Effects Of GRIM19 On Cell Apoptosis And The Sensitivity To Chemotherapy In Colon Cancer Cell

BACKGROUND&OBJECTIVESignal transducers and activators of transcription 3 (STAT3)and its pathway are involved in modulating cell proliferation, apoptosis, differentiation, and mediating malignant transformation of cells. If STAT3 and its pathway are blocked in vivo or vitro, the tumor cells will be induced to apoptosis and its proliferation will be inhibited. Now we found a gene named GRIM-19(Genes associated with Retinoid-Interferon induced Mortality 19) is a specific protein to inhibit STAT3. This study was to construct recombinant adenovirus carrying Grim19 gene and examine the effect of recombinant plasmid carrying Grim19 on cell apoptosis and the sensitivity to cisplatin in colon cancer cell.METHODSGrim19 cDNA was amplified by PCR, with the template pcxn2-Grim19 ,then cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrackCMV- Grim19 was linearized by PmeI, followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183,then identified by enzyme digestion. After linearized by PacI and transfection into HEK293 cells, recombinant adenovirus Ad-Grim19 were obtained in HEK293 cells then amplified by 3 circles. The green fluorescence in HEK293 cells was observed. Ad-Grim19 was transfected into sw480 cells. The rate of apoptosis was detected by FCM. pcxn2- Grim19 and pcxn2- stat3 were transfected into colon cancer cell line sw480 with lipofectamine2000. the efficiency of transfection was detected by Western blot and the effects of Grim19 on apoptosis in sw480 cells was examined by flow cytometry respectively. After combined treatment with cisplatin, the apoptotic effect and the bcl-xl expression were elvaluated.RESULTSThe results of enzyme digestion indicated that Ad-Grim19 was successfully constructed. The fluorescences show the expression of Ad-Grim19 in HEK293 cells. The transfection of Ad-Grim19 into sw480 cells increased the rate of apoptosis. The expression of Grim19 protein upregulated afer transfection. Cell apoptosis was induced by overexpressed Grim19 which could be decreased by cotransfection with stat3. Grim19 down-regulated the Bcl-xL protein expression and enhanced the apoptotic effect of cisplatin.CONCLUSIONRecombinant adenovirus Ad-Grim19 had been successfully constructed. Overexpression of Grim19 in sw480 colon cancer cell lines can promote apoptosis. Grim19 could accentuate cell apoptosis and the sensitivity to cisplatin in colon cancer cell.