MOB2敲低表达调控NDR1/2-YAP对肝癌细胞迁移和细胞自噬的研究

MOB2 knockdown regulates the migration and autophagy of hepatocellular carcinoma cells through NDR1/2-YAP pathway

以肝癌细胞SMMC-7721和SK-HEP1为研究对象,构建慢病毒介导的单极纺锤体1结合蛋白2 (MOB2)稳定干扰表达的细胞模型;应用RT-qPCR和Western blotting方法检测相关基因的表达水平;利用Transwell细胞迁移试验和自噬流试验检测MOB2敲低表达对细胞迁移和细胞自噬的影响。结果表明:MOB2敲低表达可抑制肝癌细胞SMMC-7721和SK-HEP1的迁移,促进细胞自噬;MOB2敲低表达可上调NDR1/2的磷酸化水平(pT444/442)和YAP的磷酸化水平(pS127YAP),下调受YAP转录调控的下游靶基因CTGF和CYR61的表达;MOB2敲低表达可上调ULK1、LC3BII,下调SQSTM1/p62,促进细胞自噬流的形成。这一研究说明MOB2敲低表达可通过促进NDR1/2的磷酸化而负调控YAP的转录激活活性,调控下游与细胞迁移和自噬相关基因的表达,影响肝癌细胞的迁移和细胞自噬过程。

Human hepatocellular carcinoma(HCC)cell lines SMMC-7721 and SK-HEP1 stably knockdown of MOB2 were established by the lentiviral-mediated MOB2 knockdown approaches.Reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR)and Western blotting assays were performed to examine the expression of indicated genes in HCC cell lines.Transwell assay and autophagic flux assay were used to evaluate the effect of MOB2 knockdown on the migration and the autophagy of the HCC cell lines.The results showed that MOB2 knockdown reduced HCC cell migration and promoted HCC cell autophagy,induced phosphorylation of NDR1/2(pT444/442)and YAP(pS127 YAP),and down-regulated the expression of connective tissue growth factor(CTGF)and cysteine rich angiogenic inducer 61(CYR61),two well characterized YAP target genes.The results also demonstrated that MOB2 knockdown up-regulated the levels of ULK1 and LC3 BII,down-regulated SQSTM1/p62,and increased autophagic flux.Data from this study indicate that MOB2 knockdown altered HCC cell migration and autophagy by increased phosphorylation of NDR1/2 and YAP,which leads to the transcriptional inactivation of YAP,and thereby consequently modulated the expression of indicated downstream genes involved in HCC cell motility and autophagy.