The Genetic Variant In The Seed Region Of Hsa-mir-499-3p (rs3746444 A>g) Is Associated With The Risk Of Human Lung Cancers:a Case-control Study

BackgroundLung cancer is now threatening worldwide. The death rate of lung cancer in America is higher than any other type of cancer in 2009, while the cancer incidence is climbing up quickly in developing countries including China. For example, the death rate of lung cancer in Guangzhou, China were 11.61/105 from 1970 to 1972, then reached 32.67/105 from 1980 to 1982, afterward raised to 41.54/105 from 1990 to 1992, and lately were 48.79/105 from 2000 to 2002, which are shocking data that urge scientists to reveal the etiology of lung cancer. It has been reported that 80% lung cancer patients are link to tobacco smoke, however, merely 10% of smokers is end in lung cancer. Therefore, not only environmental factors but also genetic impact should take into consideration while investigating the etiology of lung cancer. Though there are elucidations of genetic susceptibility on the occurence of lung cancer, it still calls for more effort to better the understanding.MicroRNAs (miRNAs) are small non-coding RNAs, functioning as gene regulators through binding to the 3'-UTR (untranslated region, UTR) of their targets. It's reported that miRNAs involve in the cellular carcinogenesis, taking examples as follows. One paper stated that hsa-mir-182 involved in the regulation of RGS17 expression through two conserved sites located in its 3' UTR region. Consistently, endogenous RGS17 expression level is regulated by hsa-mir-182 in human lung cancer cell lines. While hsa-mir-21 is a well known miRNA associated with cancer risk, recently evidences supported that hsa-mir-21 targeted 3'-UTR of PTEN in non-small cell lung cancer (NSCLC) cell lines, post-transcriptionally down-regulates the expression of tumor suppressor PTEN. Nevertheless, another research group found that, with NSCLC tissues, a mutation in the 3'-UTR of KRAS, which is let-7 complementary sites 6 (LCS6), might alter the binding of let-7 towards KRAS so that the genetic susceptibility of NSCLC is increased. The frequency of the variant allele is 5.8%, deriving from 46 human populations, so that the mutation is defined as a SNP. Lately the association between the SNP and the risk for NSCLC was defined especially for modest-smokers from New Mexico or Boston, which were the population in two independent case-control studies. Many other experiments confirmed that SNPs in the 3'-UTR of target genes also contribute to lung cancer risk.Whether the SNPs in miRNA sequence will affect cancer risk and how the SNPs interfere the regulation of miRNA have become a new topic in SNP research. Through systematically screening sequence variations in several hundred human miRNAs from >100 human tumor tissues and 20 cancer cell lines, researchers identified a series of mutations (or very rare SNPs), that specifically present in human cancers. Consecutively they found that a G>A mutation of let-7e led to a significant reduction of its expression, which locates at 19 nt downstream of pre-miRNA let-7e. The SNP could block the processing of pre-miRNA to mature miRNA and then contribute to tumorigenesis. In America, 80 patients with familial ovarian cancer were screened for genetic variants in thirty selected miRNA genes, which are predicted to regulate key ovarian cancer genes and are reported to be misexpressed in ovarian tumor tissues. Seven novel genetic variants were observed in four primary or precursor miRNA genes. Among them, one variant located in the precursor of hsa-miR-191, which locates next to the mature hsa-miR-191 at downstream, resulted in conformational changes in the predicted secondary structures and the maximal free energy of target binding were changed too, consequently altered the expression of mature hsa-miR-191. In further analysis, they found that the particular variant exists in another 4 family members, while 2 of them were again diagnosed as ovarian cancer patients, therefore a C>A might be associated with ovarian cancer heredity susceptibility.2-8 consecutive nucleotides in the 5'end of miRNA is denominated seed region, which plays key role in the miRNA processing, maturation, target recognition. Completely Watson-Crick base pair is necessary while miRNAs binding towards target genes. Now there are no more than 20 papers concerning relationship between SNP in miRNAs and cancers, moreover, in our bioinformatic prediction also confirm that rare SNPs locate in seed region even mature sequence of miRNAs. All these may indicate that the conservation of seed region is essential to living body, and a SNP locate in the seed region of miRNA might result in more severe consequence than in pri-miRNA or pre-miRNA. Such as SNP in the seed region of hsa-mir-125, which locates next to the downstream of mature hsa-mir-125, significantly influences the processing of primary miRNA (pri-miRNA) to pre-miRNA, then the expression of mature miRNA is reduced, finally translational suppressing mediated by hsa-mir-125 is blocked, Lin-28 reported to be link with breast cancer is over-accumulated, which is a putative reason that hsa-mir-125 contributes