长链非编码RNA H19通过抑制CCND2促进膀胱癌细胞增殖的分子机制

The molecular mechanism of the biological function of lncRNA H19 in bladder cancer

目的探讨长链非编码RNA(lncRNA)H19促进膀胱癌细胞增殖的分子机制。方法(1)应用实时定量逆转录PCR(RT-qPCR)分别检测膀胱癌组织中lncRNA H19和miRNA-29c-3p的表达并分析其相关性。(2)应用RNA干扰和细胞转染技术抑制膀胱癌细胞系T24及5637中H19基因的表达,其后通过细胞增殖实验检测细胞增殖情况;流式细胞仪检测细胞周期变化;RTqPCR检测细胞中H19的表达变化。(3)在膀胱癌细胞系T24和5637中共表达miR-29c-3p和H19,运用荧光素酶报告系统验证miR-29c-3p对H19及细胞周期蛋白D2(CCND2)的靶向作用,应用Western blot检测膀胱癌细胞系T24与5637中抑制H19表达后CCND2的表达变化。结果H19在膀胱癌中高表达,miR-29c-3p在膀胱癌中低表达,H19和miR-29c-3p的表达为负相关,H19与CCND2的表达为正相关。MiR-29c-3p可同时靶向H19及CCND2。在膀胱癌细胞系中过表达miR-29c-3p可以抑制H19的表达,miR-29c-3p在膀胱癌中起肿瘤抑制作用,而H19具有促进癌细胞增殖和改变细胞周期的能力,表现为癌基因样作用,抑制膀胱癌细胞系T24和5637中H19的表达可以在翻译水平减少CCND2的表达。结论 lncRNA H19通过与CCND2竞争结合miR-29c-3p,降低miR-29c-3p对CCND2的表达抑制,从而在膀胱癌中发挥肿瘤促进作用。

Objective To detect the function and mechanism of lncRNA H19 in BCa（bladder cancer）. Methods The level of H19 and miR-29 c-3 p were detected with quantitative reverse transcriptase PCR（qRT-PCR）in BCa and the correlation analysis of them was done.MTT method was used to measure proliferation capacity after knocking down the H19 in BCa cell lines T24 and 5637.Flow cytometer was used to detect the cell cycle after knocking down the H19 in BCa cell lines T24 and 5637.Expressions of H19 in BCa cell lines T24 and 5637 were detected by qRT-PCR after knocking down the H19 and overexpressing miR-29 c-3 p.We confirm the miR-29 c-3 p target genes with luciferase reporter system.And the expression of CCND2 was examined by Western blot in BCa cell lines T24 and 5637 with the down-regulation of H19.The effect of CCND2 and miR-29 c-3 p on the function of H19 in BCa was performed to explore the molecular mechanism of the biological function of lncRNA H19 in BCa. Results The level of miR-29 c-3 p was lower while H19 was higher in BCa,there was a positive correlation between H19 and CCND2 expressions,besides that,and there was a negative correlation between H19 and miR-29 c-3 p expressions.H19 and CCND2 were both the target gene of miR-29 c-3 p,other side,and miR-29 c-3 p could suppressed the BCa progression while H19 presented oncogene in BCa.CCND2 protein could be regulation by H19 in BCa. Conclusions H19 could regulate the BCa progression by interacting the CCND2 and miR-29 c-3 p,and it could be a CeRNA and an oncogene in BCa.