Effects Of PTPRQ Gene Targeted Knocking Out By CRISPR/Cas9 On The Biological Behavior Of Colorectal Cancer Cell

Objective:In this study,we aim to explore the effects of PTPRQ on biological behavior of tumor cells and the mechanisms involved by selecting human colorectal cancer cell HCT-116 as the study model.Methods:1.The recombinant plasmids of pCDgRNApuro-PTPRQ and Cas9 were transfected into human colorectal cancer cell HCT-116,and the monoclonal cells were selected by puromycin to obtain stable cell lines named HCT-116-64 and HCT-116-85 respectively.These two strains were used as experimental groups and the wild-type HCT-116-WT cell was used as control group.2.The effects of knocking out PTPRQ on the proliferation,colony formation,migration and apoptosis of HCT-116 cells were observed by cell counting kit(CCK),cell colony formation assay,cell scratch assay,transwell-migration assay and flow cytometry.3.The effects of knocking out PTPRQ on the key protein expression of PI3 K /AKT and Ras / MEK / ERK signaling pathway in HCT-116 cells were examined by Western blot.4.The changes of cell proliferation were observed after treatment with PI3 K inhibitor LY294002 or MEK inhibitor U0126.5.The effects of knocking out PTPRQ on tumor growth rate,tumor volume and tumor quality of nude mice were observed by establishment of orthotopic animal model of colorectal cancer cells HCT-116.Results:1.The expression of PTPRQ protein in HCT-116 cells was significantly decreased(> 80%,P <0.01 vs control group)after knocking out PTPRQ gene by CRISPR / Cas9,suggesting that HCT-116 cell lines knocked out PTPRQ gene were successfully constructed.2.After knocking out PTPRQ gene,the proliferation ability of HCT-116 cellswere weakened(P <0.01 vs control group),the cell colony formation ability were decreased(P <0.01 vs control group),the cell migration ability were weakened(P<0.01 vs control group),the cell tolerance to apoptosis-inducing agents 5-FU decreased and the apoptosis rate increased(P <0.01 vs control group).3.The activity of AKT and ERK increased after knocking out PTPRQ gene(P<0.01 vs control group).4.The cell proliferation ability of HCT-116-64 and HCT-116-85 were further attenuated(P <0.01 vs control group)compared with the control group after treating cells with PI3 K inhibitor LY294002 or MEK inhibitor U0126.5.The tumor formation rate was slower and the tumor volume(P <0.01 vs control group)and tumor mass(P <0.05 vs control group)were smaller compared with the control group.Conclusion:PTPRQ is involved in the regulation of the biological behavior of colorectal cancer cells,and the knockout of PTPRQ gene leads to a decrease in the degree of malignancy of tumor cells,indicating that PTPRQ may be a tumor promoting factor.Furthermore,the tumorigenic effect of PTPRQ is not mediated by the PI3 K and ERK pathway.