| BackgroundEsophageal cancer is regarded as one of the most common malignancy worldwide. Compared with other nations, there is higher incidence in China. Esophageal cancer is an extremely fatal disease. In spite of great advancement in cancer treatment, prognosis is still poor.Esophageal cancer is a complex disease involving many genes. Lots of genetic markers for tumorigenesis and progression of esophageal cancer have been identified in the case-control studies. However, the function of lots of the SNP markers hasn’t been illuminated, except the sites located in the promoter or coding regions.MicroRNAs (miRNAs) are endogenous 22nt small non-coding single stranded RNAs that bind with target mRNAs and function as post-transcriptional regulators of gene expression by either promoting mRNA degradation or translational silencing. The critical region for miRNA binding to mRNA is the ’seed region’ (2-7 nucleotides from the 5’ end of the miRNA), which most often binds to a target site in the 3’UTR of the genes by perfect Watsn-Crick complementarities. To date, approximately 10 million SNPs have been identified in the human genome, occurring on average every 100 to 300 base pairs. According to the SNP database, there are many SNP sites located in the 3’untranslated regions. Recent studies have discovered that these SNPs may influence the targeting of microRNA to protein-coding genes.The matrix outside the tumor cells and the sustentacular cells play important role in the tumorigenesis and progression. Matrix Metalloproteinases (MMPs) regulate the micro- environment through degrading the matrix. There are over 20 kinds of MMPs in human, which depend on Zn2+. Almost all of MMPs expressed abnormally in human cancers, especially MMP1, 2, 3, 9. Most of the MMPs are secreted by the tumor cells and the sustentacular cells. Lots of studies have uncovered that overexpression of MMPs correlated with progress, invasion and metastasis of cancer, so MMPs may also act as biomarkers for prognosis.Epidemiological studies have established a link between variations in MMPs genes and risk of cancer. Some SNP sites located in the promoter or coding regions were identified associated with tumorigenesis, metastasis and clinical stages. However, there is no association study focusing on the sequence variants in the 3’UTR of MMPs. In the study, we predicted the binding sits of miRNAs in the 3’UTR of MMPs by using bioinformatics approaches. Then after picking the SNP sites in these binding regions, we evaluated the relationship between the SNPs and risk of esophageal cancer in a case-control study. For the SNPs associated with ESCC, we verified the function of them in later luciferase reporter assay.Objectives1. Predicted the binding sits of miRNAs in the 3’UTR of MMPs by using bioinformatics approaches, picked the SNP sites in these binding regions and evaluated the effects of SNPs.2. Evaluated the associations between the SNPs and risk of esophageal cancer in a case-control study.3. For the SNPs associated with ESCC, we verified the biological functions of them in the cell model.Methods1. Downloaded the 3’ UTR sequences of MMP gene associated with esophageal cancer in NCBI database, used online prediction tools such as Diana, Targetscans, microCosm, miRanda, PicTar, microinspector and PolymiRTS to predict the miRNA combinding site of these sequences. Screened SNP sites in dbSNP database by BLAST-SNP, and used the total free energy (△△G) to evaluate the interaction between these SNPs and microRNA-target gene with RNAhybrid tool. If the minor allele frequency is over 10%, the SNPs could be used in the further study.2. Genotyped the SNPs by using SNPshot method in 444 ESCC patients and matched 468 healthy controls. Non-conditional logistic regression was used to revise the confounding factors such as age, sex and smoking status. We used odd ratio (OR) and 95% confidence interval (95% CI) to evaluate the associations between the SNPs and the risk of ESCC.3. RT-PCR and Western-blot were used to detect the mRNA and protein expression of MMPs in esophageal cancer tissues. The pMIR-REPORT vectors with different alleles of ESCC associated SNP sites were constructed. Then co-transfected the pMIR-REPORT vector and correlative miRNAs mimics/controls to a cell line and detected the expression of luciferase reporter gene. Compared the expression of luciferase reporter with different allele and verified the biological functions of the SNP sites. After selecting cell line with homozygous functional alleles of ESCC-associated SNP sites, transfected mimics/controls of corresponding miRNAs, detected protein expression and compared the two groups.Results1. 