miR-141-3p在肝细胞癌组织中的表达及其靶基因与肝细胞癌恶性特征的关系

Relations of miR-141-3p expression and its target gene with malignant profiles of hepatocellular carcinoma

目的观察微小RNA(miRNA,miR)-141-3p在肝细胞癌(HCC)组织中的表达及其对肝癌细胞增殖、迁移和侵袭能力的影响。方法通过生物信息学软件筛选到可能靶向调控高尔基体蛋白73(GP73)基因的hsa-miR-141-3p,并以双荧光素酶报告基因实验验证存在靶向关系。采用实时荧光定量PCR(qRT-PCR)和Western blot方法分别检测HCC细胞系、HCC组织及癌旁组织中的miR-141-3p与GP73分子的表达水平,并分析HCC组织中miR-141-3p表达与HCC临床病理学特征的关系。采用噻唑蓝(MTT)、EdU及Transwell实验观察miR-141-3p过表达对HCC细胞增殖、迁移及侵袭能力的影响。结果miR-141-3p在HCC组织中的表达水平低于癌旁组织(P<0.05),GP73mRNA及其蛋白则相反(P<0.05);HCC组织中miR-141-3p的表达水平与GP73mRNA的表达水平呈负相关,且miR-141-3p的低表达与血管侵犯、肿瘤分化等级及临床TNM分期密切相关(P<0.05)。MTT结果显示,Huh-7细胞转染miR-141-3p过表达质粒后,各时点吸光度(A)值低于空白对照组和miR-NC组(P<0.05);EdU检测结果表明,转染miR-141-3p后,Huh-7和MHCC-97H细胞的EdU阳性细胞比低于空白对照组和miR-阴性对照(NC)组(P<0.05);Traswell检测结果表明,转染miR-141-3p后,MHCC-97H细胞的侵袭细胞数和迁移细胞数低于空白对照组和miR-NC组(P<0.05);细胞学功能回复实验结果表明,miR-141-3p+GP73组的侵袭细胞数和迁移细胞数低于空白对照组和miR-NC组,但高于miR-141-3p组和miR-141-3p+GP73-NC组(P<0.05)。结论HCC组织中miR-141-3p呈低表达,且GP73mRNA表达水平与其呈负相关;低表达miR-141-3p与HCC细胞的侵袭和转移能力密切相关。过表达miR-141-3p能够显著抑制HCC细胞的增殖、侵袭和迁移,逆转miR-141-3p的部分抑制作用可通过恢复表达不含3′非翻译区(UTR)的GP73基因实现。

Objective To investigate the expression of microRNA (miRNA, miR)-141-3p in hepatocellular carcinoma (HCC) tissues and its effect on proliferation and invasion of HCC. Methods Bioinformatics tools were used to predict putative miRs with search potential downstream gene–GP73,hsa-miR-141-3p was selected for further analysis and observations. A dual-luciferase reporter assay was performed to validate GP73 as a target of hsa-miR-141-3p. Real time PCR (qRT-PCR) or Western blot was performed to measure the expression level of miR-141-3p and GP73 in HCC and adjacent tissue. MTT,EdU,and Transwell were used to measure cell proliferation and migration. Results It was identified that the expression level of miR-141-3p was significantly lower in the HCC tissues than that of adjacent tissues (P<0.05), but different in GP73 mRNA and its protein (P<0.05). Clinical analysis indicated that decreased miR-141-3p expression was significantly correlated with advanced tumor grade, advanced TNM stage, and vascular invasion (P<0.05). MTT assay showed that the absorbance (A) value of Huh-7 cells transfected with miR-141-3p overexpression plasmid was significantly decreased compared with the miR-negative control (NC) group (transfected empty plasmid) and the blank control group (P<0.05).The EdU detection results showed that the ratio of EdU positive cells in Huh-7 and MHCC-97H cells were lower than those in the blank control group and the miR-NC group after transfection of miR-141-3p (P<0.05). Transwell test found that after transfection of miR-141-3p, the number of invading cells and the number of migrated cells in MHCC-97H cells were lower than those in the blank control group and the miRNC group (P<0.05). The results of cytological function recovery experiments showed that the number of invading cells and the number of migrated cells in MHCC-97H cells of miR-141-3p+GP73 group were lower than those in blank control group and miR-NC group (P<0.05), but higher than those of the miR-141-3p group and miR-141-3p+GP73-NC group (P<0.05). Conclusions The present study revealed that miR-141-3p is reduced in HCC tissues. Additionally, GP73 expression levels are negatively correlated to miR-141-3p in HCC tissues, and that is associated with the progression of HCC. Overexpression of miR-141-3p significantly inhibit proliferation, invasion, and migration of HCC cells. Reversal of partial inhibition of miR-141-3p can be achieved by restoring the expression of the GP73 gene without the 3′ untranslated region (UTR).