Single Nucleotide Polymorphism And Functional Analysis Of Mir-196a2and Vitiligo Susceptibility

Vitiligo is a chronic acquired depigmentation disorder characterized bygeneralized or circumscribed depigmented macules resulting from the loss offunctional melanocytes. The mechanism of melanocyte destruction or apoptosishas not yet been fully elucidated. Theories concerning the cause of vitiligo havefocused on several different mechanisms: autoimmune, neural, oxidative stress,metabolic poison biochemical, and genetic hypotheses, etc. A number ofscholars believe that the development and progression of vitiligo is a result ofmulti-factor and multi-gene interaction, in which genetic variation determinesthe individual suffering from vitiligo susceptibility. Also, oxidative stress causedby hydrogen peroxide （H₂O₂） canlead to cell death and is found associatedwith the pathogenesis of vitiligo.MicroRNAs （miRNAs） are a class of small non-coding RNA consisting of²2nucleotides（nt） in length, which are transcribed first from primary transcipts（pri-miRNAs） then processed precursor sequences with one or more hairpin （pre-miRNAs）. MiRNAs are important gene regulators at the post-transcriptional level by base pairing to complementary sites in the3’untranslated region （UTR） of the target messenger RNA （mRNA）, and leadingto mRNA cleavage or translation repression. It is predicted that approximately60%of all human protein coding genes are regulated by miRNAs. So we knowthat miRNAs are of great importance in diverse physiological anddevelopmental processes.Single nucleotide polymorphisms （SNPs） is the most common geneticvariants in the human genome, which can affect gene expression, transcription,translation, and modification. It is one of the causes of disease susceptibility andleading to differences of drug-reactive in individual and ethnic. Recent evidencehas shown that miRNA mutations are correlate with various human diseases. Ithas also been reported that SNPs in pre-miRNA can affect phenotypes ordevelopment of some diseases. Recent years, the studies are mainlyconcentrated on neoplastic diseases such as lung cancer, breast cancer,hepatocellular carcinoma, and some on non-neoplastic diseases such ascongenital heart disease, coal workers’ pneumoconiosis and other researches.Therefore clearly understanding whether SNPs located in the miRNA areassociated with risk of vitiligo, and finding a vitiligo-related miRNA, discussingwhether the SNP affect on the function of melanocytes, could provide the newtheoretical basis on the mechanism of vitiligo development and clinicaltreatment of vitiligo.Methods:We screened and confirmed the SNPs that MAF>5%in pre-miRNAsthrough finding all miRNA in the public miRBase database and literatures inchinese population. We investigated the miRNA SNP with vitiligo risk of association through molecular epidemiological case-control study andPCR-RFLP experiments. Then we screened the miRNA associated with vitiligorisk, and constructed eukaryotic expression plasmids with different genotypes ofmiRNA molecules, transiently transfected the melanocytes PIG1, observed theeffect of miRNA single nucleotide polymorphism on the cell biology aftertreatment with H₂O₂though detecting the cell viability, apoptosis andultrastructural changes. We predicted the target gene to the miRNA by thebiology prediction software, and verified the relationship between the miRNAand predicted target gene through the luciferase assays. Then we detect themelanosome synthesis, the tyrosinase enzyme activity, and the level ofintracellular reactive oxygen species （ROS） after treatment with H₂O₂inmelanocytes PIG1transfected with different plasmids. Through the abovefunction experiments we can further validate the regulation of the miRNA singlenucleotide polymorphisms on target genes, as well as the impact on themelanocyte damage.Results:We determined four miRNAs from400known SNPs in human pre-miRNA:miR-146a rs2910164，miR-149rs2292832，miR-196a2rs11614913，miR-499rs3746444, indentified the genotypes of the four study sites in the819Vitiligocases and817controls. According to the stratification analyses, we could seethat the CC genotype had a significant decreased risk of vitiligo than those withthe TT/CT genotypes in the stratification analyses, but there were no similiarmain effects for the other3SNPs in the pre-miRNAs. Then the MiR-196a2rs11614913CC genotype with the TT/TC genotypes were stratifiedanalysis.This decreased risk was more pronounced among subgroups of thoseage＞20years, female, Vulgaris Type, had negative family history, with no other autoimmune diseases, had the disease for more than1year.We successfully constructed the expression plasmids pCDNA3.1-miR-196a2-T and the pCDNA3.1-miR-196a2-C. After treatment with hydrogenperoxide （H₂O₂）, the result of CCK-8showed that there was no significantdifference in the vitality of the melanocytes between the pCDNA3.1-miR-196a2-T and pCDNA3.1-miR-196a2-C expression plasmid group. The resultsof Annexin V-FITC/propidium iodide apoptosis assay showed that the earlyapoptosis rate of melanocytes transfected with the pCDNA3.1-miR-196a2-Tand pCDNA3.1-miR-196a2-C expression plasmid group was significantlylower （P <0.05）, and compared to pCDNA3.1-miR-196a2-T group, the earlyapoptosis rate of pCDNA3.1-miR-196a2-C group was also significantlyreduced （P<0.05）. In addition, as observed by transmission electron microscope,compared with the normal PIG1group, the main changes of apoptotic cellsappeared in nucleus of PIG1melanocytes with control plasmid pCDNA3.1, andthere was little change of apoptotic in the pCDNA3.1-miR-196a2-T group,whereas the ultrastructure characteristics of PIG1melanocytes with pCDNA3.1-miR-196a2-C had no significant change.We predicted target genes in the public database miRBase, in which TRP-1score is the highest and may has relationship with oxidative stress in vitiligo.Then TRP-1was used as the target gene in the study of miRNA-196a2SNP. WeSuccessfully constructed the reporter plasmid psiCHECK2-TRP-1. The resultsof luciferase activity showed that both the miR-196a2-T-and miR-196a2-Ccould combine with target gene TRP-1, thereby inhibiting the expression of theluciferase, and compared with the miR-196a2-T, the miR-196a2-C was easier tocombine with target gene TRP-1（P<0.05）. Compared with the pCDNA3.1group, the melanin synthesis of pCDNA3.1-miR-196a2-C group was significantly reduced （P<0.01）. Meanwhile, the level of ROS in the melanocytetranstected with pCDNA3.1-miR-196a2-C after treatment with H₂O₂was alsosignificantly reduced （P <0.05）.Conclusion:1.The CC genotype had a significant decreased risk of vitiligo than thosewith the TT/CT genotypes in the stratification analyses, miR-196a2SNPrs11614913T>C could decrease the risk of vitiligo.2.MiR-196a2SNP rs11614913T>C could decrease the early apoptosis rateof melanocytes, and protected the apoptosis of melanocytes after treatment withH₂O₂.3.TRP-1was the target gene of miR-196a2, and the miR-196a2-T waseasier bind to it than the miR-196a2-C. MiR-196a2SNP rs11614913T>C couldprotect the melonocytes against injuries though binding to the target gene TRP-1and decreasing the level of ROS after treatment with H₂O₂.