Correlation Of MICA And Its Receptor NKg2D With The Development Of Colon Cancer

The human major histocompatibility complex (MHC) classⅠchain-related gene A(MICA) are located about 46 kb centromeric to human leukocyte antigen B (HLA-B).MICA gene shows high polymorphism, and its allele distribution is distinct in differentpopulations. Moreover, MICA is associated with ankylosing spondylitis, celiac disease,et al. MICA protein is expressed slightly on the cell surface of normal enteric epithelium,but its expression is upregulated in infected, stressed and malignant cells. MICA acts asa ligand of NKg2D which are expressed on natural killers,γδT cells, and CD8~+ T cells.While MICA ligate with NKg2D, it will lead to lymphocyte activation. In the pastseveral years people pay more attention to the role of MICA in viral infection and tumorinvasion. A series researches have confirmed the potential significance of humanMICA-NKg2D ligand-receptor signalling in immune responses against tumours. But thepresence of MICA on many progressing tumours, including breast, lung, gastric, renal,colon, ovarian and prostate carcinomas and melanomas, suggests that MICA or NKg2Dmight be functionally impaired, thereby promoting immune evasion.Objective:The research can be devided into three parts. Firstly, polymorphism of MICA gene,expression of MICA protein on tumor cell, and concentration of soluble MICA in bloodserum were analyzed in colon cancer patients. Then expression of NKg2D on defferentlymphocyte subgroups was investigated in colon cancer patients with different stage. Inthe end, we constructed the recombinant prokaryotic expression vector pQE31-sMICA. Through the process of expression, purification and renaturation, a large quantity ofsoulbe MICA (sMICA) protein was obtained. Meanwhile, the souble MICA was coatedto the bottom of 96 well plate to form immobilized MICA (iMICA), and the effect ofsMICA and iMICA on activation, proliferation, apoptosis, cytotoxicity and IFN-γ,secreation of NKg2D positive lymphocyte was studied.Results:1. The distribution of MICA alleles displayed no siginificant difference betweencolon cancer patients and healthy controls of Han population in Yangzhou with thetyping method of PCR/SSP (P＞0.05). The expression level of MICA antigen on non-metastatic tumor was higher than the tissue with metastasis(P＜0.01) byimmunohistochemistry. The concentration of souble MICA protein was increased incolon cancer patients serum compared with normal controls by ELISA(P＜0.01), andhighest in late stage of Dukes C～D (P＜0.01).2. The percentage of NKg2D and CD56 double positive cells in colon patients wassignificantly lower than those in healthy controls (P＜0.05) by flow cytometry. NKg2Dexpression on lymphocytes from lymph nodes with metastasis decreased dramaticallycompared with corresponding peripheral blood (P＜0.01), especially the expression ofNKg2D onγδT cells (P＜0.05).3. After a large amount of soluble MICA protein was obtained through prokaryoticexpression, the effect of sMICA and iMICA on function of NKg2D positive lymphocytewas studied. The results showed that sMICA could inhibit the proliferation of NK cell inthe third and fifth day(P＜0.01), but there was no deviation of NK cell apoptosis (P＞0.05). sMICA could downregulate the expression of both NKg2D and CD69 receptorson lymphocytes in 24 hours (P＜0.05). After incubation with sMICA in 24 hours,secretion of IFN-γfrom NK cell was significantly decreased(P＜0.05), but there was noeffect on cytotoxitity of NK cell (P＞0.05). However, after incubation with iMICA in 24hours, NKg2D positive lymphocyte upregulated the CD69 expression (P＜0.01). In addition, proliferation, secretion of IFN-γ, and cytotoxicity of NK cell was increasedrespectively (P＜0.01).Conclusions:1. There was no association between MICA gene polymorphism and thedevelopment of colon carcinoma. Colon cancer patients with high levels of MICA maysuggest a good prognosis, whereas high concentration of soluble MICA in serum with apoor prognosis.2. Decreased NKG2D expression may be one of the key mechanisms responsiblefor immune evasion by tumors in colon cancer. In addition, we suggested NKg2Dexpression on peripheral lymphocytes may not be a sensitive biomarker for evaluationthe metastasis of tumor to lymph nodes.3. Soluble MICA downregulate the function of NKg2D positive lymphocyte suchas decreasion of CD69 and NKg2D expression, inhibition of the proliferation and IFN-γsecretion of NK cell. However, immobilized MICA could activate NKg2D positivelymphocytes such as upregulation of CD69 expression, enhancing the cytotoxicity,promoting the proliferation and IFN-γsecretion of NK cell.