Effect of PCK1 on migration of hepatocarcinoma cells

Abstract:

Objective：To construct overexperssion and knockout model of phosphoenolpyruvate carboxykinase1（PCK1） gene and to in－vestigate the effects of PCK1 on migration of hepatocarinoma cell. Methods：The cDNA of PCK1was cloned into plasmid pAdTrack-TO4. Recombinant adenovirus plasmid pAdTrack-PCK1 was transfected into HEK293 cells to package and generate high titer recom－binant adenoviruses encoding PCK1（AdPCK1）. The PCK1 knockout cell lines using CRISPR/Case9 system were selected in PLC/PRF/5 cells. First，the Huh7 cells were set into two groups in the overexpression model，one of which was AdGFP group as control group infected with adenovirus AdGFP，and the other of which was AdPCK1 group as experimental group infected with adenovirus AdPCK1. The knockdown model included two groups，one of which was Parental group as control group using PLC/PRF/5 cells，and the other of which was KO group as experimental group using PCK1 knockout cells. Two models were confirmed by Western blot. The effect on the migration of hepatocarcinoma cells was observed by wound assay. Further，Cdh1 were analyzed by qRT-PCR. Results：AdPCK1 was successfully constructed and Western blot showed PCK1 overexpression in AdPCK1 infected cells. Western blot showed that PLC/PRF/5 cell lines with PCK1 knockout were successfully estab－lished by the usage of CRISPR/Cas9 system. Wound assay showed PCK1 significantly inhibited the migration of hepato－carcinoma cells compared with control group. The wound clo－sure rate of AdPCK1 group was （66.300±0.383）% compared with that of （42.900±3.833）% in AdGFP control（t=10.540，P=0.000）. The wound closure rate of KO group was （59.40±5.68）% compared with that of （79.00±5.20）% in parental cell control（t=4.420，P=0.012）. In addition，qRT-PCR showed that the expressions of Cdh1 genes in EMT signal pathway were signifi－cantly inhibited by the gene of PCK1 than control. qRT-PCR showed that AdPCK1 compared with those of AdGFP control group，the relative expression of Cdh1 was 2.733±0.501（t=5.996，P=0.004），and KO group compared with those of parental cell，the relative expression of Cdh1 was 0.664±0.017（t=34.290，P=0.000）. Conclusion：PCK1 inhibited the migration of hepatocarcinoma cells through regulating the expression of Cdh1 genes in EMT pathway，suggesting that PCK1 may be involved in the initiation and progress of hepatocarcinoma.