MicroRNA-101 inhibits epithelial-mesenchymal transition in human liver carcinoma MHCC97H cells via USP22
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摘要:
目的 研究微小核糖核酸（miR）-101对人肝细胞癌（肝癌）MHCC97H细胞的上皮-间质转化功能的影响及其相关机制。 方法 MHCC97H肝癌细胞株分别转染miR-101 mimics和negative control mimic，分别作为M101组、NCM组，并设立未转染对照（control）组，采用逆转录聚合酶链反应（RT-PCR）法检测各组细胞miR-101含量。Transwell实验检测3组细胞迁移能力和侵袭能力。Western-blot法检测3组细胞vimentin、α-catenin、E-cadherin和USP22表达水平。双荧光素酶实验检测miR-101与USP22的关系。 结果 M101组miR-101的表达水平明显上调，表达水平约为control组的761倍（P < 0.05）。M101组迁移细胞数量明显低于control组的[（15.7±1.6）个比（94.1±1.8）个，P < 0.05]。M101组侵袭细胞数量明显低于control组的[（9.1±0.4）个比（51.6±0.9）个，P < 0.05]。M101组细胞vimentin蛋白表达量明显降低，α-catenin及E-cadherin蛋白表达量明显升高，USP22蛋白表达量明显降低。双荧光素酶检验结果显示USP22为miR-101的下游靶基因。 结论 miR-101可能通过降低下游靶基因USP22水平影响EMT相关蛋白表达，抑制MHCC97H肝癌细胞的上皮-间质转化功能。 -
关键词:
- 肝细胞癌 /
- 微小核糖核酸（miR）-101 /
- 上皮-间质转化 /
- 侵袭 /
- 转移 /
- 泛素特异性蛋白酶22（USP22） /
- 双荧光素酶实验 /
- 逆转录聚合酶链反应（RT-PCR）
Abstract:Objective To investigate the effect and underlying mechanism of micro ribonucleic acid (miR)-101 on the epithelial-mesenchymal transition in human hepatocellular carcinoma MHCC97H cells. Methods MHCC97H cell lines were transfected with miR-101 mimics (M101 group) and negative control mimic (NCM group), and the cell lines without transfection were used as the control group. The expression levels of miR-101 in cells of 3 groups were quantitatively measured using reverse transcription polymerase chain reaction (RT-PCR). Transwell assay was performed to evaluate the cell migration and invasion ability of 3 groups. The expression levels of vimentin, -catenin, E-cadherin and USP22 proteins in cells of 3 groups were measured by Western blot. The relationship between miR-101 and USP22 was analyzed by dual-luciferase assay. Results In the M101 group, the expression level of miR-101 was significantly up-regulated, which was approximately 761 times of that in the control group (P < 0.05). In the M101 group, the quantity of migrating cells was 15.7±1.6, significantly lower than 94.1±1.8 in the control group (P < 0.05). In the M101 group, the quantity of invasive cells was 9.1±0.4, significantly lower compared with 51.6±0.9 in the control group (P < 0.05). In the M101 group, the expression levels of vimentin and USP22 protein were significantly down-regulated, whereas the expression levels of -catenin and E-cadherin protein were significantly up-regulated. Dual-luciferase assay revealed that USP22 was the target gene of the downstream miR-101 signaling pathway. Conclusion miR-101 regulates the expression of epithelial-mesenchymal transition-related proteins and suppresses the epithelial-mesenchymal transition of hepatocellular carcinoma MHCC97H cells probably through down-regulating the expression level of USP22. -
图 1 3组MHCC97H细胞miR-101水平的比较
与control组比较，aP < 0.05
Figure 1. Comparison of miR-101 levels in 3 groups of MHCC97H cells
图 2 miR-101对MHCC97H细胞迁移能力的影响
与control组比较，aP<0.05
Figure 2. Effect of miR-101 on migration ability of MHCC97H cells
图 3 miR-101对MHCC97H细胞侵袭能力的影响
与control组比较，aP<0.05
Figure 3. Effect of miR-101 on invasion ability of MHCC97H cells
图 4 miR-101对MHCC97H细胞EMT相关功能蛋白表达的影响
与control组比较，aP<0.05
Figure 4. Effect of miR-101 on the expression of EMT functional proteins in MHCC97H cells
图 5 miR-101对MHCC97H细胞USP22蛋白表达的影响
与control组比较，aP<0.05
Figure 5. Effect of miR-101 on the expression of USP22 protein in MHCC97H cells
