miR-130b-3p通过靶向作用于INTS6促进肝细胞癌生长和转移

The miR-130b-3p promotes metastasis and proliferation of hepatocellular carcinoma by targeting INTS6

目的探讨miR-130b-3p在肝细胞癌(hepatocellular carcinoma,HCC)中的表达水平、临床意义及生物学功能,并初步探讨整合因子复合体亚基6(integrator complex subunit 6,INTS6)是否是其潜在的作用靶点。方法收集65例HCC组织和对应的癌旁组织标本,用real-time PCR检测miR-130b-3p在HCC组织和癌旁组织中的表达;用real-time PCR检测miR-130b-3p在人正常肝细胞(LO2)与肝癌细胞系中的表达; GEO数据库(GSE36915)分析miR-130b-3p在HCC和癌旁组织中的表达;分析miR-130b-3p的表达水平与HCC临床病理特征的相关性;分别应用miR-130b-3p类似物(miR-130b-3p mimics)和miR-130b-3p抑制剂(miR-130b-3p inhibitors)上调和下调MHCC-97L细胞中miR-130b-3p的表达,实验分组包括miR-130b-3p类似物组和相应的对照组以及miR-130b-3p抑制剂组和相应的阴性对照组。Transwell迁移和侵袭实验检测miR-130b-3p对HCC细胞迁移与侵袭能力的影响; MTT实验检测miR-130b-3p对HCC细胞增殖能力的影响;生物信息学工具预测INTS6是否是miR-130b-3p的下游靶点,用荧光素酶报告基因实验以及Western blot分析miR-130b-3p对潜在靶点INTS6的调节作用。结果与癌旁组织相比,miR-130b-3p在HCC组织中的表达明显升高(P <0. 001);与正常肝细胞相比,miR-130b-3p在HCC细胞系中的表达明显升高; GEO数据库(GSE36915)数据显示与癌旁组织相比,miR-130b-3p在HCC组织中表达显著上调(P=0. 035 6); miR-130b-3p的表达水平与肿瘤大小(P=0. 015)、静脉侵犯(P=0. 028)以及TNM分期(P=0. 045)显著相关;下调或者上调MHCC-97L细胞的miR-130b-3p表达后,细胞的迁移、侵袭和增殖能力显著被抑制或者增强;生物信息学分析发现,INTS6可能是miR-130b-3p的作用靶点;荧光素酶报告基因实验显示,miR-130b-3p能够负向调节INTS6-3'UTR的荧光素酶活性;此外,miR-130b-3p抑制剂(miR-130b-3p inhibitors)和miR-130b-3p类似物(miR-130b-3pmimics)分别能上调或者下调MHCC-97L细胞中INTS6的表达水平。结论 miR-130b-3p通过靶向作用于INTS6增强HCC细胞的迁移、侵袭及增殖能力,进而促进HCC的发生发展。

Objective To investigate the expression,clinical significance and biological function of miR-130 b-3 p in hepatocellular carcinoma( HCC),and to make a preliminary exploration about whether integrator complex subunit 6( INTS6) is the potential target of miR-130 b-3 p in HCC. Methods Totally 65 paired HCC tissues and adjacent non-tumor tissues were collected. Real-time PCR was used to detect miR-130 b-3 p expression in HCC tissues and adjacent non-tumor tissues. And real-time PCR was used to detect miR-130 b-3 p expression in normal hepatocyte( LO2) and HCC cell lines. In addition,the data from GEO dataset( GSE36915) were employed to analyze the expression of miR-130 b-3 p in HCC tissues. Statistical analysis was performed to explore the correlations between miR-130 b-3 p and clinicopathologic features. The expression of miR-130 b-3 p in MHCC-97 L cells was altered by miR-130 b-3 p mimics or inhibitors. The experiments groups included miR-130 b-3 p mimics group and corresponding negative control group,miR-130 b-3 p inhibitor group and corresponding negative control group. Transwell assay and MTT assay were used to study the effects of miR-130 b-3 p on migration,invasion and proliferation of HCC cells. Bioinformatics tools were applied to predict whether INTS6 was the potential target gene of miR-130 b-3 p. Luciferase reporter assay and Western blot were employed to determine the correlation of miR-130 b-3 p and its potential target gene INTS6. Results Expression of miR-130 b-3 p was significantly increased in HCC tissues compared to that in adjacent non-tumor tissues( P < 0. 001). Expression of miR-130 b-3 p was also increased in HCC cell lines compared to that in normal hepatocyte cells( P < 0. 001). GSE36915 data indicated that miR-130 b-3 p was increased in HCC tissues compared to that in adjacent non-tumor tissues( P = 0. 035 6). Expression of miR-130 b-3 p was significantly correlated with tumor size( P = 0. 015),portal vein infiltration( P = 0. 028) and TNM stage( P = 0. 045). Overexpressed miR-130 b-3 p notably enhanced migration,invasion and proliferation of MHCC-97 L cells,while miR-130 b-3 p inhibitors had the contrary effects on MHCC-97 L cells. Bioinformatics analysis suggested that INTS6 might be the target of miR-130 b-3 p. Furthermore,luciferase reporter assay indicated that the luciferase activity of INTS6-3’-UTR was negatively regulated by miR-130 b-3 p. Consistently,Western blot showed that miR-130 b-3 p negatively regulated the expression of INTS6 in HCC cells. Conclusion The miR-130 b-3 p may suppress migration,invasion and proliferation of HCC cells by targeting INTS6.