The Effect Of YAP-siRNA On The Growth And Apoptosis Of Huh-7Cells

Objiective Employ the sequence-sepcific siRNA silencing the YAP gene and toobserve the effection of YAP gene on proliferation and apoptosis in human hepatoma cellline Huh-7and gene expression in HCC tissues. Investigate the mechanism of the YAPgene affects the growth of liver cancer cells, and provide a theoretical basis for genetherapy.Methods1.Structure of YAP mRNA was analysed by software to select targetedinterference sites. The YAP-siRNA was transfected into Hepatocellular carcinoma lineHuh-7by positive ion liposome Lipofectamine2000.Transfect efficiency was assessed byflow cytometry；2.After human liver cancer cell line Huh-7was transfected by YAP smallinterfering RNA (siRNA)，the mRNA and protein of YAP were determined by RT-PCRand Western blot assay respectively(p<0.05), Verify the effect of siRNA silence；3.The cellgrowth inhibition was assesses by MTT.Besides，apoptosis and cell cycle were measuredby flow cytometry;4.63cases of liver cancer tissue samplewere divided into groups of small hepatocellular carcinoma and non-small livercancer tumor by diameter less than2cm.Realtime PCR method were used to measure theexpression level of YAP specimens.Results1.Negative control FAM-siRNA was successfully transfected into humanliver cancer cell line Huh-7.The best transfection efficiency(98.6%) was detected by FCMwith cell density at1.5*10~5/well and siRNA concentration with80pmol in6well play.2.After YAP-siRNA were successfully transfected into Huh-7cell48h, detected byRT-PCR，YAP mRNA levels in cells was significantly inhibited,contrasted with negativecontrol and blank control,the expression of YAP protein were obviously decreased72hafter YAP-siRNA transfection.3.YAP-siRNA could effectively inhibit the growth ofHuh-7cells and accelerate apoptosis in vitro(p<0.05).4YAP expression level of the non-small liver cancergroup was3.61times that of the group ofsmall hepatocellular carcinoma(p<0.05). Conclution YAP-siRNA is successfully constructed and efficiently transfected intoHuh-7cells. YAP-siRNA can effectively silence the overexpression of YAP inhepatocellular carcinoma cell lines Huh-7. Using YAP-siRNA transfection could inhibit thegrowth of Huh-7cells and accelerate apoptosis, but it could not change cell cycle.YAP expression levels is positively correlated with tumor volume.