HHIP基因去甲基化对HCC827肺腺癌细胞Hedgehog信号通路及增殖影响

Effect of HHIP Gene Demethylation on Hedgehog Signaling Pathway and Proliferation of Lung Adenocarcinoma HCC827 Cells

[目的]探讨HHIP基因去甲基化后对HCC827肺腺癌细胞Hedgehog信号通路及其增殖的影响。[方法]应用细胞转染技术获得HHIP高表达的HCC827细胞,并将HCC827细胞分为空白对照组、过表达转染组及5-氮杂-2′脱氧胞苷(5-Aza-2′-de-oxycytidine,5-AzaCd R)处理组。采用甲基化特异性聚合酶链式反应和亚硫酸盐测序PCR检测5-Aza-CdR处理前后HCC827细胞HHIP基因启动子区甲基化状况,应用Real-time qPCR及Western-blot方法检测各组细胞的Hedgehog信号通路相关基因及细胞周期素D2(CCND2)mRNA及蛋白表达水平,CCK8分析细胞增殖的变化。[结果] HHIP基因去甲基化或过表达质粒转染后,HHIP基因表达明显上调,其Hedgehog信号通路相关基因PTCH、SHH基因表达均明显下调,CCND 2表达上调(P均<0.01),HCC827增殖能力出现降低(P<0.01)。[结论]高甲基化抑癌基因HHIP基因去甲基化后,可抑制HCC827腺癌细胞Hedgehog信号通路的活化并抑制其增殖,这可能与其Hedgehog信号通路负性调节因子HHIP基因恢复表达相关。

[Objective]To investigate the effect of HHIP gene demethylation on Hedgehog signaling pathway and proliferation of lung adenocarcinoma HCC827 cells.[Methods]HCC827 cells with high expression of HHIP were obtained by cell transfection technique,and the HCC827 cells were divided into control group,overexpressed transfection group and 5-Aza-2′-de-oxycytidine(5-Aza-CdR)treatment group.By using methylation-specific polymerase chain reaction(MSP)and sulfite sequencing PCR(BSP)techniques,methylation status of HHIP gene promoter region in HCC827 cells before and after 5-Aza-CdR treatment was analyzed.The qRT-PCR and Western blot methods were used to detect the mRNA and protein expression levels of Hedgehog signaling pathway related genes and cyclin D2(CCND2)in each group.The changes of cell proliferation were analyzed by CCK8.[Results]After HHIP gene demethylation or overexpression plasmid transfection,the expression of HHIP gene was significantly up-regulated,and the expression of PTCH,SHH genes in Hedgehog signaling genes were significantly down-regulated,CCND 2 expression was upregulated(P<0.01),HCC827 proliferation ability was decreased(P<0.01).[Conclusion]The demethylation of HHIP gene inhibits the activation of Hedgehog signaling pathway and proliferation of in lung adenocarcinoma HCC827 cells,which may be related to the re-expression of the negative regulatory factor HHIP gene in the Hedgehog signaling pathway.