Screening Of UVB-related MicroRNAs And Preliminary Functional Analysis

Rencently, a class of non-coding RNAs called microRNAs (miRNAs), play an an important role in monitoring development phase changes of individual, in regulation of specific cell proliferation, differentiation and other aspects of the process. The role of miRNAs in UVB induced damages and diseases are gradually discovered by scientists.UVB relative miRNAs are found by miRNAs array, which is a start for studying UVB sensentive miRNAs. Therefore, targets genes of miRNAs were verified by bioinformatics and experiments. Furthermore, we analysed the role of miRNAs in epidermal carcinoma.The results provide the theory and experiment basis for the study of the UV-induced damage in the molecular mechanism and the role of miRNA in epidermal carcinoma.Aim:To investigate the differentially expressed miRNAs and their target genes in NIH3T3 cells after UVB irradiation, which are useful for further studies on functions of miRNAs in UVB induced signal transduction pathway. Furthermore, to comfirm the target genes of hsa-miR-365 from structure, preparing for the study on function, to study the expression level of hsa-miR-365 in epidermal carcinoma and investigate the role of hsa-miR-365 inhibitor in A431 cells in vitro, which established the relationship between miR-365 and epidermal carcinoma and pointed out a new direction for studying epidermal carcinoma.Methods:MTT, Flow Cytometry and AO/EB staining were used to detect cell morphological changes in NIH3T3. MicroRNA microarray was used to examine the differentially expressed miRNAs in NIH3T3 cells after UVB irradiation, and the discovered miRNAs were confirmed by real time RT-PCR assay. Targets predictions were performed by Microcosm and TargetScan and Potential target genes of these miRNAs were classified into different function categories with the GOstat software (http://gostat.wehi.edu.au/cgi-bin/goStat.pl).Hsa-miR-365 target genes were predicted by availble data TargetScan. After hsa-miR-365 inhibitor were transfected A431 cells by Lipofectamine2000, Real-time qPCR, western blot were used to test the expression level at RNA and protein level. And Luciferase activty assay were performed to confirm the targets of hsa-miR-365 after hsa-miR-365 tansferred A431 cells.Hsa-miR-365 expression level was test by real time RT-PCR assay in A431 cells. Corning Transwell Inserts and Boyden chamber were used to test the migration and invasion of A431 cells respectively. Meanwhile, pate clone forming assay and flow cytometry were used to examed the cell proliferation and apoptosis.Results:the results of MTT showed that there was a great change in 50J/m2 UVB dose; when NIH3T3 cells were continued to culture 2h,4h,6h and 12h after 50J/m2 UVB irradiation, the change of survival rate was rapid in these piont. Morever, the results of flow cytometry showed there was an G1 arrest and an apoptosis peak at 12h after 50J/m2 UVB irradiation. AO/EB straining showed that typital apoptosis at 12h after 50J/m2 UVB irradiation. In some cells stained with EB, the nuclei exhibit bright condensed chromatin or fragmented chromatin, some cells showed apoptotic bleb phenomenon.MicroRNA microarray showed that 30 miRNAs were differentially expressed, accounting for 11%, in which 27 up-regulated,3 down-regulated. Interestingly, the express level of mmu-miR-365 and mmu-miR-21 were more than six times, while mmu-miR-465 showed low levels of expression at all time points. The expression levels of mmu-miR-let-7a, mmu-miR-24, mmu-miR-376b and mmu-miR-21 were accordant with the results from real time RT-PCR. Single pathway analysis showed that miR-24 involed in MAPK signaling pathway, miR-21 involved in Jak-STAT signaling pathway.To predict the target genes of miR-365 using TargetScan and other softwares, combined with GO analysis, we decided to confirmed the targets genes NFIB,BCL2 and CDK6 by experiment methods. After hsa-miR-365 inhibitors were transfected to A431 cells, we found that there was no change in the three target genes at RNA level, but their protein level increased compared with the control and negative control. Luciferase activty assay showed that there was a significant difference in the luciferase activty assay with miR-365 inhibitors among the three groups (p<0.05).The expression level of hsa-miR-365 in A431 cells is more than 15 times as high as control.The results of vitro invasion assay showed that A431 cells with hsa-miR-356 inhibitors had significantly reduced in invasiveness as compared with control and negative control(F=111.709, P＜0.001). Furthermore, A431 cells with hsa-miR-356 inhibitor also caused a significant decrease of motility compared with control and negative control by use of Corning Transwell Inserts(F=41.732, P<0.001). All of these results showed low expression of miR-365 partially led to a downregulated migration and invasion of A431 cells. In addition, the ability to form colonies in plate of A431 cells with hsa-miR-365 inhibitors was significant reduced compareing that in control and negative control (F=30.077, P<0.001). However, the cell cycle distribution detected by flow cytometry is not significantly different among each other. These results revealed lower expression of miR-365 partially suppress proliferatin of A431 cells (P> 0.05).Conclusions:Our research reveals the radiation between UVB radiation and miRNAs. We found the UVB sensentive miRNAs, which provides a basis for studying the regulation of miRNA expression in UVB radiation. Furthermore, we confirmed that NFIB is a direct target of miR-365, repressing the NFIB protein expression, not NFIB RNA. Hsa-miR-365 highly expressed in epidermal cancer cells A431 and hsa-miR-365 inhibitors significantly reduce skin cancer cell migration and invasion in vitro, laying a foundation for studying the function of skin cancer.