Characterization Of PTPN12 Knock-out A549 Stable Cell Line

PTP-PEST is a protein tyrosine phosphatase(PTP)encoded by the PTPN12(Protein Tyrosine Phosphatase Non-receptor Type12)gene.It is a member of the non-receptor PTP subfamily PTPN12 located autosomal 7q11.23,mainly in the cytoplasm,are widely expressed highly in hematopoietic and non-hematopoietic cells,such as the accumulation of extensive and thymus,spleen and liver hematopoietic cells involved in the regulation of immune cell migration.PTP-PEST exerts various regulatory functions by interacting with different substrates,participating in multiple steps of cell proliferation and migration processes,and acting as an important regulator of tumor cells.At present,many studies have shown that the high expression of PTP-PEST can inhibit the occurrence of several cancer tumors,among which the effect on triple negative breast cancer(TNBC)is more obvious.Our previous research found that the expression of PTP-PEST in lung adenocarcinoma is also very high,so it is speculated that PTPN12 plays a key role in the occurrence and development of lung adenocarcinoma.In order to study the biological function and mechanism of PTP-PEST in lung adenocarcinoma,the research team used CRISPR/Cas9 gene editing technology to knock out the PTPN12 gene in lung adenocarcinoma cell A549,which was successfully verified by gene sequencing and immunoblotting.Two stable cell lines with A549 PTPN12 knockout were obtained.In this study,the biological properties of these two cells were characterized.Through XTT cell proliferation experiment,cell digestion counting experiment,cell scratch experiment and Transwell cell invasion experiment,it was found that after knocking out the PTPN12 gene,the proliferation,migration and invasion ability of A549 cells have been enhanced to some extent.The cell hanging drop experiment and soft agar clone formation experiment showed that the aggregation ability and clone formation ability of A549-PTPN12-KO cells were also significantly enhanced.In addition,we also proved that the tumor formation ability of the A549-PTPN12-KO cell line in vivo was significantly stronger than that of wild-type A549 cells through a tumor xenograft nude mouse model.Finally,the application of immunoblotting technology revealed the molecular mechanism of PTP-PEST functioning through the Akt-ERK signaling pathway.In summary,through a series of in vitro and in vivo experiments,we characterized the A549 lung cancer cell line with PTPN12 gene knockout at the cellular and animal levels,which laid the foundation for revealing the role of the PTPN12 gene in lung adenocarcinoma.At the same time,it also provides a very valuable cell model for further studying the biological function on PTP-PEST.