LincRNA00858靶向miR-3126-5p影响肝癌细胞的增殖、迁移和侵袭

LincRNA00858 regulates miR-3126-5p to affect proliferation,migration and invasion of hepatocellular carcinoma

目的探讨长链非编码RNA(LncRNA)Linc00858通过调控靶向小分子RNA-3126-5p(miR-3126-5p)影响肝癌细胞增殖、迁移和侵袭的作用机制。方法通过实时荧光定量PCR(qRT-PCR)法检测Linc00858在肝癌组织、癌旁正常组织、人正常肝细胞LO2和人肝癌细胞系中的表达情况及两者相对关系。筛选出人肝癌细胞HUH-7和MHCC-97H作为研究对象,分别转染Linc00858过表达和干扰Linc00858表达的重组慢病毒,使用q RT-PCR测定转染效果,通过CCK-8实验、Transwell实验检测肿瘤细胞的增殖、迁移及侵袭能力,Western blot实验检测Linc00858对上皮-间质转化和细胞周期相关的蛋白表达水平的影响;生物信息学预测和双荧光素酶报告实验确定两者的结合关系。调整Linc00858的表达水平后,采用qRT-PCR法检测肝癌细胞中miR-3126-5p表达水平的变化,将miR-3126-5p mimics和miR-3126-5p inhibitor分别进行转染,通过上述方法再次检测肝癌细胞增殖、迁移和侵袭能力及相关蛋白表达情况。结果Linc00858在肝癌组织和细胞中的表达量高于癌旁正常组织和LO2;过表达Linc00858后HUH-7细胞增殖、迁移及侵袭能力明显上升,细胞周期蛋白E1(cyclin E1)、周期素依赖性激酶2(CDK2)和波形蛋白(Vimentin)的表达明显上升,P27和钙粘附蛋白E(E-cadherin)的表达明显下降;沉默Linc00858可以抑制MCHH-97H细胞增殖、迁移及侵袭能力,cyclin E1、CDK2和Vimentin的表达明显下降,P27和E-cadherin的表达明显上升;Linc00858靶向调控miR-3126-5p的表达,miR-3126-5p mimics能明显逆转过表达Linc00858对HUH-7细胞的增殖、迁移及侵袭能力以及相关蛋白表达水平;miR-3126-5p inhibitor可以逆转沉默Linc00858对MHCC-97H细胞的增殖、迁移及侵袭能力以及相关蛋白表达水平。结论Linc00858通过负向调控miR-3126-5p来促进肝癌细胞的增殖、侵袭和迁移。

Objective To investigate the effect of long-stranded non-coding RNA(LNCRNA)Linc00858on the proliferation,migration and invasion of hepatoma cells by regulating targeted small molecule RNA-3126-5P(MIR-3126-5P).Methods Real-time fluorescent quantitative PCR(qRT-PCR)was used to detect the expressions of Linc00858 in hepatocellular carcinoma(HCC),normal para-cancerous tissues,normal human hepatocytes LO2 and human hepatoma cell lines.Human hepatoma cell lines HUH-7 and MHCC-97H were transfected with Linc00858-overexpressed plasmid and Linc00858-expressing recombinant lentivirus,respectively.The transfection effect was determined by q RT-PCR,and proliferation of tumor cells was detected by CCK-8 and Transwell assay.Western Blot assay was used to determine the effects of link00858 on epithelial-mesenchymal transformation and expression of cell cycle related proteins.Bioinformatics and double luciferase assay were used to determine the association of Linc00858 with miR-3126-5p.After the level of Linc00858 expression was adjusted,the expression of miR-3126-5p in hepatoma cells was detected by qRT PCR.miR-3126-5p mimics and miR-3126-5p inhibitor were transfected respectively;then the proliferation,migration,invasion and expression of related proteins were analyzed by the above methods.Results Linc00858 expression was higher in cancerous tissues than in LO2 and normal tissues.Proliferation,migration and invasion of HUH-7 cells were increased after overexpression of Linc00858.The protein expressions of Vimentin,Cyclin E1 and CDK2 increased obviously,while those of E-cadherin and P27 decreased obviously.Proliferation,migration and invasion of MCHH-97H cells were inhibited by silencing Linc00858.The expression levels of Cyclin E1,CDK2 and Vimentin-related proteins decreased significantly,while those of E-cadherin and P27 increased significantly.Linc00858 regulated miR-3126-5p expression,miR-3126-5p mimics significantly reversed proliferation,migration and invasion of HUH-7 cells induced by Linc00858 overexpression.miR-3126-5p inhibitor reversed the impact of Linc00858 silencing on proliferation,migration and invasion ability of MHCC-97H cells and related protein expression level.Conclusions Linc00858accelerated proliferation,invasion and migration of hepatoma cells through the negative regulation of microRNA3126-5p.