The Mechanism Research Of CRIP1(Cysteine-Rich Intestinal Protein 1)on Promoting The Proliferation Of Colorectal Cancer Cells By Fas/FasL Signaling Pathway

Backgrouds and Objective:Colorectal cancer(colorectal carcinoma,CRC)is the top three common malignant tumor in China,and is also one of the leading causes of death in cancer patients.There is an upward trending in incidence of recent years.Although 90%of patients can be cured by surgical treatment in the early stage of colorectal cancer,but most of the patient are definitedly diagnosised very late and have a poor prognosis.CRIP1 is a memeber of the LIM family,protein molecular weight 8 kDa,and belongs to small molecular weight proteins.Researches show that CRIP1 is hignly expressed in breast cancer,endometrial cancer,prostate cancer,and pancreatic cancer,and has a significant influence on the prognosis of diseases,meanwhile,it is still unclear whether CRIP 1 has an expression and function in CRC or not.This research aims to exploring the effect of CRIP1 on the occurrence and development of CRC and its regulatory mechanism,and strives to find new a target or method for the colorectal cancer effective curing.Methods:1.Analysised CRIP1 expression in colorectal cancer through real-time quantitative PCR to detect CRIP1 mRNA level,western blot to test its protein expression level in colorectal cancer cells,and immunohistochemical staining to explore the differential expression in colorectal cancer tissues and normal tissues;2.Constructed CRIP1 vector,and verify it by western blot.To detect the effect of CRIP1 on the proliferation ability of SW620 cells,we adopted CCK8 and EDU proliferation experiments after overexpressing CRIP1 by transient transfection.Then using flow cytometry instrument to detect the cell cyclling and apoptosis,cell apoptosis is showed by Tunel imaging experiments at the same time.On account of the apoptosis is weak in tumor cells,here we used 5-fluorouracil to induce an obvious trend;3.Constructed CRIP1 siRNA,and verified it by western blot.The continuing methods are the same as point 2;4.Using western blot to detect how CRIP1 regulating apoptosis related proteins,particularly Fas.FasL(Fas Ligand)regulation detection used enzyme linked immunosorbent assay(ELISA),then we adopted flow cytometry and Tunel apoptosis cell imaging experiments for recovery test;5.To explore controlling mechanism of CRIP1 to Fas,we used of real-time quantitative PCR(RT-PCR)and western blot to detect the influence of CRIP1 to Fas on mRNA and protein level,and then reseached the interactive mechanism between CRIP1 and Fas protein by immune coprecipitation experiment(CO-IP).6.Statistical analysisData was analyzed by SPSS 19.0 statistical software(SPSS,Chicago,USA).Quantitative data was collected by three independent experiments,which represented as mean±standard deviation.Results:1.real-time quantitative PCR(RT-PCR)and western blot detected CRIP1 have been expressed on mRNA and protein level in colorectal cancer cell lines,besides,it has expression in colorectal cancer tissus by immunohistochemical staining;2.After overexpressing CRIP1 in SW620 and knocking-down it in HCT116,CCK8 and EDU proliferation experiment proved CRIP1 can promote colorectal cancer cell proliferation;Flow cytometry instrument detection and Tunel apoptosis cell image experimental demonstrated CREP1 can inhibit apoptosis of human colorectal cancer cells,but not obviously effects on cell cycling;3.Western blot experiments showed CRIP1 negative regulated apoptosis related proteins,especially Fas,but enzyme-linked immunosorbent(ELISA)proved CRIP1 has no obvious impact on FasL;Flow cytometry instrument and Tunel apoptosis cell imaging method has also proved that the negative regulation of CRIP1 to Fas,consequently inhibiting apoptotic pathway in recovery test;4.Real-time quantitative PCR(RT-PCR)show CRIP1 has no obvious influence on Fas in mRNA level,but immune coprecipitation experiment(CO-IP)proved the interaction between the two proteins,and the degradation of Fas by CRIP1 through ubiquitination.Conclusions:1.CRIP1 has been expressed in colorectal cancer cell lines on both mRNA and protein level,and has a high expression in colorectal cancer tissues;2.CRIP1 promotes colorectal cancer cell proliferation;3.CRIP1 promotes colorectal cancer cell proliferation by inhibiting cell apoptosis;4.CRIP1 regulates apoptosis by inhibiting Fas/FasL apoptosis signaling pathway;5.CRIP1 affects Fas/FasL apoptosis signaling pathway by ubiquitining Fas.