Establishment Of High-Throughput Model By Inhibiting YAP-EAD Transcription Activity For Anti-HCC And Compounds Screening

Hepatocellμlar carcinoma(HCC),as a common cancer,is hard to be detected in time,however,with high lethallty rate.So finding novel target spot,exploiting innovative drugs is important.Many signal patfhways are involved in the pathogenesis of cancer.Hippo pathway plays an essential role in the growth of organs.Meantime,this pathway is highly relative to cancers,especially HCC.Signal cascade system in Hippo pathway will regμlate the phosphorylation of different sites in YAP,and regμlate the process of YAP getting into the nucleus.Then bind with TEAD,forming YAP-TEAD transcription complex,then the transcription can be activated,and then the some protooncogenes in Hippo pathway,e.g.CTGF will be translated and expressed.The over-expression of CTGF will lead to the over-growth of organ,then cancer following.Therefore,the expression level of CTGF can reflect the transcription activity of YAP/TEAD.So this project is divided into three parts:Part I:We constructed the high-throughput screening model inhibiting YAP/TEAD transcription activity and finished screening.We inserted the promoter region of CTGF into the mμltiple cloning site in pGL4.17,constructed the pCTGF-Luc plasmid.Transfected it into the HepG2,through G418,completed the construction of steady cell lines.We have already screened over 700 different compounds from diverse areas.Part II:The functional experiments of screened compounds.C1O can improve the fluorescence value,which means they can up=regμlate the expression of CTGF,so may facilitate tissue regeneration;C2071,N60 obviously decrease the fluorescence value.Then we selected out the most sensitive cell lines for Hippo pathway.We made the functional experiments to prove its ability to against the proliferation,migration and invasion of liver cancer cell.Part Ⅲ:The exploration of its anti-tμMor mechanism.Through WB,PCR,flow cytometry,etc,we ascertained some mechanism.However,because of the complexity of Hippo pathway,we didn’t find out all the steps in mechanism,but at least,we know the compound can produce its anti-tμMor effect through Hippo pathway.