lncRNA LINC00240通过靶向miR-656-3p调控胶质瘤细胞发生发展的分子机制研究

Molecular mechanism of lncRNA LINC00240 in regulating the pathogenesis and development of glioma cells by targeting miR-656-3p

目的探讨LINC00240调控胶质瘤细胞发生发展的分子机制。方法培养正常人类神经胶质细胞HEB和胶质瘤细胞系LN18、SW1088、Hs683,将胶质瘤细胞LN18随机分为si-NC组、si-LINC00240组、pcDNA-NC组、pcDNA-LINC00240组、miR-NC组、miR-656-3p组si-LINC00240+anti-miR-NC组、si-LINC00240+anti-miR-656-3p组。实时荧光定量PCR(RT-qPCR)检测LINC00240、miR-656-3p的表达水平;蛋白质印迹(Western blot)法检测磷酸化p65(p-p65)、磷酸化核因子kappaB抑制蛋白α(p-IкBα)表达;四甲基偶氮唑盐比色法(MTT)检测细胞活性;流式细胞术检测细胞凋亡;双荧光素酶报告实验检测LINC00240和miR-656-3p的靶向关系。结果与正常人类神经胶质细胞HEB相比,胶质瘤细胞系LN18、SW1088、Hs683中LINC00240表达水平升高,miR-656-3p表达水平降低(P<0.05)。LINC00240低表达或miR-656-3p高表达,胶质瘤细胞LN18活性降低,LN18细胞凋亡率升高(P<0.05)。LINC00240靶向miR-656-3p;LINC00240低表达,p-p65、p-IкBα表达水平降低(P<0.05)。低表达miR-656-3p可以逆转LINC00240低表达对LN18细胞增殖、凋亡的影响。结论抑制LINC00240表达可能通过上调miR-656-3p抑制NF-κB信号通路,从而抑制胶质瘤细胞增殖,促进细胞凋亡。

Objective To investigate the molecular mechanism of lncRNA LINC00240 in regulating the pathogenesis and development of glioma cells by targeting miR-656-3p.Methods Normal human glial cells HEB and glioma cell lines LN18,SW1088,Hs683 were cultured in vitro,and glioma cells LN18 were randomly divided into si-NC group,si-LINC00240 group,pcDNA-NC group,pcDNA-LINC00240 group,miR-NC group,miR-656-3p group,si-LINC00240+anti-miR-NC group,si-LINC00240+anti-miR-656-3p group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression levels of LINC00240 and miR-656-3p,and Western Blot was used to detect the expression levels of phosphorylated p65(p-p65),phosphorylated nuclear factor kappaB inhibitor proteinα(p-IкBα),and MTT was used to detect cell activity,and flow cytometry was used to detect apoptosis,and dual luciferase report test was used to detect the targeting correlation between LINC00240 and miR-656-3p.Results Compared with those in normal human glial cell HEB,the expression levels of LINC00240 in glioma cell lines LN18,SW1088 and Hs683 were significantly increased,however,the expression levels of miR-656-3p were significantly decreased(P<0.05).In LINC00240 low expression or miR-656-3p high expression,cell viability was significantly decreased,but,cell apoptosis rate was significantly increased(P<0.05).LINC00240 targeted miR-656-3p.In LINC00240 low expression,the expression levels of p-p65 and p-IкBαwere significantly decreased(P<0.05).The low expression of miR-656-3p could reverse the effects of LINC00240 low expression on the proliferation and apoptosis of LN18 cells.Conclusion Inhibiting the expression of LINC00240 may inhibit the NF-κB signaling pathway by up-regulating miR-656-3p,thereby inhibiting the proliferation of glioma cells and promoting cell apoptosis.