Generation And Characterization Of PTPN12 Knock-Out HepG2 Stable Cell Line

Liver cancer is a common malignant tumor.The incidence rate of primary liver cancer is the fourth in the world,and the mortality rate is the third.China is a major country of liver cancer,accounting for 45%-55% of the world’s incidence rate,seriously threatening patients’ health and life.In recent years,the overall treatment level of liver cancer in China has reached the level of developed countries in Europe and America.However,the reasons for recurrence and metastasis of liver cancer have not been fully elucidated.Therefore,exploring the molecular mechanism of liver cancer recurrence and metastasis is of great significance for developing new treatments and improving patients’ prognosis.Protein tyrosine phosphatase PTP-PEST is encoded by the PTPN12 gene,and its abnormal expression or decreased activity is closely related to a variety of diseases.Currently,there are very limited reports on the role of PTPN12 in the occurrence and development of liver cancer.Through analysis of the GEPIA and Kaplan-Meier Plotter databases,we found that high expression of PTPN12 is associated with a shortened overall survival period in hepatocellular carcinoma(LIHC)patients,indicating that high expression of PTPN12 is a prognostic risk factor for LIHC.This finding has prompted us to carry out related mechanism research at the cellular level.In this study,the plasmid of PX459-PTPN12-sg RNA1 and PX459-PTPN12-sg RNA2 preserved in the laboratory were co-transfected into HepG2 cells.HepG2 cell lines with PTPN12 knockout were successfully obtained by purinomycin screening,Western Blot,PCR and gene sequencing.Through clonal formation experiment,CCK-8 experiment,live cell counting experiment,cell suspension drops experiment,periodic detection and other experiments,we found that the proliferation ability of HepG2 cells was significantly enhanced after knockout of PTPN12 gene.By cell scratch and Transwell invasion experiments,we found that the migration and invasion ability of HepG2 cells was also significantly enhanced after deletion of PTPN12 gene.The subsequent rescue experiment also validated the above results.However,the results of subcutaneous xenograft experiments showed that there was no significant change in tumorigenic ability in immunodeficient mice implanted with PTPN12 gene knockout cells compared with HepG2 wild-type cells.RNA-Seq results showed that PTPN12 gene deletion affected Pathways related to EMT,cell growth and proliferation,and cell migration and invasion.Finally,Western blotting was used to verify relevant signaling Pathways,and we found that PTPN12 gene affects cell proliferation,migration and invasion by regulating JNK,Cadherin,FAK and other signaling Pathways.In summary,we successfully generated HepG2 cells with PTPN12 knockout.The characterization results in vitro showed that the proliferation,migration and invasion of HepG2 cells were significantly enhanced after PTPN12 knockout,but there was no significant change in the tumor-forming ability of HepG2 cells.The present study preliminarily revealed the role and molecular mechanism of PTPN12 in HepG2 cells,which laid a foundation for further study of the biological function of PTPN12 in liver cancer.