Mechanisms Of Activation Of Hippo/YAP Pathway By Myocyte Enhancer MEF2D In Hepatocellular Carcinoma

OBJECTIVE: Hepatocellular carcinoma(HCC)ranks among the top three of the highest morbidity and mortality diseases in china.And the poor prognosis,recurrence and low survival are the common problems for clinical treatment.It is reported that the over-activated Hippo pathway has close relationship with the occurrence of HCC while the clear molecular mechanism has not been elucidated.Our study will strengthen the understanding about the regulatory of Hippo/YAP pathway and provide new target for clinical therapy,which has great scientific significance and considerable clinical value.METHODS: Firstly,we verify that MEF2 D could bind to the upstream of AMOTL2 promoter through ChIP assay and next use the luciferase reporter experiment to identify MEF2 D could bind to the MEF2 cis-acting element of AMOTL2 and inhibit the transcript of AMOTL2.And the hepatocellular carcinoma cell lines such as Huh7,HepG2,PLC/RPF/5 were used for detecting the protein and mRNA expression level of AMOTL2 after over-expressed and silenced MEF2 D treatment via western blotting and qPCR.In order to prove that MEF2 D could activate Hippo pathway and promotes the proliferation of HCC cells,we detect the protein expression of YAP and phosphorated YAP via western blotting and the cell proliferation at 24,48 and 72 h via CCK8 treatment in over-expressed MEF2 D Huh7 cells.We also recorded the 450 nm OD value at 24 h via CCK8 treatment in MEF2D-silenced Huh7 cells.In order to verify if MEF2 D could affect the Hippo pathway activity through negatively regulating the transcript of AMOTL2,we set up experiment groups as following: Lv-shMEF2 D,Lv-shAMOTL2,Lv-shMEF2D+ Lv-shAMOTL2 and Control.We recorded the 450 nm OD value at 0h and 24 h via CCK8 treatment on all the samples.RESULTS: ChIP and luciferase reporter experiment results showed that MEF2 D could bind to the MEF2 cis-acting element within the upstream of AMOTL2 promoter.In overMEF2D-expressed cells,the western blotting and q-PCR results showed the decrease in the protein and mRNA expression level of AMOTL2 while the western blotting results was opposed in MEF2D-silenced cells and the YAP expression level were increased as well.The phosphorated YAP expression level has decreased in Huh7 cell but increased in HepG2 and PLC/RPF/5 cells after MEF2D-over-expressed treatment,and the P-YAP/YAP phosphorylation ration are decreased.The OD value of MEF2D-over-expressed Huh7 cells at 24,48 and 72 hours was significantly higher than that of NC group(P < 0.05);the OD value of MEF2D-silenced Huh7 cells at 24 hours was significantly lower than that of NC group(P < 0.05),that is,MEF2 D could promote cell proliferation.The OD value of Lv-shMEF2 D group and Lv-shMEF2D+ Lv-shAMOTL2 group at 24 hours was significantly lower than that of control group(P < 0.05),and the cell viability was also significantly decreased(P < 0.05);however,the OD value of Lv-shMEF2D+ Lv-shAMOTL2 group at 24 hours was significantly higher than that of Lv-shMEF2 D group(P < 0.05),and the cell viability was significantly increased(P < 0.05),that is,MEF2 D could promote cell proliferation by inhibiting the expression of AMOTL2.CONCLUSION: Our study identified that MEF2 D could bind to the MEF2 cis-acting element of the upstream of AMOTL2 promoter and activate the Hippo/YAP pathway in hepatocellular carcinoma which results in the proliferation of hepatocellular carcinoma cell.