of the proteins was analyzed by enhanced migration. chemiluminescence (ECL). 2.4. shRNA Transfection. In short, the specific oligonucleo- 2. Materials and Methods tides targeting HULC were synthesized by Genepharmacy 2.1. Cell Lines and Culture. The human osteosarcoma cell ′ Technology (China): sense: 5 -GATCCGCCACATGAAC lines, including Saos-2, U-2OS, and MG63, were selected GCCCAGAGATTTTCAAGAGAAATCTCTGGGCGTTC as subjects for subsequent analysis. All cells were purchased ′ ′ ATGTGGTTTTTTG-3 and antisense: 5 -AATTCAAAA from the American Type Culture Collection (ATCC) AACCACATGAACGCCCAGAGATTTCTCTTGAAAAT (Manassas, VA, USA). CTCTGGGCGTTCATGTGGCG-3 . After that, the plas- mids and control were transfected into Saos-2 and MG63 2.2. RNA Extraction and Quantitative Real-Time PCR (qRT- cells. RT-qPCR was performed to confirm the knockdown PCR). Total RNA was extracted by TRIzol reagent (Invitro- efficiency of HULC. gen Inc., Carlsbad, CA, USA). Subsequently, the RNA was reversely transcribed to the cDNA. The M-MLV reverse 2.5. Cell Viability Assay. The cell viability was detected using transcriptase (Promega, Madison, WI, USA) was used for 4 Cell Counting Kit-8 (CCK-8). Briefly, 1×10 cells were reverse transcription reaction. SYBR-Green Real-Time Mas- seeded into 96-well plates and cultured for 1, 2, 3, and 4 d. ter Mix (Toyobo, Tokyo, Japan) was applied for qRT-PCR Subsequently, osteosarcoma cells were incubated with −ΔΔCt reaction. Finally, the data were calculated with 2 ° CCK-8 at 37 C for 1 h. The absorbency at 450 nm of the cells method. was measured with microplate reader Thermo Plate (Rayto ′ ′ HULC P1: 5 -AACCTCCAGAACTGTGAT-3 and Life and Analytical Sciences Co., Ltd., Germany). ′ ′ HULC P2: 5 -CATAATTCAGGGAGAAAG-3 2.6. Cell Migration and Invasion Assays. For migration assay, ′ ′ β-Actin P1: 5 -CTTCCTTCCTGGGCATGGAG-3 and 2×10 cells and 100 μl serum-free medium were injected ′ ′ β-actin P2: 5 -GGAACGCTTCACGAATTTGC-3 into upper chamber. For Transwell invasion assay, Matrigel-coated chamber was used for invasion assay, and 3 2.3. Western Blot. The cells were treated with RAPA buffer (Beyotime, Jiangsu, China) on ice for 30 min. BCA kit ×10 cells were plated in 100 μl serum-free medium in the (Pierce, Rockford, IL, USA) was applied for the measurement upper. In both assays, 500 μl of the medium and 20% FBS of protein concentration. Subsequently, total proteins were were added to the lower chamber. Subsequently, the cells Number of invasive cells sh-NC sh-HULC Journal of Oncology 5 SAOS-2 MG63 5 5 4 4 3 3 2 2 1 1 0 0 (a) (b) SAOS-2 MG63 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 24 48 72 96 24 48 72 96 Time (h) Time (h) pcDNA3.1 pcDNA3.1 pcDNA3.1-HULC pcDNA3.1-HULC (c) (d) 2.0 1.5 1.0 0.5 0.0 pcDNA3.1-HULC pcDNA3.1 (e) (f) Figure 4: Continued. OD value (450 nm) Relative expression of HULC pcDNA3.1 pcDNA3.1-HULC OD value (450 nm) Relative expression of HULC pcDNA3.1 Tumor weight (g) pcDNA3.1-HULC pcDNA3.1 pcDNA3.1-HULC 6 Journal of Oncology 9 14192429 34 39 44 Days pcDNA3.1 pcDNA3.1-HULC (g) Figure 4: Overexpression of HULC increased the proliferation of osteosarcoma cells. (a, b) HULC expression in cells was detected by RT- qPCR in SAOS-2 (a) and MG63 cells (b). (c, d) CCK-8 assay was applied to measure the cell proliferation of SAOS-2 (c) and MG63 cells (d). (e) Mouse tumorigenesis assay was conducted. (f, g) The tumor weight (f) and volume (g) were detected. P <0:05. were cultured at 37 C and 5% CO for 24 h. After that, the knockdown notably inhibited the migration of osteosarcoma cells were fixed with 100% methanol for 30 min, and then, cell lines, including SAOS-2 (Figure 3(a)) and MG63 the cells in upper chamber were removed. Finally, the cells (Figure 3(c)). HULC knockdown markedly inhibited the were stained with 0.5% crystal violet (Sigma, St. Louis, MO, invasion of osteosarcoma cell lines, including SAOS-2 USA) for 20 min for cell count. (Figure 3(b)) and MG63 (Figure 3(d)). 