Effect Of Phyllanthus Compound ? On IGF-1R Signaling Pathway In Hepatocellular Carcinoma Huh7 Cells

ObjectiveTo explore Phyllanthus compound II the new mechanism of the effect of Phyllanthus compound II by inhibiting the activation of IGF-1R signaling pathway to play an anti-hepatocarcinoma effect,in order to provide a new experimental basis for its clinical application.Methods1.The establish of hepatocarcinoma Huh7 cell line that its IGF-1R was inhibited expressBased on the principle of siRNA target design,three pairs of nucleotide interference sequences were designed and synthesized for IGF-1R sequence in Huh7 cells by siRNA gene silencing technique.These sequences were cloned into the lentivirus pLVX-shRNAl vector,then extract shRNA expression of the recombinant plasmids,and confirmed that the sequence is exactly the same as the design sequence by using enzyme cleavage.The highly purified recombinant plasmids were extracted and transfected into 293T cells with the virus packaging plasmids.The virus was packaged and the virus solution was collected,concentrated and purified.Selecting Huh7 cells infected with the virus to obtain stable cell lines.The differences of IGF-1R gene expression among anti-IGF-1R Huh7(I1,12)cells,Huh7-NC cells and Huh7 cells were detected by western blot.2.The effect of Phyllanthus compound IHon the proliferation of Huh7 cells and anti-IGF-1R Huh7 cellsThe inhibitory effect of different concentrations of Phyllanthus compound II on the proliferation of Huh7 cells and anti-IGF-1R Huh7 cells was detected by MTT assay at 48h,and the half inhibitory concentration was calculated.Detect the inhibitory effect on Huh7 cells and anti-IGF-1R Huh7 cells in the blank group,control group,the high dose group(3.Omg/ml)and low dose group(1.5mg/ml)of Phyllanthus compound ?,5-fluorouracil group(30?g/ml)with four holes in each group at 24h and 48h.3.The effects of Phyllanthus compound ? on the apoptosis.of Huh7 cells and anti-IGF-1R Huh7 cellsFlow cytometry instrument Annexin V-FITC/PI method was used to detect the apoptotic rate in Huh7 cells and anti-IGF-1R Huh7 cells in the blank group,control group,the high dose group(3.Omg/ml)and low dose group(1.5mg/ml)of Phyllanthus compound ?,5-fluorouracil group(30?g/ml)with three holes in each group for 48h.4.The effects of Phyllanthus compound ?on the expression of genes of Huh7 cells and anti-IGF-lR Huh7 cellsQ-PCR was us'ed to detect the effect of gene expression of IGF-1R signaling pathway in Huh7 cells and anti-IGF-1R Huh7 cells in the blank group,control group,the high dose group(3.Omg/ml)and low dose group(1.5mg/ml)of Phyllanthus compound ?,5-fluorouracil group(30?g/ml)with three holes in each group for 48h.Results.1.The expression of IGF-1R protein in Huh7-NC cells,? cells and 12 cells was significantly lower than that in Huh7 cells(P<0.05).The expression of IGF-1R protein was the lowest in I1 cell group,so ? cells were selected as stable transfected cell lines,That is,anti-IGF-1R Huh7 cell line was successfully constructed.2.The half inhibitory concentration of Phyllanthus compound ? act in Huh7 cells for 48h was 2.575mg/ml,and it was 1.686?g/ml when in anti-IGF-1R Huh7 cells.Phyllanthus compound ? has a significant inhibitory effect on the proliferation of Huh7 cells and anti-IGF-1R Huh7 cells(P<0.05),and the inhibijtory effect was more obvious with the increase of dose.The inhibitory effect on on the anti-IGF-1R Huh7 cells was more obvious than that of Huh7 cells after Phyllanthus compound ? act 24 hours(P<0.05);the inhibitory effect on on the anti-IGF-1R Huh7 cells was stronger than that of Huh7 cells after Phyllanthus compound II act 48 hours,and the two groups had significant difference(P<0.05).3.The early apoptotic rate of Huh7 cells after Phyllanthus compound ?act 48 hours was significantly higher than that of the control group(P<0.05),the results showed that Phyllanthus compound ? could induce the early apoptosis of Huh7 cells.4.Compared with the control group,after 48h for Phyllanthus compound ?act on Huh7 cells and anti-IGF-1R Huh7 cells,the mRNA expression of IGF-1R gene and PI3K,AKT3,N-RAS,K-RAS,C-RAF,ERK1 genes in Huh7 cells and anti-IGF-1R Huh7 cells were significantly decreased(P<0.05).Compared with Huh7 cells,the mRNA expression levels of IGF-1R,PI3K and AKT3 genes in anti-IGF-1R Huh7 cells were lower after the treatment of Phyllanthus compound ?(P<0.05).Conclusion1.Phyllanthus compound ? can significantly inhibit Huh7 cell proliferation and induce Huh7 cell apoptosis,the mechanism of action were related to inhibition of IGF-1R signaling pathway activation.2.In the case of siRNA inhibition of Huh7 cell IGF-1R expression,the use of Phyllanthus Compound ?,can play a synergistic effect of anti-cancer.