Expression Of AIM2 In Colorectal Cancer And Its Role And Mechanism Of Proliferation And Apoptosis

Background:Absent in melanoma 2(AIM2),as DNA sensor in innate immunity,played an important role in tumorigenesis and progress of tumor.Studies shows abnormal expression of AIM2 in many solid tumor,exogenous AIM2 expression was shown to reduce proliferation of human breast cancer cell and murine fibroblast cells.A better understanding of the biological function of AIM2 in CRC is important to identify therapies that could be used in the treatment of this disease.Objective:In this study we aimed to identify the AIM2 expression in colorectal tissues in Chinese patient cohort,even more,to evaluated the relationship between AIM2 expression and clinic pathological parameters.Besides,we also aimed to identify the role of AIM2 expression on colorectal cell,and tried to find out the mechanism,so we may discover a potential therapeutic target in CRC.Materials and Methods1.Paired samples of CRC tissues and corresponding non-cancerous colonic tissues from 64 CRC patients taken from surgical excision specimen were included in this study.The expression of AIM2 was analyzed by quantitative real-time PCR assay(qRT-PCR).2.We used immunohistochemical staining assay to evaluate the expression of AIM2 in protein level in 68 pairs of colorectal specimen,and we analyzed the relationship between AIM2 expression and clinic pathological parameters.3.We reconstituted the expression of AIM2 in human HCT116 CRC cells,which lack functional AIM2,by transfection with AIM2-bearing lentiviral vector,and we confirmed the expression of AIM2 in HCT-116 colorectal cell by qRT-PCR and western blotting assay.4.Compared HCT-116 colorectal cell transfected with AIM2 vectors and Empty vector,and study the effect of AIM2 expression on the biological behavior of colorectal cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,cell apoptosis assay,cell cycle assay,and Transwell invasion assay.5.We treated HCT-116 cells with insulin-like growth factor-1(IGF-1),which activates the PI3K/AKT pathway,and the level of p-AKT was assessed by Western Blotting.6.The apoptosis rate of HCT-116 colorectal cell which activated by IGF-1 was analyzed by cell apoptosis assay,and compared with the apoptosis rate of HCT-116 colorectal cell without IGF-1,try to find out the mechanism of AIM2 effect in the apoptosis of colorectal cell.Result1.The result of qRT-PCR suggested that the expression of AIM2 was significantly decreased in colorectal cancer tissues compared with the corresponding non-cancerous colonic tissues(P<0.01).2.The immunoreactivity score and positive rate of colorectal cancer tissues was also significantly lower than corresponding non-cancerous colonic tissues.3.The of colorectal cancer tissues was associated with the tumor grade of colorectal cancer.The immunoreactivity score is decreased in the one which tumor grade is lower.4.The result of qRT-PCR and Western Blotting suggested that the expression of AIM2 in HCT-116 colorectal cancer cell transfected with AIM2 vectors was significantly increased compared with empty vectors in mRNA and protein level.5.The result of MTT assay suggested the relative to empty vector cells,the viability of AIM2-HCT116 cells decreased significantly,and the growth of cells was slower,which implied the AIM2 expression can inhibit the proliferation of colorectal cancer cells.6.The result of cell apoptosis assay suggested that AIM2-expressed HCT116 cells exhibited a significantly higher rate of apoptosis compared with the empty vector cells(P<0.01).7.The distribution of cells in different phases of the cell cycle was analyzed by flow cytometry.In AIM2-expressed HCT116 colorectal cancer cells,the proportion of cells in G1 phase and G2/M phase was increased(P<0.05),whereas the proportion in S phase was decreased(P<0.01)relative to control-transfected cells.8.The invasion ability of AIM2-expressed HCT116 had no difference with empty vector cells analyzed by Transwell invasion assay(P>0.05).9.Results of Western Blotting analysis showed that protein level of p-AKT was nearly the same in AIM2-expressed HCT116 cells and in empty vector cells.Upon activation with IGF-1,the level of p-AKT of two groups was significantly increased,while the p-AKT level was significantly lower in AIM2-expressed HCT116 cells compared with empty vector cells,indicating that the expression of AIM2 resulted in significant suppression of AKT activation.10.Without IGF-1,AIM2-HCT116 cells presented a higher apoptosis rate than the negative control cells,whereas after treatment with IGF-1 the apoptosis rate of AIM2-HCT116 cells was not significantly different from that of the negative control.These data suggest that AKT activation inhibits the capacity of AIM2 to induce apoptosis in CRC cells.Conclusion:1.The expression of AIM2 was significantly decreased in colorectal cancer tissues,and the AIM2 expression was associated with the tumor grade of colorectal cancer.2.The AIM2 expression can inhibit the proliferation of colorectal cancer cells and promote apoptosis of colorectal cancer cell.Inhibit the cell cycle transform to S phase.3.AIM2 inhibits the progress of colorectal cancer cells via the PI3K/AKT pathway.