2.7. Xenograft Tumor Formation. Male BALB/c nude mice (6 3.3. Overexpression of HULC Increased the Proliferation of weeks) were obtained from HFK Biosciences and main- Osteosarcoma Cells. Overexpression experiment was per- tained under pathogen-free conditions. The experiment formed to detect the effect of HULC in osteosarcoma cell was approved by the Shandong Provincial Hospital Affili- proliferation. In SAOS-2 (Figure 4(a)) and MG63 cells ated to Shandong First Medical University. For tumor prop- (Figure 4(b)), we successfully overexpressed HULC using agation analysis, the mice accepted the subcutaneous overexpression vectors. Overexpression of HULC signifi- injection including 2×10 HULC overexpression cells. After cantly increased cell proliferation of SAOS-2 (Figure 4(c)) 5 week, the weight of the tumors was measured. and MG63 cells (Figure 4(d)). Then, we injected the cultured cells into nude mice for tumorigenesis. As shown in 2.8. Statistical Analysis. SPSS 20.0 (SPSS Inc., Chicago, IL, Figure 4(e), HULC overexpression markedly raised tumor USA) was applied for data analyses. The Kaplan-Meier size. As observed by statistical data, HULC overexpression method and log-rank test were applied for the visualization markedly raised tumor weight (Figure 4(f)) and volume of survival curves. Moreover, one-way analyses of variance (Figure 4(g)). and two-tailed Student’s t-tests were applied for difference analysis. P <0:05 was considered statistically significant. 3.4. HULC Impeded PTEN to Activate AKT-PI3K-mTOR Pathway in Osteosarcoma Cells. In order to investigate the 3. Results interaction between HULC and PTEN, we overexpressed HULC, and western blot was applied to detect the expression 3.1. lncRNA HULC Was Increased in Osteosarcoma Tissue. of PTEN and other proteins (Figure 5(a)). We found that First, the expression of lncRNA HULC abundance was overexpression of HULC could result in decreased expres- examined in osteosarcoma tissue and normal tissue using sion of PTEN protein (Figure 5(b)). Moreover, excessive RT-qPCR. The expression of HULC in tumor tissues was HULC increased the phosphorylation of AKT higher than normal tissues (Figure 1(a)). Similarly, the (Figure 5(c)), mTOR (Figure 5(d)), and PI3K (Figure 5(e)), expression of HULC was also increased in tumor cell lines while overexpressed PTEN fully abrogated the HULC’s Saos-2, MG63, and U-2OS in contrast to normal cell (hFOB action. This suggested that HULC interacts in vivo with sig- 1.19) (Figure 1(c)). naling pathways that promote cancer formation. However, 3.2. HULC Knockdown Inhibited the Malignant Behaviors of the overexpression of PTEN cancels the role of HULC. Osteosarcoma Cells. Then, the effect of downregulation Together, PTEN determined the carcinogenic function of HULC on osteosarcoma cells was investigated. First, HULC HULC in liver cancer cells. was knockdown in osteosarcoma cell lines successfully (Figures 2(a) and 2(b)). In CCK-8 assay, we found that 3.5. Overexpressing HULC Enhanced the Malignant HULC knockdown significantly reduced cell proliferation Behaviors of Osteosarcoma Cells. To explore whether the in osteosarcoma cell lines (Figures 2(a) and 2(d)). We used effect of HULC on osteosarcoma cells depends on PI3K/ Transwell assay to detect cell migration and invasion. HULC Akt/mTOR signaling pathway, we added LY294002, an Tumor volume (mm ) Journal of Oncology 7 0.8 0.6 𝛽 -Actin 0.4 PTEN 0.2 AKT p-AKT 0.0 PI3K p-PI3K mTOR p-mTOR (a) (b) 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 (c) (d) 1.5 1.0 0.5 0.0 (e) Figure 5: HULC increases activity AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. (a) Protein expression was detected by western blot. (b) Overexpression of HULC could result in PTEN downregulation. (c–e) HULC advanced the phosphorylation of AKT (c), PI3K (d), and mTOR (e). P <0:05. Protein expression of p-AKT/AKT pcDNA3.1 pcDNA3.1 Protein expression of pcDNA3.1-HULC p-mTOR/mTOR pcDNA3.1-HULC HULC + PTEN HULC + PTEN pcDNA3.1 Protein expression of Protein expression of PTEN pcDNA3.1-HULC p-PI3K/PI3K HULC + PTEN pcDNA3.1 pcDNA3.1 pcDNA3.1-HULC pcDNA3.1-HULC HULC + PTEN HULC + PTEN 8 Journal of Oncology 1.5 𝛽 -Actin ⁎ 1.0 AKT p-AKT 0.5 PI3K p-PI3K 0.0 mTOR p-mTOR HULC HULC + LY294002 (a) III III 150 150 100 100 50 50 0 0 I II III III III (b) I II III 80 ⁎ I II III III III (c) Figure 6: Overexpressing HULC enhanced the malignant behaviors of osteosarcoma cells. I = pcDNA3:1, II = pcDNA3:1 − HULC, and III = pcDNA3:1 − HULC + LY294002. (a, b) Cell migration (a) and invasion (b) assay in SAOS-2 cells was detected by Transwell assay. (c, d) Cell migration (c) and invasion (d) assays in MG63 cells were measured by Transwell assay. P <0:05. inhibitor of PI3K signaling pathway. LY294002 