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[1] Chen JS, Li HS, Huang JQ, et al. MicroRNA-379-5p inhibits tumor invasion and metastasis by targeting FAK/AKT signaling in hepatocellular carcinoma[J]. Cancer Lett, 2016, 375(1):73-83. DOI: 10.1016/j.canlet.2016.02.043. [2] Li SP, Xu HX, Yu Y, et al. LncRNA HULC enhances epithelial-mesenchymal transition to promote tumorigenesis and metastasis of hepatocellular carcinoma via the miR-200a-3p/ZEB1 signaling pathway[J]. Oncotarget, 2016, 7(27):42431-42446.DOI: 10.18632/oncotarget.9883. [3] Guo J, Huang X, Wang H, et al. Celastrol induces autophagy by targeting AR/miR-101 in prostate cancer cells[J]. PLoS One, 2015, 10(10):e0140745. DOI: 10.1371/journal.pone.0140745. [4] Zheng M, Jiang YP, Chen W, et al. Snail and Slug collaborate on EMT and tumor metastasis through miR-101-mediated EZH2 axis in oral tongue squamous cell carcinoma[J]. Oncotarget, 2015, 6(9):6797-6810. doi: 10.18632/oncotarget [5] Wang J, Ou J, Guo Y, et al. TBLR1 is a novel prognostic marker and promotes epithelial-mesenchymal transition in cervical cancer[J]. Br J Cancer, 2014, 111(1):112-124. DOI: 10.1038/bjc.2014.278. [6] Jiang W, Gu W, Qiu R, et al. MiRNA-101 suppresses epithelial-to-mesenchymal transition by targeting HMGA2 in pancreatic cancer cells[J]. Anticancer Agents Med Chem, 2016, 16(4):432-439. doi: 10.2174/1871520615666150507122142 [7] Guo F, Parker Kerrigan BC, Yang D, et al. Post-transcriptional regulatory network of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions[J]. J Hematol Oncol, 2014, 7:19. DOI: 10.1186/1756-8722-7-19. [8] Zhao S, Zhang Y, Zheng X, et al. Loss of microRNA-101 promotes epithelial to mesenchymal transition in hepatocytes[J]. J Cell Physiol, 2015, 230(11):2706-2717. DOI: 10.1002/jcp.24995. [9] 陈斌, 沈顺利, 彭宝岗.微小RNA let-7a对CD133+肝癌干细胞增殖和转移的影响[J/CD].中华细胞与干细胞杂志(电子版), 2015, 5(3):44-47. DOI:10.3877/cma.j.issn.2095-1221. 2015.03.010.Chen B, Shen SL, Peng BG. Effect of microRNA let-7a on the proliferation and metastasis of CD133+ liver cancer stem cells[J/CD]. Chin J Cell Stem Cell(Electr Edit), 2015, 5(3):44-47. DOI: 10.3877/cma.j.issn.2095-1221.2015.03.010. [10] Guo F, Cogdell D, Hu L, et al. MiR-101 suppresses the epithelial-to-mesenchymal transition by targeting ZEB1 and ZEB2 in ovarian carcinoma[J]. Oncol Rep, 2014, 31(5):2021-2028. DOI: 10.3892/or.2014.3106. [11] Bao RF, Shu YJ, Hu YP, et al. MiR-101 targeting ZFX suppresses tumor proliferation and metastasis by regulating the MAPK/Erk and Smad pathways in gallbladder carcinoma[J]. Oncotarget, 2016, 7(16):22339-22354. DOI: 10.18632/oncotarget.7970. [12] Wei X, Tang C, Lu X, et al. MiR-101 targets DUSP1 to regulate the TGF-beta secretion in sorafenib inhibits macrophage-induced growth of hepatocarcinoma[J]. Oncotarget, 2015, 6(21):18389-18405. doi: 10.18632/oncotarget [13] Qian K, Liu G, Tang Z, et al. The long non-coding RNA NEAT1 interacted with miR-101 modulates breast cancer growth by targeting EZH2[J]. Arch Biochem Biophys, 2017, 615: 1-9. DOI: 10.1016/j.abb.2016.12.011. [14] Hu H, He Y, Wang Y, et al. MicorRNA-101 silences DNA-PKcs and sensitizes pancreatic cancer cells to gemcitabine[J]. Biochem Biophys Res Commun, 2017, 483(1) : 725-731. DOI: 10.1016/j.bbrc.2016.12.074. [15] Ye Z, Yin S, Su Z, et al. Downregulation of miR-101 contributes to epithelial-mesenchymal transition in cisplatin resistance of NSCLC cells by targeting ROCK2[J]. Oncotarget, 2016, 7(25):37524-37535. DOI: 10.18632/oncotarget.6852. [16] Zhao H, Tang H, Huang Q, et al. MiR-101 targets USP22 to inhibit the tumorigenesis of papillary thyroid carcinoma[J]. Am J Cancer Res, 2016, 6(11):2575-2586. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126274/ [17] Tang B, Liang X, Tang F, et al. Expression of USP22 and Survivin is an indicator of malignant behavior in hepatocellular carcinoma[J]. Int J Oncol, 2015, 47(6):2208-2216. DOI: 10.3892/ijo.2015.3214. [18] Tang B, Tang F, Li B, et al. High USP22 expression indicates poor prognosis in hepatocellular carcinoma[J]. Oncotarget, 2015, 6(14):12654-12667. doi: 10.18632/oncotarget [19] Zhai R, Tang F, Gong J, et al. The relationship between the expression of USP22, BMI1, and EZH2 in hepatocellular carcinoma and their impacts on prognosis[J]. Onco Targets Ther, 2016, 9:6987-6998. doi: 10.2147/OTT -
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