13 SNPs were identified in 3’UTR of ESCC relevant MMPs. MMP8 (rs12284255), MMP2 (rs7201) and MMP10 (rs470168) were selected for further study.2. The three SNPs were genotyped by using SNPshot assay in 444 ESCC patients and 468 healthy controls. The genotype distributions of the three SNPs all conformed to H-W equilibrium in controls and were coincident with that of CHB (Chinese Han in Beijing) in HapMap database. However, they were different from that in CEU (Utah residents with Northern and Western European ancestry from the CEPH collection), ASW (African ancestry in Southwest USA) and YRI (Yoruban in Ibadan, Nigeria). Then the association between the three sites and risk/clinic stage of ESCC were evaluated. The results showed that site MMP8 (rs12284255) and MMP2 (rs7201) were both associated with risk of ESCC. The association was more significant under codominant genetic model for the two sites (P=0.001, P<0.001). Genotype AA of site MMP8 (rs12284255) was protective for ESCC (OR: 0.31, 95%CI: 0.15-0.62), however Genotype CC of site MMP2 (rs7201) increased the risk for ESCC (OR: 2.29, 95%CI: 1.44-3.64). When took account of smoking status, the association was lost for both sites in the non-smokers.The haplotype results showed that there was no linkage disequilibrium between site MMP10 (rs470168) and MMP8 (rs12284255) (D=0.423, r2=0.135). So the two sites might be not in a LD block. Further association study also showed there was no association between haplotypes and ESCC.Site MMP8 (rs12284255) and MMP2 (rs7201) were also associated with the clinic stage of ESCC. The frequency of allele A of site MMP8 (rs12284255) was 0.26 in stage I/IIa and 0.17 in stage IIb/III/IV. Genotype CA was associated with early stage of ESCC (OR: 0.48, 95% CI: 0.31-0.74). The frequency of allele C of site MMP2 (rs7201) was 0.32 in stage I/IIa and 0.45 in stage IIb/III/IV. Genotype CA was associated with a later stage of ESCC (OR: 2.85, 95% CI: 1.65-4.23).3. In the part two, the biological function of site MMP2 (rs7201) and MMP8 (rs12284255) were evaluated.(1) Biological function study of MMP2 (rs7201)The mRNA/protein expression of MMP2 in samples with different genotypes of site MMP2 (rs7201) were measured. The results showed that there was no significant difference between three groups on the mRNA expression level. However, the protein expression was higher in genotype CC than genotype CA or AA. The difference between genotype CC and AA was significant statistically (P<0.05). Then selected esophageal carcinoma cell line KYSE150 with low expression of miR-520g for further study. After cotransfecting with miR-520g mimics/controls and pMIR-REPORT-C/pMIR-REPORT-A, the expression of luciferase was obviously low in“miR mimics + pMIR-REPORT-A”group, compared with“miR mimics + pMIR-REPORT- C”group (P<0.05). The results revealed that the affinity of allele A to miR-520g was much higher than allele C.The genotype of site MMP2 (rs7201) for esophageal carcinoma cell line TE-1 was AA. We transfected Mimics/controls of miR-520g to KYSE450 cell respectively and detected the protein expression of MMP2. The results showed the expression of MMP2 decreased obviously after transfection with mimics of miR-520g, however, that remained invariable after transfection with controls.So different alleles of site MMP2 (rs7201) might influence the combination of miR-520g to 3’UTR of MMP2 gene.(2) Biological function study of MMP8 (rs12284255)Detected the mRNA/protein expression of MMP8 in samples with different genotypes of site MMP8 (rs12284255). The results showed that there was no significant difference between three groups on the mRNA expression level. However, the protein expression was higher in genotype CC than genotype CA or AA. The difference between genotype CC and AA was significant statistically (P<0.05).Esophageal carcinoma cell line KYSE150 was picked for further study, because of low expression of miR-495. After cotransfecting with miR-495 mimics/controls and pMIR-REPORT-G/pMIR-REPORT-T, the expression of luciferase was obviously low in“miR mimics + pMIR-REPORT-T”group, compared with“miR mimics + pMIR- REPORT-G”group (P<0.05). The results revealed that tha affinity of allele T (corresponding to C allele of rs12284255 in the sense strand) to miR-495 was much higher than allele G (corresponding to C allele of rs12284255 in the sense strand). So different alleles of site MMP8 (rs12284255) might influence the combination of miR-495 to 3’UTR of MMP8 gene.Conclusions1. For the three SNPs located in 3’UTR of MMPs, we conducted a case-control design in Chinese Han population in Chongqing. These results demonstrated there was statistical significance of MMP8 (rs12284255) and MMP2 (rs7201) polymorphisms in the risk and clinical stage of esophageal squamous cell carcinoma.2. Different alleles of rs7201 could affect the ability of miR-520g combining to the target sites in MMP2. The affinity of A allele was higher than C allele. This polymorphism could participate the pathogenesis of ESCC by affecting this combination.3. Different alleles of MMP8 (rs12284255) could affect the ability of miR-495 combining to the target sites in MMP8. The affinity of T allele (corresponding to A allele of rs12284255 in the sense strand) was higher than G allele (corresponding to C allele of rs12284255 in the sense strand). This polymorphism could also participate the pathogenesis of ESCC.Above all, we discovered a set of SNP sites in the 3’UTR of MMPs genes. After a case-control study in a Chinese Han population in Chongqing, we found two sites MMP8 (rs12284255) and MMP2 (rs7201) were associated with risk and clinic stage of ESCC significantly. The biological functions of the two ESCC-associated SNP sites were studied in the later study. The results might be valuable for early detection of ESCC, however the association needs verification in larger populations or in other ethnic populations. The study might also elucidate possible mechanism of abnormal expression of MMPs in ESCC from a sight from miRNA. |
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