Expression Of RACK1in Colorectal Adenocarcinoma And Its Significance

Objective: In recent years, people found receptor for activated protein kinaseC-1(RACK1) involved in the regulation of a wide variety of tumor. This study aims toresearch the expression of RACK1protein in different stages of colorectal cancercanceration process, and through establishing of stable transfection RACK1coloncancer cell lines, analyze their biological behaviour after knock-in and knock-downthe RACK1in colon cancer cell lines, in order to primarily explore the relationshipbetween the expression of RACK1and colorectal carcinogenesis.Methods: Immunohistochemical staining was performed to detect the RACK1expression in colorectal adenocarcinoma, colorectal adenoma, colorectalinflammatory polyp and normal colorectal mucosal tissues. Western blot was used todetect the RACK1expression in normal colon tissues and colon cancer cells includingHCT116, HRT18, SW620, SW480and HT29. then RACK1expression plasmid(PcDNA3-GFP-RACK1) and its blank plasmid (PcDNA3-GFP) were transfectedto one colon cancer cell line with lower expression of RACK1, and RACK1interference plasmid (PSilencer3.1-RACK1) and its blank plasmid (PSilencer3.1)were transfected to one colon cancer cell line with higher expression of RACK1byLiposome-mediated gene transfection, Western blot was used again to detect theexpression level of RACK1and determine the transfection efficiency. Aftertransfection, MTT assay tested celluar serum-dependent growth and flow cytometrywas used to analyze the cell cycle distribution.Results:1The expression of RACK1in colin cancer tissue and cells and its clinicsignificanceImmunohistochemistry showed that RACK1expression in23normal colorectalmucosal tissues,20colorectal inflammatory polyp,20colorectal adenoma and92colorectal adenocarcinoma was progressively increased(P<0.05), the expression ofRACK1in30colorectal high differentiation adenocarcinoma,30colorectal moderately differentiation adenocarcinoma and20colorectal poorly differentiatedadenocarcinoma was progressively decreased(P<0.05), Moreover, the colorectaladenocarcinoma with higher differentiation showed higher level than colorectaladenocarcinoma with poorer differentiation(r=0.213, P=0.011). RACK1expressionlevels did not correlate with the patients’ age and gender, regional lymph nodemetastasis and Duke stage(P>0.05). Western blot showed that The expression ofRACK1in colon cancer cells(HCT116, HRT18, SW620and HT29) was higher thannormal colorectal tissues(P<0.05).2Building stable transfection colon cancer cellsRACK1expression plasmid (PcDNA3-GFP-RACK1) and its blank plasmid(PcDNA3-GFP) were transfected to SW480, after screening by G418two weeks,Western blot results showed that the most obvious RACK1expression up-regulationwas3and34clones, which were used as subjects in follow-up experiments. RACK1interference plasmid (PSilencer3.1-RACK1) and its blank plasmid (PSilencer3.1)were transfected to HRT18, after screening by G418two weeks, Western blot resultsshowed that the most obvious RACK1expression down-regulation was20and30clones, which were used as subjects in follow-up experiments.3Effect of biological behaviour after knock-in and knock-down the RACK1incolon cancer cell linesMTT result showed that, SW480-3and SW480-34was growing faster than vectortransfected cells and untransfected cells after two days(P<0.05); while compared withvector transfected cells and untransfected cells, there was obivious inhibiting effect inHRT-20and HRT-30after three days. cell cycle distribution that was detected by flowcytometry showed RACK1expression increased, the colon cancer cells in G0/G1Phase was decreased(P<0.05), while the cells in S phase was increaced(P<0.05).Conclusion:1RACK1expression was progressively increased during the colorectalcarcinogenesis, The expression level of RACK1had correlation with differentiateddegree of colorectal adenocarcinoma, and RACK1upregulation may play a role incolorectal adenocarcinoma.2Upregulated expression of RACK1may promoted the growth of colon cancer cells,and influenced cell cycles.