Role And Mechanism Of Glypican-1 In The Proliferation Of Hepatoma Cells

Part 1: Clinical study on the expression of Glypican-1 in samples of hepatocellular carcinomaObjective:By detecting the expression of Glypican-1(GPC1)in hepatocellular carcinoma(HCC)and its adjacent tissues,to analyze the correlation of abnormal GPC1 expression between the clinicopathological characteristics and survival of HCC,and to find the significance of abnormal GPC1 expression in HCC.Methods:GPC1 expression in HCC and para-tumor was detected by the TCGA database on the UALCAN website,and the correlation of abnormal GPC1 expression with the survival and prognosis of HCC.Four fresh HCC and para-tumor specimens were collected,and GPC1 expression in HCC and para-tumor specimens was detected by Western Blot.111 cases of HCC and para-tumor tissue samples were collected,and GPC1 expression in HCC and para-tumor tissue samples was detected by IHC.111 patients were divided into two groups according to the GPC1 expression level,and the correlation of GPC1 expression between the clinicopathological characteristics and survival of HCC was statistically analyzed.Results:Western blot results indicated that GPC1 expression level was higher in the 4 pairs of HCC than that in its para-tumor tissues(P<0.05).The IHC results indicated that GPC1 expression level was higher in the 111 cases of HCC than that in its para-tumor tissue samples(P<0.05).Statistical analysis showed that GPC1 expression level was inversely proportional to postoperative overall survival and disease-free survival(P<0.05).Conclusion: GPC1 expression level in HCC is higher than that in para-tumor,and the expression level of GPC1 is inversely proportional to the prognosis of HCC,suggesting that GPC1 may be involved in the progression of HCC.Part 2:Study of the role and mechanism of GPC1 in the proliferation of hepatoma cellsObjective:The biological function and mechanism of GPC1 in hepatoma cells were investigated by gene silencing technology.Methods:Lentiviruses carrying GPC1 interference sequences were constructed.Knock down the expression of GPC1 in hepatoma cell lines Hep G2,Huh7,and HCC-LM3 by lentiviral transfection.The silencing efficiency of GPC1 was verified by q-PCR.The proliferation of Hep G2,Huh7,and HCC-LM3 cells was detected by colony formation assay,CCK-8 and EDU staining.The migration ability of Huh7 and HCC-LM3 cells were detected by wound healing assay,and the invasion ability of Huh7 and HCC-LM3 cells were detected by Transwell.The effect of GPC1 on the proliferation of Hep G2 and HCC-LM3 cells was verified in vivo by subcutaneous tumor formation in nude mice.The effect of GPC1 on the cell cycle of Hep G2 and HCC-LM3 cells was detected by FCM,and the effect of GPC1 on cell cycle-related proteins and proteins in the regulation of PI3K/AKT related-pathway was detected by Western blot.Results:After silencing the expression of GPC1 in Hep G2/Huh7 and HCC-LM3.Colony formation assay,CCK-8 and Edu staining experiments showed that the proliferation ability of Hep G2 and HCC-LM3 cells was reduced(P<0.05),while that of Huh7 was partially reduced;Wound healing assay showed that the migration ability of Huh7 and HCC-LM3 cells was significantly reduced(P<0.001);Transwell invasion test showed that the invasion ability of Huh7 and HCC-LM3 cells was significantly reduced(P<0.001).The results of subcutaneous tumor formation in nude mice indicated that Hep G2 and HCC-LM3 cells’ proliferation ability in vivo was significantly decreased after silencing the expression of GPC1(P<0.05).The Ki-67 staining showed that Ki-67 expression level in the GPC1 silence group was significantly lower than that in the control group.Low expression of GPC1 significantly increased the cell subsets in G2/M phase arrest(P<0.05),down-regulated the promoting cyclins cyclin D1,cyclin B1 and CDK1,and up-regulated the inhibitory cyclins p21 and p27(P<0.05).The proteins in the regulation of PI3K/AKT related-pathway of p-Akt,p-Bad,p-GSK-3β,β-catenin and c-Myc were down-regulated and Bax,p-PTEN and PTEN were up-regulated(P<0.05).Conclusion: After silencing GPC1 expression,the proliferation of hepatoma cells in vitro and in vivo was inhibited.GPC1 may promote hepatoma cells proliferation by regulating cell cycle and PTEN/Akt/β-catenin signaling pathway.GPC1 is expected to become a potential target for precision treatment of HCC.