Tumor Suppressive Effect And Possible Molecular Mechanism Of PKHD1 On Intrahepatic Cholangiocarcinoma Via Notch Signaling Pathway

Objective: The expression of PKHD1 gene in bile duct carcinoma(CC)and adjacent normal tissues was detected,and the effect of CRISPR/Cas9 knockout of PKHD1 on the cellular behavioral ability of hccc-9810 and RBE cell lines in human intrahepatic bile duct carcinoma was studied.The proteins associated with Notch pathway were determined to observe the tumorigenic ability of cell lines in the knockout group to explore the potential mechanism of PKHD1 in vitro and in vivo.Methods: Adopting q RT-PCR in detecting the expression of PKHD1 in CC and adjacent normal liver tissues;utilizing CRISPR/Cas9 technique in constructing PKHD1 gene knockout of HCCC-9810 and RBE cell lines;verifing the effect of knocking out of PKHD1 gene by PCR sequencing;detecting the proliferation of Hucct-1 by CCK-8;detecting migration and invasion potentials of cells through wound healing assay and Transwell migration/ invasion assays,and assessing related proteins expression of the Notch pathway by Western blot;establishing a model of subcutaneous implant tumor in nude mice and to observe the growth of the tumor,30 days after which the mice were executed to detect the growth volume and quality of tumors.Western blot was used to detect the expression of PKHD1 and MMP9 protein in tumor-bearing tissues of nude mice.Results: The expression of PKHD1 in CC tissues was significantly lower than that in adjacent tissues(P < 0.01).Stable HCCC-9810 and RBE cell lines with PKHD1 gene knockout were successfully constructed.Compared with wild-type cells,the expression of Notch,Jagged-1,NICD and PTEN in downstream cells were significantly increased after PKHD1 gene was knocked out(P < 0.05).The growth of cells in the knockout group was significantly faster than that of the negative control group and the blank control group(P<0.01),however,there was no significant difference between the control groups(P>0.05).The speed of cell migration was faster after PKHD1 silenced in wound healing assay.The number of cells passing through Transwell chamber in knockout group was significantly higher than that in negative control group and blank control group(P < 0.01).Comparably,there was no significant difference between the control groups(P > 0.05).The results of subcutaneous implantation in nude mice showed that the growth rate,final volume and mass of subcutaneous implantation tumors in knockout groups were significantly higher than those in blank control group and blank virus group,and the expression of PKHD1 protein in transplanted tumors was significantly decreased.Conclusions: The expression of PKHD1 gene is reduced in tissue of intrahepatic cholangiocarcinoma.Targeted knockout of PKHD1 in HCCC-9810 and RBE cells can significantly promote the proliferation,invasion and migration of human intrahepatic cholangiocarcinoma cells and the growth of tumors in vivo,which may be resulted from the activation of NOTCH pathway.