inhibited the 4. Discussion phosphorylation of AKT, mTOR, and PI3K (Figure 6(a)). Overexpressing HULC enhanced cell migration and inva- Of all bone cancers, osteosarcoma is the second leading sion of SAOS-2 cells (Figure 6(b)) and MG63 cells cause of death in children [2]. Osteosarcoma is a fibrogenic (Figure 6(c)), while LY294002 reversed the effects of overex- malignant bone tumor that can directly or indirectly form pressing HULC on the cell migration and invasion. tumor bone-like tissue and bone tissue during development, MG63 SAOS-2 HULC HULC + LY294002 Protein expression levels Invasion Migration Invasion Migration Number of migrated cells p-AKT/AKT Number of migrated cells p-PI3K/PI3K p-mTOR/mTOR Number of invasive cells Number of invasive cells Journal of Oncology 9 which is a common primary tumor of bone [3]. Osteosar- gested that HULC interacts in vivo with signaling pathways coma has atypical clinical symptoms in the early stage of that promote cancer formation. However, the overexpres- onset, which is easy to be confused with other traumatic sion of PTEN cancels the role of HULC. In addition, the swelling and pain diseases. Due to the invasive growth of present study found that LY294002 inhibited the phosphor- osteosarcoma and early rapid proliferation, the cure rate ylation of AKT, mTOR, and PI3K. Overexpressing HULC and prognosis of osteosarcoma are poor. Recent studies have enhanced cell migration and invasion of SAOS-2 cells and shown that the occurrence, development, and biological MG63 cells, while LY294002 reversed the effects. A large characteristics of osteosarcoma are the result of polygenic number of literature reports pointed out that PI3K/Akt/ and multifactorial abnormalities dominated by oncogene mTOR signaling pathway is an important signaling pathway activation or tumor suppressor gene inactivation [18]. in cells, which is widely involved in the regulation of cell Therefore, to study the occurrence and development of oste- functions such as cell proliferation, apoptosis, and invasion osarcoma, we need to understand the changes of its gene [27]. The abnormal activation of PI3K/Akt/mTOR signaling level. For the treatment of osteosarcoma, there are mainly pathway has been confirmed in a variety of cancer cells. radiotherapy, chemotherapy, and combination therapy, but Miao et al. believed that PI3K/Akt/mTOR signaling pathway the prognosis is still not improved [5]. Despite numerous is the key to driving tumor cell proliferation and invasion, and emp1 can promote glioblastoma cell proliferation by studies, the pathogenic mechanisms of osteosarcoma are still not fully understood. activating PI3K/Akt/mTOR signaling pathway [28]. Previous reports have shown that lncRNA is involved in In conclusion, this study supported that the expression the formation and development of a variety of tumors [19], of lncRNA HULC was increased in osteosarcoma, which mainly because it is involved in important cellular processes enhanced the progression of osteosarcoma in vivo and such as regulating genome expression, transcription, and in vitro. translation [20]. lncRNA can participate in the pathological process of tumor cell proliferation, metastasis, and invasion Data Availability [21, 22]. As the lncRNA located on human chromosome 6p24.3, HULC is primarily located in the cytoplasm and The data that supports the findings of this study is available can play an important role in various physiopathological on reasonable request from the corresponding author. processes by binding to the ribosome. Previous reports have shown that HULC was involved in various processes of tumor formation and metastasis. Plasma HULC is consid- Conflicts of Interest ered to be a biomarker for detecting liver cancer [23]. In gas- The authors do not have conflicts of interest to declare. tric cancer, high expression of HULC promotes cell proliferation, inhibits apoptosis of cancer cells, and enhances tumor metastasis [24]. Xu et al. found that HULC regulates References PTPRO/NF-κB signaling pathway that promotes the devel- opment of lung squamous cell carcinoma [25]. The present [1] S. Wang, F. Ma, Y. Feng, T. Liu, and S. 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Journal of OncologyHindawi Publishing Corporation

Published: Sep 30, 2022

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