沉默环指蛋白187表达调控自噬对肝癌细胞恶性生物学行为的影响

Effect of silencing ring finger protein 187 on malignant biological behaviors of hepatocellular carcinoma

目的探讨环指蛋白187(RNF187)调节自噬水平产生对肝癌细胞恶性生物学行为的影响。方法通过生物信息学手段分析RNF187在肝癌中的mRNA水平。分别使用小干扰RNA(siRNA)阴性对照和靶基因进行转染的Huh7细胞作为未经转染(NC)组和敲低RNF187组;以上两组细胞转染24 h后添加二甲基亚砜(DMSO)的细胞分别作为NC+DMSO组和敲低RNF187+DMSO组;行siRNA靶基因转染24 h后添加巴弗洛霉素A1(BFA)的细胞作为敲低RNF187+BFA组。通过CCK8细胞增殖实验、细胞划痕实验、Transwell实验和蛋白质印迹法探究RNF187对肝癌细胞恶性生物学行为和自噬水平的调控。结果与正常肝脏组织样本比较,RNF187在肝癌样本中的mRNA水平升高,差异有统计学意义(P<0.05)。与NC组相比,敲低RNF187组细胞在48 h和72 h的吸光度以及12 h和24 h的划痕愈合率均降低,差异具有统计学意义(均P<0.001)。敲低RNF187组24 h后的穿膜细胞数(39.50±5.57)个低于NC组的(128.25±17.35)个,差异具有统计学意义(t=9.74,P<0.001)。与NC组相比,敲低RNF187组总LC3和Beclin-1的相对表达量均增加,而磷酸化哺乳动物雷帕霉素靶蛋白的相对表达量降低,差异具有统计学意义(均P<0.05)。与敲低RNF187+DMSO组相比,敲低RNF187+BFA组的自噬流水平、48 h和72 h的吸光度以及24 h的划痕愈合率均增加,差异具有统计学意义(均P<0.001)。敲低RNF187+BFA组24 h的穿膜细胞数(119.00±2.65)个多于RNF187+DMSO组的(57.67±2.52)个,差异具有统计学意义(t=29.09,P<0.001)。结论RNF187在肝癌细胞中高表达,敲低RNF187可通过升高细胞自噬水平来抑制肝癌细胞恶性生物学行为。

Objective To investigate the effect of ring finger protein 187(RNF187)on cell pro-liferation,migration and invasion of hepatocellular carcinoma(HCC).Methods Messenger RNA(mRNA)level of RNF187 in HCC was analyzed by bioinformatics.Huh7 cells transfected with small interfering RNA(siRNA)of negative control or target gene respectively were classified as non-transfection(NC)group and RNF187 knockdown group.After 24 hours of transfection,the above two groups dimethyl sulfoxide(DMSO)were used as NC+DMSO group and RNF187 knockdown+DMSO group.24 hours after transfection with siRNA of target gene,the cells dealt with bafliomycin A1(BFA)were set as RNF187 knockdown+BFA group.The regulation of RNF187 on malignant biological behavior and autophagy level of HCC cells were explored by cell counting kit-8(CCK8)proliferation assay,cell scratch assay,transwell assay and western blot.Results Compared with normal liver tissue,the mRNA level of RNF187 was higher in HCC tissue(P<0.05).Compared with NC group,the absorbance at 48 h and 72 h and the scratch healing rate at 12 h and 24 h of RNF187 knockdown group were all lower,the differences were statistically significant(all P<0.001).The number of transmembrane cells in RNF187 knockdown group(39.50±5.57)at 24 h was lower than that in NC group(128.25±17.35),the differences were statistically significant(t=9.74,P<0.001).Compared with NC group,the relative expression of total LC3 and Beclin-1 in RNF187 knockdown group all increased,while the relative expression of phosphorylated mammalian target of rapamycin decreased,the difference were statistically significant(all P<0.05).Compared with RNF187 knockdown+DMSO group,the autophagy flow level,the 48 h and 72 h absorbance,the scratch healing rate at 24 h in RNF187 knockdown+BFA group were higher,the differences were statistically significant(all P<0.001).The number of transmembrane cells in RNF187 knockdown+BFA group(119.00±2.65)was more than that in RNF187 knockdown+DMSO group(57.67±2.52),the differences were statistically significant(t=29.09,P<0.001).Conclusion RNF187 is highly expressed in HCC tissue and knockdown of RNF187 inhibits the malignant biological behavior of HCC by enhancing the level of autophagy.