miRNA-1297通过靶向BH3结构域凋亡诱导蛋白促进肝细胞癌的进展

miRNA-1297 promotes the progression of hepatocellular carcinoma by targeting BID

目的该研究旨在探讨miR-1297在肝细胞癌中的表达水平,及其对肝细胞癌癌细胞增殖和凋亡的影响。方法分析109对肝癌组织和配对的癌旁组织中miR-1297的表达情况。从上述队列患者的生存资料中分析miR-1297的表达与肝癌患者预后的关系。应用qRT-PCR技术检测肝细胞癌中miR-1297上调表达的机制。应用CCK8试验测定miR-1297在肝细胞癌发生发展中的作用。用Annexin V/丙碘化物染色检测HCC细胞凋亡。用蛋白质免疫印迹分析法研究与BH3结构域凋亡诱导蛋白(BID)的表达。结果相对癌旁正常组织和人正常肝细胞,miR-1297在肝癌组织和肝癌细胞中表达上升。Kaplan-Meier分析提示癌组织中高表达miR-1297的肝癌患者预后较差。在SMMC-7721和HepG2转染miR-1297表达抑制剂可抑制肝癌细胞增殖,促进其凋亡。生物信息学分析提示BID为miR-1297直接调控靶基因;荧光素酶报告基因检测提示miR-1297可与BIDmRNA 3'UTR结合;蛋白质印迹分析证实miR-1297可抑制肝癌细胞BID蛋白的表达。进一步研究提示,抑制miR-1297表达显著提高线粒体中tBID和细胞色素C的表达水平。拯救实验证实在肝癌细胞中miR-1297通过靶向BID蛋白抑制肝癌细胞凋亡。结论证实miR-1297通过靶向BID促进肝癌的进展,提示其可能是肝细胞癌患者潜在的预测预后的临床标志物和治疗靶点。

Objective The aim of this study was to investigate the expression of microRNA-1297(miR-1297)in hepatocellular carcinoma(HCC)and its effect on proliferation and apoptosis of HCC cells.Methods The expression of miR-1297 in HCC was analyzed in 109 pairs of HCC tissues and matched para-carcinoma tissues.The correlation between miR-1297 expression and prognosis of HCC patients was analyzed from the survival data in above cohort.The mechanism of miR-1297 upregulation in HCC was detected using chromatin immunoprecipitation-quantitative real-time PCR.The roles of miR-1297 in HCC development were examined using a cell counting kit-8 assay.Apoptosis of HCC was detected using Annexin V/propidium iodide staining.The expression of BH3-interacting death agonist(BID)protein was examined using Western blot analysis.Results Compared with para-carcinoma tissues or normal human hepatocyte cell lines,the level of miR-1297 was increased in HCC tissues and HCC cells.Kaplan-Meier analysis indicated that the prognosis of HCC patients with high level of miR-1297 was poor.Transfection of miR-1297 expression inhibitors in SMMC-7721 and HepG2 inhibited the proliferation and promoted the apoptosis of HCC cells.Bioinformatics analysis indicated that BH3 domain apoptosis-inducing protein was the target gene directly regulated by miR-1297.Luciferase reporter gene detection indicated that miR-1297 could bind to the 3'UTR of BID.The data from Western blot analysis confirmed that miR-1297 could inhibit the expression of BID protein in HCC cells.Further studies suggested that the inhibition of miR-1297 level significantly increased the expression of tBID and cytochrome C in mitochondria.The results from rescue experiments confirmed that miR-1297 inhibited apoptosis of hepatocellular carcinoma cells by targeting BID protein.Conclusion Our results suggest that miR-1297 promotes cancer progression by targeting BID,indicating that miR-1297 may be a potential prognostic predictor and therapeutic target for HCC patients.