| Background and objective:TIPE2is a newly discovered regulator of inflammation and can thus play a negative regulatory role in inflammation combined with caspase-8. It is a negative regulator of TLR and TCR pathway required to maintain immune homeostasis as well. There is no report about TIPE2in the mechanism of colon cancer. Colon inflammatory disease is a major cause inducing and promoting colon cancer tumorogenesis and development. TLR4signaling pathway mediated immune escape and tumor development of colon cancer cells by which chronic colon inflammation leads to colon cancer initiation and development. Following TLR4pathway activation, caspase-8can induce release of a large number of bioactive inflammatory cytokines. Our previous study found that TIPE2expression level was significantly increased in colon cancer tissue. In this paper, by analyzing the expressions of TIPE2, TLR4and their relationship in colon cancer tissues and measuring caspase-8activity, we explored TIPE2regulatory role of inflammation in the pathogenesis of colitis-associated tumorogenesis in depth.Methods:Immunohistochemical method was performed to detect the expression of TIPE2and TLR4in the tissue sections obtained from62colonic samples which were confirmed by pathology after resection of colon cancer between2008and2012and25normal colonic tissues achieved by colonoscopy biopsy. None of the samples received preoperative chemotherapy, radiation therapy, or immune therapy. In the62cases,40cases were male, the rest were female. They were21-84years old, the median age was61,10cases well-differentiated,35cases middle differentiation,17cases poorly differentiated; as for Duke’s stage,15cases A period,15cases B period,16cases C period,16cases D period. The TIPE2and TLR4polyclonal antibody were diluted at concentration of1:50,1:100respectively, and the rest steps were performed strictly according to the instruction of the antibody kit, phosphate buffer (PBS) was selected to replace the primary antibody as a negative control. The Spearman’s related analysis was used to analyze relationship between TIPE2and TLR4expressions. SiRNA interference technology reduced the expression level of TIPE2in colon cancer cell line HT-29, and real time PCR proved interference efficiency. We measured the caspase-8activity in the following four groups:TIPE2knockdown cells, TIPE2knockdown and TLR4activation cells, TLR4activation cells and wild-type cells, thereby further explored the role of TIPE2to TLR4signaling pathway in colon tumorogenesis and development.Results:1. The expression of TIPE2and TLR4in colon cancer:The positive expression of TIPE2was mainly located in cytoplasm of colon cancer cells, and there was no expression in the nucleus. Furthermore, the positive expression rate of TIPE2in colon cancer was69%, which was significantly higher than that in normal colon tissues24%(χ2=17.547, P<0.01). The expression of TLR4was mainly located in the membrane of colon cancer cells, and there was no expression in the nucleus. There was negative expression in most of the normal bowel mucosal cells. The positive rate of TLR4expression in colon cancer tissue was60%. significantly higher than that in the normal colon mucosa tissues20%(χ2=9.072, P<0.05).2. Spearman’s related analysis was used to analyze the relationship between TOPE2and TLR4expression in colon cancer tissues and found that in serial sections of the same sample, TIPE2strong expression area was accompanied by strong expression of TLR4and TIPE2weak expression parts often associated with TLR4weak expression (r=0.7354).3. Caspase-8activities in the four groups was analyzed by ANOVA and found that caspase-8activity in TIPE2knockdown cells was higher than that in wild-type cells (P<0.05), caspase-8activity in TLR4activation cells higher than that in wild-type cells (P<0.05), caspase-8activity in TIPE2knockdown and TLR4activation HT-29cells higher than that in TIPE2knockdown cells (P<0.05).Conclusions:1. TIPE2expression was positive correlation with that of TLR4in colon cancer.2. TIPE2can inhibit caspase-8activity in colon cancer cells and TIPE2can regulate TLR4inflammatory effect negatively and inhibit further amplification of cascade reaction by binding to caspase-8in colon cancer, thereby participates in the process of tumorogenesis and development of colon cancer. |
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