NR2C2介导的长链非编码RNA LINC00675靶向miR-665调控肝癌细胞生物学行为的机制研究

A study on mechanism of NR2C2 mediated long non-coding RNA LINC00675targeting of miR-665 in regulating the biological behavior of HCC cells

目的探讨研究长链非编码RNA LINC00675对肝癌细胞生物学行为的影响及其分子机制。方法通过qRT-PCR检测LINC00675在肝癌组织以及细胞中表达量,并检测LINC00675在肝癌细胞HepG3与Huh7细胞质及细胞核中的表达分布。通过在HepG3与Huh7中稳定转染慢病毒过表达LINC00675建立细胞模型。通过CCK-8与流式细胞学检测LINC00675对肝癌细胞增殖、凋亡行为的影响,并通过检测细胞模型中葡萄糖摄取量以及乳酸产量变化监测细胞Warburg效应的变化,运用带有AGO2抗体的RNA免疫沉淀技术评估LINC00675与mRNA的潜在结合能力。使用LncBase Predicted v.2 DIANA工具预测LINC00675下游mRNA靶基因。运用RNA pull-down技术检测LINC00675与miR-665结合的可能性。运用荧光素酶实验验证LINC00675与miR-665的靶向结合,共转染实验验证miR-665介导了LINC00675对肝癌生物学行为的影响。通过使用JASPAR数据库预测LINC00675上游调控基因。通过构建NR2C2过表达以及敲低细胞模型,并使用qRT-PCR实验检测细胞模型中的LINC00675表达量的变化。运用荧光素酶实验验证LINC00675启动子与NR2C2的靶向结合位点。结果LINC00675在肝癌细胞系中低表达,且主要表达于细胞质中。过表达LINC00675抑制肝癌细胞的增殖能力与Warburg效应,并促进肝癌细胞凋亡。NR2C2介导的LINC00675在肝癌细胞中靶向结合miR-665,负向调控miR-665的表达,并通过结合miR-665来调控肝癌细胞的生物学行为。结论NR2C2介导的LINC00675通过靶向结合miR-665调控肝癌细胞的增殖和凋亡的生物学行为。

Objective To investigate the effect of long non-coding RNA LINC00675 on the biological behavior of HCC cells and its molecular mechanism.Methods The expression of LINC00675 in HCC tissues and cells was detected by qRT-PCR,and the expression and distribution of LINC00675 in the cytoplasm and nucleus of HCC cells HepG3 and Huh7 were detected.The cell model was established by stably transfecting lentivirus overexpression of LINC00675 in HepG3 and Huh7.The effects of LINC00675 on the proliferation and apoptosis of HCC cells were detected by CCK-8 and flow cytometry,and the changes of Warburg effect were monitored by detecting the changes of glucose uptake and lactic acid production in the cell model,and RNA immunoprecipitation with AGO2 antibody was used to evaluate the potential binding ability of LINC00675 and mRNA.LINC00675 downstream mRNA target genes were predicted by LncBase Predicted v.2 DIANA tool.The possibility of LINC00675 binding to miR-665 was examined by RNA pull-down technology.Luciferase assay was used to verify the targeted binding of LINC00675 to miR-665,and the co-transfection assay was used to verify that miR-665 mediated the effect of LINC00675 on the biological behavior of HCC.The LINC00675 upstream regulatory genes were predicted by using JASPAR database.The NR2C2 overexpression and knockdown cell model were constructed,and the changes of LINC00675 expression in the cell model were detected by qRT-PCR.In addition,the targeted binding site of the LINC00675 promoter to NR2C2 was verified by luciferase assay.Results LINC00675 was low expressed in hepatoma cell lines and mainly expressed in cytoplasm.Overexpression of LINC00675 inhibited the proliferation and Warburg effect of hepatoma cells,and promoted the apoptosis of hepatoma cells.NR2C2 mediated LINC00675 binded miR-665 in hepatoma cells,negatively regulated the expression of miR-665,and regulated the biological behavior of hepatoma cells by binding miR-665.Conclusion NR2C2 mediated LINC00675 regulates the biological behavior of proliferation and apoptosis of hepatoma cells by targeting the binding to miR-665.