PTPRN2 Promotes The Invasion And Metastasis Of PGCCs And Their Daughter Cells Through Cofilin1 And TRAF2

Objective:Our study confirmed that cobalt chloride(CoCl2),radiation and chemotherapy agents can induce polyploid giant cancer cells(PGCCs)from various tumors.Polyploid tumor giant cells have the characteristics of tumor stem cells,which are slow to self-renew and have the ability to resist various adverse stimuli.In addition,PGCCs can generate daughter cells with high invasive and proliferation capacity through asymmetric division,which may be closely related to tumor recurrence,metastasis and invasion after chemotherapy.At present,the most common first-line chemotherapy for colorectal adenocarcinoma is 5-fluorouracil and oxaliplatin.Previous experiments have confirmed that PTPRN2 is highly expressed in PGCCs and their daughter cells induced by cobalt chloride.In this study,5-fluorouracil and oxaliplatin were used to induce PGCCs in human colorectal cancer cell lines HCT116 and LoVo.Moreover,we explored the molecular mechanism of PTPRN2 regulating the highly invasive and proliferation abilities of PGCCs and their progeny cells,and further provided new ideas and therapeutic targets for the treatment of colorectal adenocarcinoma.Methods:(1)Oncomine,GEPIA and UALCAN databases were used to analyze the mRNA expression of PTPRN2 in colorectal adenocarcinoma;The Human Protein Atlas database was used to analyze the protein expression and localization of PTPRN2 between colorectal cancer and normal colorectal tissues.Western blot assay was performed to detect the expression of PTPRN2 protein in normal colon cells and several colorectal cancer cell lines.(2)UALCAN databases was used to explore DNA methylation in the promoter region of PTPRN2.MethSurv database was used to analyze the correlation between methylation of CpG island in PTPRN2 gene and prognosis of colorectal cancer patients.CBioPortal database was used to analyze PTPRN2 gene changes in colorectal cancer and its effect on prognosis of patients.(3)TIMER,TIGIBA and GEPIA databases were used to observe the relationship between PTPRN2 and tumor immune cells in the microenvironment and whether it affects the prognosis of colorectal cancer patients.GO analysis and KEGG pathway analysis of PTPRN2 were performed by using LinkedOmics database and GSEA tool.(4)HCT116 and LoVo cells were treated with 5-fluorouracil and oxaliplatin for 48h to induce polyploid tumor giant cells and their daughter cells as experimental group.Western blot assay was performed to detect the expression levels of PTPRN2 protein in HCT116 and LoVo cells in control group and PGCCs and their daughter cells.The expression and localization of PTPRN2 in the control group and the experimental group were compared by immunocytochemical staining assay.(5)The migration,proliferation and invasion abilities of PGCCs and their daughter cells were compared with those of the control group and experimental group,before and after PTPRN2 knockdown by plate cloning experiment,scratches-healing experiment,Transwell migration and invasion experiment and Ki67 cell staining assay.(6)The PTPRN2 in PGCCs and their daughter cells was knocked down by small interfering RNA(siRNA),and the expression of downstream proteins regulated by PTPRN2 protein was detected by Western Blot assay.(7)The interaction between PTPRN2 protein and TRAF2,Twistl and CyclinDl proteins was detected by immunoprecipitation assay.Flow cytometry was used to detect the cell cycle distribution of the control group and the experimental group as well as the knockdown of PTPRN2 by the small interfering RNA.Results:(1)The mRNA expression level of PTPRN2 in colorectal adenocarcinoma was significantly higher than that in normal colorectal tissues.In addition,PTPRN2 was associated with pathological stage and metastatic grade in colorectal cancer.Immunohistochemical staining confirmed that PTPRN2 protein expression in colorectal adenocarcinoma was higher than that in normal colorectal tissue.Compared with normal IEC6 cells,PTPRN2 protein expression was elevated in colorectal cancer cells,especially in HCT116 and LoVo cells.(2)Promoter methylation of PTPRN2 was significantly reduced in colorectal adenocarcinoma compared with normal colorectal tissue.In colorectal adenocarcinoma,hypomethylation of 23 and 11 CpG sites in PTPRN2 gene was associated with poor prognosis,respectively.In addition,the PTPRN2 gene was altered by 2.6%in patients with colorectal adenocarcinoma,and their overall survival were higher than in the unaltered group.(3)PTPRN2 was widely involved in the regulation of various immune molecules in colorectal adenocarcinoma patients.In a tumor microenvironment rich in Type 1 helper and regulatory T cells,high expression of PTPRN2 was associated with poor prognosis in patients with rectal adenocarcinoma.In addition,KEGG analysis showed that PTPRN2 was mainly involved in the enrichment of insulin secretion,adhesion connection,synaptic transmission,hormone synthesis and secretion,cGMP-PKG signal,cAMP signal and FoxO signal pathway,etc.(4)5-fluorouracil and oxaliplatin were used to induce the formation of PGCCs in HCT116 and LoVo cells,and PGCCs generated progenitor cells after the static recovery period.The abilities of migration,invasion,proliferation and Ki67 staining intensity of PGCCs and their daughter cells were higher than those of the control group.PTPRN2 expression was increased in PGCCs and their daughter cells compared with the control group.(5)The malignant biological behavior of PGCCs and their daughter cells was significantly reduced after the expression of PTPRN2 was knocked down by siRNA.Compared with the negative control group,the expression of Cofilin1 protein,TRAF2 protein,EMT biological process,JAK2/STAT3/CyclinD1 pathway,PI3K/AKT pathway and MAPK pathway related proteins in PGCCs and their daughter cells with PTPRN2 knockdown were also decreased.In addition,immunoprecipitation assay revealed that PTPRN2 interacted with TRAF2,Twistl and CyclinDl.Conclusions:The mRNA and protein expression levels of PTPRN2 in colorectal adenocarcinoma were significantly higher than that in normal colorectal tissue,and were related to the pathological stage and grade of colorectal cancer.High expression of PTPRN2 was associated with poor prognosis of colorectal cancer patients with cancer,and it may affect prognosis of patients through DNA hypomethylation,gene alteration and tumor immune cells.PTPRN2 protein was significantly up-regulated in PGCCs and their daughter cells,which promoted the migration,proliferation and invasion of colorectal cancer by regulating the EMT biological process,JAK2/STAT3/CyclinDl pathway,PI3K/AKT pathway,MAPK pathway,Cofilin1 protein and TRAF2 protein.