to the pathogenesis of breast cancer. A SNP in seed region of hsa-miR-499-3p (rs3746444 A>G) will correspondingly produce 3 types of genotype, which distribute significantly different between cases and controls of Nanjing, China, and researchers infer that the SNP alter function of hsa-miR-499-3p then cause cancer.Search the bioinformatics database, there are 4 SNPs in the mature miRNA with Minor allele frequency (MAF) being more than 0.05, which are hsa-miR-499-3p, hsa-miR-608, hsa-miR-196a2 and hsa-miR-146a respectively, including the rs3746444 (A>G) SNP is in the seed region of hsa-miR-499-3p. Furthermore, target prediction uncovers that hsa-miR-499-3p might target the 3'-UTR of NBS1, which is a DNA damage repair gene we are interested in and get foundation from National Natural Science Fundation of China. In summary, the study is to investigate the association between the rs3746444 SNP and lung cancer of southern Chinese populationObjectThis study is to find the association between the rs3746444 (A>G) SNP and lung cancer of southern Chinese population. Through meta-analysis, systematically discussion on the relationship between the SNP and cancers is made. With bioinformatic prediction, try to explain the reasons for the association between the genetic variation and cancer risk. To provide biomarker for lung cancer prevention, diagnose and therapy, furthermore, it could be a reference for other cancer research.Methods1. With a case-control model, we recruited 526 lung cancer cases from Guangzhou and the surrounding cities. Matching by age (±5 years), sex, without blood relationship with lung cancer cases, 526 cancer-free controls were randomly selected from 10,000 residents of Guangzhou. By bioinformatic predictting and references analyzing, we select the rs3746444 (A>G) SNP. Using PCR-RFLP to genotype rs3746444, employ SAS 9.13 to analyze characteristic of population, single risk factors selection, the association between the SNP and lung cancer risk, as well as the gene-environment interaction in unlogistical regression model.2. Through meta-analysis, the combined effect could help to systematically evaluate relationship between the SNP and cancers.3. With bioinformatic prediction, we try to explain the reasons for the association between the rs3746444 genetic variation and cancer risk.Results1. This study included 526 lung cancer patients and 526 cancer-free controls. Among the 526 cases, there were 195 (37.1%) cases of adenocarcinoma, 180(34.2%) of squamous cell carcinoma, 21(4.0%) of large cell carcinoma, 65(12.4%) of small cell lung cancer, and 65(12.4%) of mixed-cell or undifferentiated carcinoma. According to the IASLC staging classifications, there were 73(13.9%) cases of stage I, 50(9.5%) of stage II, 173(32.9%) of stage III, and 230(43.7%) of stage IV. The SNP of rs3746444 produce 3 types of genotype (AA,AG and GG), which distribute significantly different between cases and controls, with P<0.0001. For the cases and cancer-free controls, the differences in distributions of age, sex were not statistically significant (p=0.084 and p=0.322 respectively). The cases were more likely to be smokers (p=0.014), but no difference appear among drinkers and non-drinkers, people have family history of cancer or not (p=0.178 and p=0.174 respectively). Compared with the AA genotype, AG heterozygote marginally higher the risk of lung cancer (adjusted odds ratios [OR] =1.32, 95%CI =1.01-1.81), while the GG homozygote had a 1.22-fold increased risk of lung cancer (OR=2.22, 95%CI=1.52-3.23). There was a significant trend for an allele dose effect on risk of lung cancer (P-trend=3.04×10-5). Stratification analysis show that the increase risk of lung cancer is obvious in population older than age 60 (OR=2.36, 95%CI=1.60-3.49); female (OR=2.49, 95%CI=1.51-4.12); never smokers (OR=2.12, 95%CI=1.43-3.15); never drinkers (OR=1.73, 95%CI=1.28-2.34); with family history of cancer (OR=1.57, 95%CI=1.19-2.07).2. Through meta-analysis of 5 papers, individuals carrying the rs3746444 G allele comparing with A allele, that is (AG+GG) vs. AA, will increase cancers risk (OR=1.15,95%CI=1.05-1.26, P=0.002).3. With bioinformatic prediction, the maximal free energy of mutated G allele is lower than the A allele while hsa-miR-499-3p targeting toward ADAMTS9 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9) which is reported to be a tumor suppressor previously, with the maximal free energy (mfe) -31.1 kcal/mol for G allele while -26.4 kcal/mol for A allele. The lower is the mfe, the more stable is the target binding so that the ADAMTS9 is to be cleavaged more easily. Furthermore, by bioinformatic analysis, we found that the expression of ADAMTS9 in breast cancer tissue is lower than in their normal counterparts, and the expression of ADAMTS9 is also decreased in lung cell lines after exposure to asbestos. However, the mutation located in the seed sequence of hsa-miR-499-3p contributes to the alteration of the expression of ADAMTS9 is still to see.ConclusionThe genetic variant in the seed region of hsa-miR-499-3p (rs3746444 A>G) could increase the risk of lung cancer, and the SNP is a potential genetic biomarker for lung cancer prevention.