| Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which has done serious harm to the pig industry and also results in significant economic loss. PRRSV is a single-stranded positive RNA virus which has a strong cell tropism of porcine alveolar macrophages (PAM).The frist strain of PRRSV was isolated from PAM. PAM is considered as the major sites of viral replication during acute infection period, and the immune defense cells of the lung that first contact with pathogenic microorganisms.MicroRNA (miRNA) is an endogenous small non-coding RNA, playing the regulation role in posttranscriptional regulation. By binding to the 3’untranslated region (3’-UTR) of target genes, it can inhibit the translation or induce the degradation of target genes. An increasing number of studies have shown that miRNA plays important regulation roles in the development and differentiation of immune cells,the non-specific and specific immunity, and the development of diseases, but the specific mechanism remains to be further studied.In the study, using the PAM from Pietrain and Qingping pigs at 9h (early),36h (middle),60h (later) after infected with the PRRSV as the research object, and uninfected Pietrain and Qingping PAM as control. We constructed 12 miRNA libraries and sequenced them by Solexa high-throughput sequencing technology, and validate the sequencing results by experiments. The results are as follows:(1) 97,729,281 and 83,323,798 of original small RNA sequences are obtained from the Pietrain and Qingping PAM respectively. By removing the connector, pollution sequences and low-quality reads (less than 18nt),94,402,050 and 80,765,398 clean reads were obtained.(2) Through bioinformatic analysis,254 and 251 known porcine miRNAs of were identified in Pietrain and Qingping PAM respectively.298 and 169 new candidate miRNAs were predicted in Pietrain and Qingping PAM respectively.(3) The expression of 117,21,39 miRNA is significant difference at 9h,36h,60h after Pietrain PAM infected with PRRSV compared with the homochronous PAM uninfected with PRRSV. There are 6,11,23 significantly different miRNAs in early, middle and later period after Qingping PAM infected with PRRSV compared with the homochronous PAM uninfected with PRRSV, respectively. The number of different miRNAs achieves its maximum at 9h after Pietrain PAM were infected with the virus, but the number is least at the same time in Qingping PAM, suggesting that these two breeds are obvious difference in immune regulation mechanism against the PRRSV infection.(4) 30 (9h vs 36h),6 (36h vs 60h) significantly different miRNA were found among three periods after infection in Pietrain PAM, and 17(9h vs 36h), 11(36h vs 60h) were found in Qingping PAM. The results indicate that the critical period of virus infection is at 9h to 36h after infection and the larger number of different miRNAs appears during this period.(5) Nine miRNAs are screened out to analyze the expression by real-time fluorescent quantitation PCR. The results verify the accuracy of sequencing in Pietrain and Qingping.Tissue expression profile of three miRNA were analyzed in 11 tissues of Large White pigs.(6) The differential expression analysis of 7 miRNAs in four pig breeds (Landrace, Pietrain, Meishan, Qingping) PAM indicated that the expression of miRNAs existed significant difference in the four pig breeds PAM (uninfected or 9h/36h after infection). In addition, the size of expression variation is different among the four pig breeds PAM infected with/without PRRSV, which suggested that the innate immune was different among different breeds.(7) The target predicted by bioinformatics method was validated by dual luciferase reporter assay, the result showed that the luciferase activity of report protein was significantly decrease (P﹤0.05) after cotransfection with miR-210, which could suggest that MX1 might be the target gene of miR-210. |
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