MiR-373-3p靶向YWHAZ抑制肝细胞癌增殖和转移能力的研究
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摘要
[目的]探讨miR-373-3p在肝细胞癌中的生物学作用和分子机制。[方法]用miR-373-3p模拟物或miR-373-3p抑制剂转染肝细胞癌细胞系。通过MTT、克隆形成和Transwell实验鉴定miR-373-3p对肝细胞癌细胞增殖和迁移能力的影响。Western blot和实时荧光定量逆转录聚合酶链反应(real-time quantitative reverse transcription PCR,qRT-PCR)检测相关基因蛋白和mRNA的表达水平。采用双荧光素酶法验证miR-373-3p与YWHAZ的关系。[结果]QRT-PCR检测显示,miR-373-3p在肝癌组织中的表达明显低于癌旁组织,且miR-373-3p的低表达与肝癌患者的肿瘤大小、血管浸润和不良预后显著相关。体外细胞学实验结果显示,过表达miR-373-3p能够显著抑制肝癌细胞的增殖和侵袭能力,而下调miR-373-3p的表达则作用相反。双荧光素酶报告、qRT-PCR和Western blot实验证实,miR-373-3p能够直接靶向YWHAZ的3′-非翻译区(UTR)且抑制YWHAZ在肝癌细胞中的表达。过表达的YWHAZ能够抵消miR-373-3p对肝细胞癌细胞增殖和转移的抑制作用。[结论] Mi R-373-3p可能是一种新的潜在的肝癌治疗靶点。
[Purpose]To investigate the biological function and molecular mechanism of miR-373-3p in hepatocellular carcinoma. [Methods] Hepatocellular carcinoma(HCC) cells were transfected with miR-373-3p mimics or miR-373-3p inhibitor. Proliferation and migration were identified by MTT assay,colony formation and transwell assays. Protein and mRNA expression levels of associated genes were measured by Western blot and real-time quantitative reverse transcription PCR(q RT-PCR). Dual luciferase assay was used to determine the relation of miR-373-3p to YWHAZ gene. [Results] The results of qRT-PCR assay demonstrated that expression of miR-373-3p was significantly lower in HCC tissues compared with adjacent liver tissues. The lower miR-373-3p expression significantly associated with tumor size,vascular infiltration and poor prognostic outcome in HCC patients. In vitro,ectopic overexpression of miR-373-3p significantly inhibited cell proliferation and invasion capability,while down-regulated miR-373-3p had reversed effects. Furthermore,luciferase reporter gene assay,qRT-PCR,and Western blot assays demonstrated that miR-373-3p targeted 3′-untranslated region(UTR) of YWHAZ gene and inhibited its expression in HCC cells. Overexpression of YWHAZ partially abolished the tumor suppressing effects induced by upregulating miR-373-3p in HCC cells. [Conclusion] MiR-373-3p might represent a novel potentially therapeutic target for HCC.
[Purpose]To investigate the biological function and molecular mechanism of miR-373-3p in hepatocellular carcinoma. [Methods] Hepatocellular carcinoma(HCC) cells were transfected with miR-373-3p mimics or miR-373-3p inhibitor. Proliferation and migration were identified by MTT assay,colony formation and transwell assays. Protein and mRNA expression levels of associated genes were measured by Western blot and real-time quantitative reverse transcription PCR(q RT-PCR). Dual luciferase assay was used to determine the relation of miR-373-3p to YWHAZ gene. [Results] The results of qRT-PCR assay demonstrated that expression of miR-373-3p was significantly lower in HCC tissues compared with adjacent liver tissues. The lower miR-373-3p expression significantly associated with tumor size,vascular infiltration and poor prognostic outcome in HCC patients. In vitro,ectopic overexpression of miR-373-3p significantly inhibited cell proliferation and invasion capability,while down-regulated miR-373-3p had reversed effects. Furthermore,luciferase reporter gene assay,qRT-PCR,and Western blot assays demonstrated that miR-373-3p targeted 3′-untranslated region(UTR) of YWHAZ gene and inhibited its expression in HCC cells. Overexpression of YWHAZ partially abolished the tumor suppressing effects induced by upregulating miR-373-3p in HCC cells. [Conclusion] MiR-373-3p might represent a novel potentially therapeutic target for HCC.
引文
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[17]Huang Q,Gumireddy K,Schrier M,et al.The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis[J].Nat Cell Biol,2008,10(2):202-210.
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[19]Voorhoeve PM,Le SC,Schrier M,et al.A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors[J].Cell,2006,124(6):1169-1181.
[20]Nishimura Y,Komatsu S,Ichikawa D,et al.Overexpression of ywhaz relates to tumor cell proliferation and malignant outcome of gastric carcinoma[J].Br J Cancer,2013,108(6):1324-1331.
[21]Chen CH,Chuang SM,Yang MF,et al.A novel function of YWHAZ/β-catenin axis in promoting epithelial-mesenchymal transition and lung cancer metastasis[J].Mol Cancer Res,2012,10(10):1319.
[22]Watanabe N,Komatsu S,Ichikawa D,et al.Overexpression of YWHAZ as an independent prognostic factor in adenocarcinoma of the esophago-gastric junction[J].Am J Cancer Res,2016,6(11):2729.
[23]Tang Y,Lv P,Sun Z,et al.14-3-3B promotes migration and invasion of human hepatocellular carcinoma cells by modulating expression of MMP2 and MMP9 through PI3K/AKT/NF-κB pathway[J].PLoS One,2016,11(1):e0146070.
