Characterization Of MPZL1 Knock-out HeLa Stable Cell Line

Cervical cancer is the leading cause of cancer morbidity and mortality in women worldwide,second only to breast cancer in mortality.The onset of cervical cancer is insidious and difficult to detect in the early stage.As the disease progresses,it has entered the advanced stage when obvious symptoms appear.Advanced cervical cancer cells metastasize to various parts of the body through direct spread,lymphatic metastasis,and vascular metastasis,and the molecular mechanisms are not fully understood.Therefore,it is of great significance to improve the new molecular mechanism of cervical cancer metastasis to promote new treatment methods and improve the prognosis of patients.Human myelin P0 protein related protein（PZR）is an immunoglobulin encoded by the MPZL1 gene,which can interact with SH2 domain-containing tyrosine phosphatase2（SHP-2）specifically binds to regulate cell adhesion,proliferation,differentiation and migration.Previous studies have shown that PZR can promote the fibronectindependent migration of mouse MEF cells and the invasion and metastasis of hepatoma cells,while the role of PZR in cervical cancer metastasis remains unclear.We used the PX458-MPZL1-sgRNA1 and PX459-MPZL1-sgRNA2 recombinant vectors constructed in the previous stage to co-transfect HeLa cells,and obtained HeLa stable cell lines with MPZL1 knockout after screening and identification.we verified that the migration ability of HeLa cells was significantly reduced after wound healing experiments.But the deletion of MPZL1 gene did not affect its invasion ability,proliferation ability and intercellular adhesion through experiments such as clone formation experiments,wound healing experiments,hanging drop experiments,CCK-8,transwell invasion experiments,and cell cycle detection.And then,we also demonstrated that tumor-forming ability of HeLa-MPZL1-KO cell line was significantly weaker than that of wild-type HeLa cells by tumor xenografting NYG immunodeficient mouse model in vivo,and it was found by histopathological section and HE staining microscope observation.The deletion of MPZL1 gene can lead to the disappearance or fragmentation of the nuclei of tumor cells,resulting in abnormal tissue structure and necrosis of tumor cells.Subsequently,through RNA-seq analysis,the differential gene search and the analysis of the signal molecule interaction network were carried out.Finally,the deletion of MPZL1 did not affect the expression of signaling molecules such as SHP-2,ERK and FAK in HeLa cells by western blot analysis.Since these signaling pathways involve many upstream and downstream signaling molecules,how the loss of MPZL1 affects these signaling pathways remains to be further investigated.In summary,we first constructed HeLa cells with a stable knockout of MPZL1 gene and characterized them at the cellular and animal levels through in vitro and in vivo experiments,laying a foundation for revealing the role of MPZL1 gene in cervical cancer.It also provides a very valuable cell model for further study of the biological function of PZR.