| Mycoplasma gallisepticum(MG) is the one of the most important pathogenic microorganism in avain Mycoplasmas, which causes Chronic Respiratory Disease (CRD) in chickens. The disease is widely distributed in all over countries, causing serious economic loss for the poultry industry.microRNA is an abundant class of highly conserved non-coding, small(18-25 nucleotides) RNAs, posttranscriptional regulation of gene expression via identifying sequence-specific of the targets. Host miRNAs is differential abundance following pathogen infections, which varies at different stages of infection and type of pathogen involved. Therefore, it is important to study the change of miRNAs and the targets function of the host before and after the pathogen infection. It not only helps us reveal the pathogenesis of disease, but also provide theoretical basis for the gene therapy.It was reported that gga-miR-99a and gga-miR-146c paly a key role in regulating inflammatory response. In addition, our lab found that the expression of gga-miR-99a is significantly down-regulated and gga-miR-146c is significantly up-regulated by infecting chicken embryos with MG and deep sequencing.This research detected the expression of gga-miR-99a and miR-146c in different stages of infection lung tissues through Q-PCR. Then, their target genes were analyzed by bioinformatics and verified by experiments. We also studied the targets function by over-expression and the RNA interference. It clarified the regulation mechanism of gga-miR-99a and miR-146c after infected MG. The main results are as follows:(1) We detected the expression of gga-miR-99a in different stages of infection lung tissues via q-PCR. The expression of gga-miR-99a is extremely significant increased(p<0.01) in 13d,14d,16d,17d and that is extremely significant decreased(p<0.01) in 12d,15d,19d,20d.(2) The targets of gga-miR-99a was predicted by TargetScan and miRDB, SMARCA5 was selected as the regulate target of gga-miR-99a.In addition, the target predicted by bioinformatics method was validated by dual lucif erase reporter assay, the result showed that compared with the NC group, the mutant group and the blank group, the luciferase activity of report gene was extremely significant decreased(p<0.01). Mean while, gga-miR-99a was over-expressed in DF-1 cell, the relative expression of SMARCA5 is extremely significant decreased(p<0.01) comparing with the NC group and gga-miR-99a was suppressed in DF-1 cell, the relative expression of SMARCA5 is extremely significant increased(p<0.01) comparing with the NC group. The relative expression of SMARCA5 in the infection lung tissue of chicken embryos was extremely significant increased(p<0.01) at 12d, and it was extremely significant increased decreased(p<0.01) at 16d, the expression trend was opposite to that of gga-miR-99a at the same period. The above results showed that SMARCA5 is the target of gga-miR-99a.(3) gga-miR-99a was transfection into DF-1 cells, the cell proliferation of DF-1 was no changed after 24h,48h, but 72h later, the cell proliferation of DF-1 was significantly decreased(p<0.05), which indicated that gga-miR-99a could significantly suppresse the proliferation of DF-1.At the same time, we detected the cell cycle of DF-1 after transfecting gga-miR-99a, the G1 phase was significantly increased(P<0.05) and the G2 phase was no changed(p>0.05). Because the ratio of cell in Gl phase was increased, the cell could not get into S phase(synthetic phase), so the cell proliferation of DF-1 was suppressed.(4) We detected the expression of gga-miR-146c in different stages of infection lung tissues via Q-PCR. The expression of gga-miR-146c is extremely significant decreased(p<0.01) in 13d,14d and that is extremely significant increased(p<0.01) in 18d, 19d,20d and which is significantly increased(p<0.05) in 16d,17d.(5) The targets of gga-miR-146c was predicted by TargetScan and miRDB, MMP16 and TRAF7 were selected as the regulate targets of gga-miR146c.In addition, the targets predicted by bioinformatics method were validated by dual luciferase reporter assay, the result showed that compared with the NC group, the mutant group and the blank group, the luciferase activity of report gene was extremely significant decreased(p<0.01). Meanwhile, when gga-miR-146c was over-expressed in DF-1 cell, the relative expression of MMP16 and TRAF7 were extremely significant decreased(p<0.01) comparing with the NC group and after gga-miR-146c was suppressed in DF-1 cell, the relative expression of MMP16 and TRAF7 were extremely significant increased(p<0.01) comparing with the NC group. The above results showed that MMP16 and TRAF7 were the targets of gga-miR-146c.(6) gga-miR-146c was over-expressed in DF-1, the relative expression of TLR6, TLR2, MyD88, TNF-a were extremely significantly increased(p<0.01), the relative expression of Apoal was no significantly changed(p>0.05). Otherwise, gga-miR-146c was suppressed in DF-1, the relative expression of Apoal was extremely significantly increased(p<0.01), the relative expression of TLR6, TLR2, MyD88, TNF-a were no significantly changed(p>0.05). |
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