Microrna146a In Uvb-induced Acute Light Damage In The Mechanism Of Action Study

BackgroundWith the destruction of ozone layer ,the damage of ultraviolet radiation in sunlight, especially for the 290-320nm wavelength of ultraviolet(UVB) is much more serious.Absorption of UVB by the DNA of skin cells causes DNA photodamage, predominantly cyclobutane pyrimidine dimers (CPDs) and (6-4)-photoproducts (6-4PPs). These photoproducts are important inducers of photodamage, and normally they will be repaired or removed by the nucleotide excision repair (NER) pathway. But if they can't be removed in time, tumors will occur.MicroRNAs (miRNAs) are about 22-nucleotides, short, noncoding RNAs that are thought to regulate gene expression through sequence-specific base pairing with the 3'-untranslated region (3'-UTR) of target mRNAs.A large number of literature shows that miRNAs involved many biological processes including development, differentiation, proliferation and apoptosis. They occupy about 5% of human genome, but can regulate 30% genes' expression. Recently, several studies have reported that the dysregulated miRNA expression caused by enviromental carcinogens(such as irradiation).Bioinformatics is one of the core areas of natural science in the 21st century, which is focused on Genomics and Proteomics.Specifically,starting from the nucleic acid and protein sequences, it analyzes biological information of the structure and function in the sequence.Our group previously analyzed the miRNA expression profiles caused by UVB irradiation in the mice skin.Our result shows that miRNA146a was down-regulated by UVB irradiation Significantly.The miR-146 family is composed of two members, miR146a and miR146b that are located on chromosomes 5 and 10 respectively. Evidence showed that miR146a might be involved in the innate immune response. Liu studied bronchial epithelial cells and found that miRNA146a can regulate the apoptosis induced by cytokine,which including IL-1β, IFN-γand TNF-α.The mechanism may be related with STAT3 and gene Bcl-XL.Studies proved that STAT3 expressed unusually in many skin diseases.So, we believe that maybe some adjustment mechanism may existive among miRNA146a, UVB radiation and STAT3 signal transduction pathway.ObjectiveTo verify the expression of miR146a after UVB irradiation, find out some meaningful miR146a downstream target genes by Bioinformatics and focus the relationship between miR146a and JAK/STAT3 after up-regulating miR146a .Methods1. Targetscan and Gostat-analysis were employed to predicate the targets of miR146a , then analyze the function of predicted biological pathways.2. The C57BL/6 mice were selected as the experimental animals. Mice were divided into dose-effect group (30,60,90,180 and 270 mJ/cm2,24h) and time-effect group (1,12,24 and 48h,180mJ/cm2). HE was used to observe the histopathological change; IHC was employed to semi-quantitative STAT3 and p-STAT3; Realtime-PCRwas used to detect the expression of miR146a in different groups.3. HaCaT cells were transfected with miR146a-mimics to up-regulate the expression of miR146a in cells. Different doses of UVB (0, 30, 60, 90mJ/cm2) radiation was used to induce the acute photodamage.Flow cytomery was used to detect the cell apoptosis , MTT ws used to detect the cell activity,Realtime-PCR was employed to detect the miR146a expression, Western Blot was used to detect protein expressin of STAT3 and p-STAT3. Result1. Bioinformatics analysis showed that, miRNA146a involved many biological processes, including protein binding, development process, metabolism of the cell cycle, ect. The target genes of miRNA146a , such as traf6, smad4, are very important genes involved in immune events of photodamage induced by UVB radiation. We conclude that miRNA146a may play an important role in regulating in the UVB-induced acute photodamage.2. In dose-effect group, HE staining showed, the inflammation of mouse skin increase gradually in UVB dose. In time-effect group, the inflammation was the most serious 1h after UVB radiation, and then it reduces gradually.3. The result of IHC showed: with the increasing of dose, the expression of STAT3 increase gradually in UVB dose, which was associated with the intensity of inflammation; in time-effect group, the expression of STAT3 was down-regulated gradually with the reduction of inflammation improved. So was the expression of p-STAT3, but the phenomenon did not exist in normal mouse skin. It was confirmed by correlation analysis in which the expression of miRNA146a and STAT3, p-STAT3 acts as a negative correlation.(r=-0.92,p<0.01; r=-0.981, p<0.01)4. After UVB radiation, the expression of miRNA146a in each group is 0.01158±0.00098,0.01083±0.00104,0.00872±0.00031,0.00851±0.00033,0.00810±0.00057,0.00770±0.00031, which was a negative correlation with the UVB dosage(r=-0.83,p<0.05). In time-effect group, the expression of miRNA146a in each group is 0.00730±0.00036,0.00805±0.00035,0.00810±0.00057,0.00837±0.00039,compared with the control group, the expressionwere all down-regulated(p<0.05).5.With increasing UVB dosage, the expression of protein STAT3 increases gradually, and so dose the p-STAT3 expression. After up-regulating miRNA146a expression after adding miRNA146a-mimics, the expressions of protein STAT3 and p-STAT3 declined, compared with the mimics negative group. We detected the expression of miRNA146a in each group after different UVB dosage radiation, the result showed, the expression of miRNA146a in each dose group declines ,compared with the control group.The results were statistically significant(p<0.01).ConclusionUVB radiation down-regulates the expression of miR146a, but the protein expression of STAT3 and p-STAT3 increases. After up-regulating miR146a, the expression of protein STAT3 and p-STAT3 declines, which suppress, the cell apoptosis induced by UVB radiation. Therefore, by the JAK/STAT3 Signal transduction. miR146a may play a key role in the photodamage induced by UVB radiation.