[2]Chen WQ,Cui FQ,Fan CS,et al.Strategies of primary prevention of liver cancer in China:expert consensus(2018)[J].China Cancer,2018,27(9):660-669.[陈万青,崔富强,樊春笋,等.中国肝癌一级预防专家共识(2018)[J].中国肿瘤,2018,27(9):660-669.]
[3]Arzumanyan A,Reis HM,Feitelson MA.Pathogenic mechanisms in HBV-and HCV-associated hepatocellular carcinoma[J].Nat Rev Cancer,2013,13(2):123-135.
[4]Song SF,Li Y,Li K,et al.Quality of life and related influencing factors of patients with primary liver cancer[J].China Cancer,2017,26(9):745-748.[宋韶芳,李燕,李科,等.广州市原发性肝癌患者生存质量及影响因素分析[J].中国肿瘤,2017,26(9):745-748.]
[5]Altekruse SF,Mcglynn KA,Dickie LA,et al.Hepatocellular carcinoma confirmation,treatment,and survival in Surveillance,Epidemiology and End Results registries,1992-2008[J].Hepatology,2012,55(2):476-482.
[6]Chen D,Wang KB,Li JZ,et al.Prediction of serum VEG-FR-2 concentration on the short-term effects of TACE in patients with hepatocellular carcinoma[J].China Cancer,2017,26(6):476-481.[陈丹,王凯冰,李加桩,等.血清VEGFR-2浓度变化预测肝癌患者TACE近期疗效的研究[J].中国肿瘤,2017,26(6):476-481.]
[7]Yates LA,Norbury CJ,Gilbert RJC.The long and short of MicroRNA[J].Cell,2013,153(3):516-519.
[8]Negrini M,Gramantieri L,Sabbioni S,et al.MicroRNA involvement in hepatocellular carcinoma[J].Anticancer A-gents Med Chem,2011,11(6):500-521.
[9]Li Y,Ren M,Zhao Y,et al.MicroRNA-26a inhibits proliferation and metastasis of human hepatocellular carcinoma by regulating dnmt3b-meg3 axis[J].Oncol Rep,2017,37(6):3527.
[10]Mahati S,Xiao L,Yang Y,et al.MiR-29a suppresses growth and migration of hepatocellular carcinoma by regulating CLDN1[J].Biochem Biophys Res Commun,2017,486(3):732-737.
[11]Fu X,Wen H,Jing L,et al.MicroRNA-155-5p promotes hepatocellular carcinoma progression by suppressing PTENthrough the PI3K/AKT pathway[J].Cancer Sci,2017,108(4):620-631.
[12]Liu Z,Wang C,Jiao X,et al.MiR-221 promotes growth and invasion of hepatocellular carcinoma cells by constitutive activation of NFκB[J].Am J Transl Res,2016,8(11):4764.
[13]Zhang JJ,Wang CY,Hua L,et al.MiR-107 promotes hepatocellular carcinoma cell proliferation by targeting Axin2[J].Int J Clin Exp Pathol,2015,8(5):5168-5174.
[14]He H,Yang Q,Zuo Y,et al.MicroRNA-494-3p promotes cell growth,migration and invasion of nasopharyngeal carcinoma by targeting sox7[J].Ann Oncol,2015,26(Suppl9):150-150.
[15]Wei F,Cao C,Xu X,et al.Diverse functions of miR-373in cancer[J].J Transl Med,2015,13(1):162.
[16]Wu AB,Li JM,Wu KP,et al.MiR-373-3p promotes invasion and metastasis of lung adenocarcinoma cells[J].Chinese Journal of Lung Cancer,2015,18(7):427.[吴爱兵,李金媚,吴昆鹏,等,MiR-373-3p促进肺腺癌细胞的侵袭转移[J].中国肺癌杂志,2015,18(7):427-435.]
[17]Huang Q,Gumireddy K,Schrier M,et al.The microRNAs miR-373 and miR-520c promote tumour invasion and metastasis[J].Nat Cell Biol,2008,10(2):202-210.
[18]Zhang X,Xiaofeng LI,Tan Z,et al.MicroRNA-373 is upregulated and targets TNFα1 in human gastric cancer,contributing to tumorigenesis[J].Oncol Lett,2013,6(5):1427-1434.
[19]Voorhoeve PM,Le SC,Schrier M,et al.A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors[J].Cell,2006,124(6):1169-1181.
[20]Nishimura Y,Komatsu S,Ichikawa D,et al.Overexpression of ywhaz relates to tumor cell proliferation and malignant outcome of gastric carcinoma[J].Br J Cancer,2013,108(6):1324-1331.
[21]Chen CH,Chuang SM,Yang MF,et al.A novel function of YWHAZ/β-catenin axis in promoting epithelial-mesenchymal transition and lung cancer metastasis[J].Mol Cancer Res,2012,10(10):1319.
[22]Watanabe N,Komatsu S,Ichikawa D,et al.Overexpression of YWHAZ as an independent prognostic factor in adenocarcinoma of the esophago-gastric junction[J].Am J Cancer Res,2016,6(11):2729.
[23]Tang Y,Lv P,Sun Z,et al.14-3-3B promotes migration and invasion of human hepatocellular carcinoma cells by modulating expression of MMP2 and MMP9 through PI3K/AKT/NF-κB pathway[J].PLoS One,2016,11(1):e0146070.