Effect Of Id2 On The Proliferation,Migration And Invasion Of HCT116 Colorectal Carcinoma Cells

Objective To investigate the effects of inhibitor of differentiation 2(Id2)on the proliferation,invasion and metastasis of HCT116 colorectal carcinoma cells,and explore the possible mechanisms.Methods Total cell proteins and RNA were extracted from six colorectal cancer cells HCT116,HT29,SW480,SW620,LoVo,and CaCo2 and analyzed the expression of Id2 by western blot and q RT-PCR experiments.The colorectal carcinoma cell line HCT116 with the highest expression of Id2 was selected for further research.HCT116 was transiently transfected with shId2 plasmid as the experimental group HCT116-shId2,while HCT116-shcon cell line was established as the negative control group.The transfection efficiency was observed according to the green fluorescence expression in 48 hours.The inhibition of Id2 was detected by western blot and q RT-PCR.Then Real time cellular analysis(RTCA)and clone formation assay were performed to detect the change of cell proliferation and clone formation abilities.The cell cycle wasanalyzed by flow cytometry.The abilities of cell invasion and metastasis were observed by wound healing assay,transwell migration and invasion assay.The proteins expression was determined by Western blot assay.Results 1.Among the six colorectal cancer cell lines,HCT116 cells showed high expression of Id2 protein and mRNA;2.The cell proliferation curve showed that the growth of HCT116 cells was significantly inhibited because of Id2 gene knockdown(p<0.05).The number of clone in HCT116-shId2-7 group(49.7 ± 11.0)was significantly less than that in HCT116-shcon group(150.7 ± 17.4)(p<0.05),and the size of clones also became smaller.It suggested that the clonogenic ability was significantly suppressed in Id2-knockdown HCT116 cells.According to the cell cycle analysis by flow cytometry,it was found that the proportions of G0/G1 phase cells of HCT116-shcon and HCT116-shId2 were respectively 45.17%±2.5% and52.52%±2.65%(p<0.05).The proportions of S phase cells were respectively 36.93%±1.71% and 30.63%±1.45%(p<0.01),and the proportions of G2/M phase cells were respectively 17.99%±4.06% and 16.84%±3.49%(p> 0.05).Further analysis showed that the expression of CyclinB and CyclinD1 were downregulated while Id2 gene was knowdown.It indicated that Id2 inhibit the expression of CyclinB and CyclinD1,which blocked the cell cycle in G0/G1 phase and suppressed HCT116 cell growth.3.The wound healing rates of HCT116-shcon group and HCT116-shId2-7 group were respectively 75.3%±8.1% and 35.5%±9.7% for 24 h culture.There are significant differences between the two groups(p<0.05).According to the Transwell migration experiment,the number(109.0 ± 11.1)of transmembrane cells in HCT116-shId2-7group was obviously reduced compared with the HCT116-shcon group(155.3 ± 7.6)(p<0.05).In the Transwell invasion experiment,the number of transmembrane cells in the HCT116-shcon group and HCT116-shId2-7 group were respectively 75.3±12.6and 46.7±6.0(p<0.05).It was showed that the cell migration and invasion abilities were suppressed in HCT116 cells with Id2 geneknockdown,and the expression of invasion and metastasis-related proteins MMP2 wassignificantly downregulated,and TIMP1 was upregulated(p< 0.05).MMP7 and MMP9 showednochange.Conclusion 1.Knockdown of Id2 geneinhibited the proliferation of HCT116 cells by decreasing the expression of Cyclin B and Cyclin D1 molecules.2.The cell migration and invasion were inhibited by the downregμlation of MMP2 and upregulation of TIMP1 in Id2-knockdown HCT116 cells.