The Regulatory Role Of MicroRNAs In The Expression Of CD80and CD86and Their Receptors With Clinical Significance In Gastrointestinal Carcinoma

IntroductionActivation of T lymphocytes is essential for anti-cancer, anti-infection, andautoimmune disease. The activation of T cells needs not only the first signal induced byinteraction between the T-cell receptor (TCR) and the major histocompatibility complex(MHC)-peptide complex, but also the second signal that is induced by the combinationof B7-family costimulatory molecules with their receptors. Costimulatory moleculesand their reporters can generate stimulatory or inhibitory signal to regulate the activityof T cells, so as to control immune response. The co-stimulatory molecules CD80andCD86expressed on APC cells, but no or lowly expressed in most tumors. They promoteimmune escape of tumor cells mediated by ineffective activation of T cells. In a coloncarcinoma mouse model, immunoescape mechanism was found to be mediated by lowexpression of CD80on tumor cells, which is similar to the regulatory mechanismmediated by the CD80expression on immature DC cells. MicroRNA (miRNA), anendogenous non-coding small RNA of~22nucleotides, plays an important role in thepost-transcription regulation of the gene expression by binding to the3’-untranslatedregion (3’-UTR) of its target messenger RNAs (mRNAs). It has been reported that thedysregulated miRNAs in most tumors can influence the tumor microenvironment byregulating cell differentiation, proliferation, and apoptosis. Part I, The regulatory role of dysregulated microRNAs in theexpression of CD80and CD86and their receptors in humancolorectal carcinomaObjective: To investigate the regulatory role of dysregulated microRNAs on theexpression of CD80and CD86and their receptors in the colorectal carcinoma.Methods: First, the real-time PCR was used to detect the expression of CD80andCD86mRNA in6pairs of colorectal cancer tissues and adjacent tissues. Then, miRNAchip technology was used to detect miRNAs expression in6pairs of colorectal cancertissues and adjacent tissues. Furthermore, real-time PCR was used to validate theexpression of miRNAs. The dysregulated miRNAs that can bind to the3’-UTR of CD80,CD86, CD28or CTLA-4gene were predicted by bioinformatics softwares. Finally, dualluciferase reporter gene assay system was used to analyze the regulatory role of thepotential miRNAs on the expression of CD80, CD86, CD28and CTLA-4.Results:(1) The results showed that there was no significant difference of theexpression of CD80and CD86mRNA between colorectal cancer tissues and adjacenttissues.(2) There were thirty upregulated miRNAs and eight downregulated miRNAs incolorectal cancer tissues as compared with adjacent tissues. The overexpressed miRNAsmiR-21and miR-34a were predicted to be able to bind with CD803’-UTR. Theoverexpressed miRNAs miR-21, miR-31, miR-34a, and miR-181b may bind with CD863’-UTR. The overexpressed miRNAs miR-31, miR-34a, miR-181b, miR-20b, andmiR-142-3p may bind with CD283’-UTR. The downregulated miRNAs miR-140-3p,miR-143, miR-145, and miR-378may bind with CTLA-43’-UTR.(3) We found that theactivities of luciferase were obviously inhibited by the overexpressed miRNAs miR-21,miR-31, miR-20b, or miR-142-3p by binding with CD80, CD86, or CD283’-UTR,respectively. Moreover, the activities of luciferase were obviously suppressed by thedownregulated miR-378and miR-145by binding with CTLA-43’-UTR.Conclusion: The tumor immune escape is mediated by the upregulated miRNAsmiR-21, miR-31, miR-20b, and miR-142-3p through inhibiting the expression of CD80,CD86, and CD28, and/or by the downregulated miRNAs miR-378and miR-145thatcan suppress the expression of CTLA-4. These findings broadened our understanding of the mechanism of tumor immune escape.Part II, Polymorphisms within microRNA-binding sites inCD80gene and risk of gastric cancerObjective: To study the single nucleotide polymorphisms (SNPs) in the3’-UTR ofCD80gene and the correlation with gastric cancer development.Methods: We collected peripheral blood samples of183patients with gastriccancer and348cases of normal. Firstly, the SNPs in the3’-UTR of the CD80gene wereobtained from dbSNP database. Then, the online softwares were used to predict thepotential miRNA binding-sites in the CD803’-UTR. The SNPs that located in putativemiRNA binding sites and had a minor allele frequency (MAF) of not less than10%were selected as candidate miRSNPs. The candidate miRSNPs were genotyped by usingpolymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)analysis, and the correlation of the miRSNPs with the risk of gastric cancer wasstatistically analyzed.Results:(1) There are21SNPs in the3’-UTR of CD80gene, and the minor allelefrequencies (MAF) of the SNPs rs1599795, rs1599796, rs17281703, rs57271503, andrs7628626are greater than10%. Among them, only the SNP rs1599795located in thebinding sites of three miRNAs and was analyzed in this study.(2) The logisticregression analysis results discovered that the TA genotype (OR=1.44,95%CI=0.98-2.11) and the A allele carriers (p=0.036) were associated with a significantincreased risk of cancer, as compared with the TT genotype.(3) We also found asignificant association between the genotype of SNP rs1599795and theclinicopathological features, including tumor size, tumor location, depth of invasion,lymph node metastasis, distant metastasis, and TNM staging.Conclusion: The miRSNP rs1599795in CD803’-UTR was related to the incidenceof gastric cancer. This SNP was also found to be related to the clinicopathologicalparameters of gastric cancer, suggested that it may be involved in tumor development.These results further support the evidence that CD80plays an extremely important rolein the occurrence and development of gastric cancer. SummaryIn this study, we found that the immune escape of colorectal cancer was mediatedby the upregulated miRNAs miR-21, miR-31, miR-20b, and miR-142-3p throughinhibiting the expression of CD80, CD86, and CD28, and/or by the down-regulatedmiRNAs miR-378and miR-145that can suppress the expression of CTLA-4.Futhermore, the SNP rs1599795in CD803’-UTR was related to the incidence of gastriccancer. This SNP was also found to be related to the clinicopathological parameters ofgastric cancer, suggested that it may be involved in tumor development. Our findingsmay contribute a lot to understanding the role of CD28-B7costimulatory molecules inthe occurrence and development of gastrointestinal cancer.