The Effects Of ShRNA-mediated Silencing Of PKHD1 On Proliferation,Invasion And Metastasisof The Hucct-1 Human Intrahepatic Cholangiocarcinoma Cells

Objective:To explore the effect of silencing the PKHD1 gene by short hairpin RNA(sh RNA)on the proliferation, invasion and migrationof the Hucct-1 humanintrahepatic cholangiocarcinoma cells.To analysis the possible molecular mechanismof PKHD1 silencing in the proliferation, invasion and migrationof humanintrahepatic cholangiocarcinoma cells.Methods: Designing the short hairpin RNA(sh RNA) sequence which can effectively inhibit the expression of PKHD1 for constructing the recombinant lentiviral vector of PKHD1-sh RNA(LV3-PKHD1,LV3NC),and then transfected into human intrahepatic cholangiocarcinoma cells Hucct-1,respectively.The silence efficiency of PKHD1 was routinely detected by Western blot and q RT- PCR. Western blot were used to detect the apoptosis pathway –related proteins expression in each group of cells, such as PI3K、Akt、p-Akt、NF-κB. The proliferation potential of transfected Hucct-1 cells was assessed by the cell counting kit-8(CCK8)colofimetric assay. The Migration and invasion potentials were assess ed by wound healing assay and transwell migration/ invasion assays. Scanning electron microscopy was used to observe the morphological changes of primary cilia on the surface of Hucct-1 cells after stable silencing of PKHD1.Results:Following transfection of LV3-PKHD1 into Hucct-1 cells, the m RNA and the protein expression of PKHD1 were significantly decreased,and the protein expression of PI3K、p-Akt、NF-κB were significantly increased.Cell growth was significantly faster in the silencing group than the negative control group and blank control group(P < 0.01),but no significant difference(P > 0.05)between the two control groups.The speed of cell migration were faster after PKHD1 silencedin wound healing assay.The numbers of cell passing the membrane were significantly higher in the silencing group than the negative control group and blank control group(P<0.01), but no significant difference(P>0.01)between the two control groups.The primary cilia on the surface of Hucct-1 cells were decreased in the number and length after PKHD1 silenced under the scanning electron microscopy.Conclusion:sh RNA-mediated silencing of PKHD1 exerts promotion effects on Hucct-1proliferation, invasion and migration, this effect may be achieved via the activation of PI3K/Akt pathway. PKHD1 silencingcan cause the changes of length and number of theprimary cilia on the surface of Hucct-1 cells,and the structure and function of the primary cilia may be the indirect reasons for the development of biliary epithelial cells into intrahepatic cholangiocarcinoma.