MicroRNAs Screening And Regulatory Network Analysis For Prrs Resistance In Tongcheng Pigs

Porcine reproductive and respiratory syndrome(Porcine Reproductive and Respiratory Syndrome, PRRS), commonly known as blue ear disease, to the popular wide geographical spread fast, and high prevalence of one of the world’s major swine diseases, caused mainly pregnant sows reproductive failure and cause of all ages especially piglets pig respiratory diseases. PRRS is caused by porcine reproductive and respiratory syndrome virus(Porcine Reproductive and Respiratory Syndrome Virus, PRRSV), PRRSV is a single-strand RNA virus, with a volatility of the host and can cause immune suppression, to carry out the veterinary clinic control the disease has caused great difficulties, there is no effective prevention and control approach. Tongcheng is the excellent local varieties with strong resistance, fresh meat, etc., during the HP-PRRS outbreak, located in Tongcheng County of Hubei Province territory but no case is due to infection and HP-PRRS Death(Our previous observations), it is speculated Tongcheng on HP-PRRS has some resistance. Micro RNA(mi RNA) as a class of endogenous genes encoding a length of about 20-24 nucleotides noncoding single-stranded RNA molecules in prokaryotes and widely distributed in the mammalian body generally involved in a variety of life processes Regulation, has been reported mi R-181, mi R-125 b, etc. can inhibit the proliferation in a host of PRRSV, PRRSV infection in the body if there is a large number of mi RNAs involved in antiviral yet it somehow. In this study, Tongcheng pigs and Large White as materials after artificial infection before infection PRRSV and alveolar macrophages(Porcine Alveolar Macrophages, PAMs) high-throughput small RNA sequencing, both before and after the screening of cultivars and varieties infect PAMs differentially expressed mi RNAs and analyze the regulatory networks, laying the foundation for further screening Tongcheng pigs against PRRSV of mi RNAs. The main contents and results of this study are as follows:(1) after artificial infection PRRSV, lung disease and inflammation Tongcheng pigs than the Large White Light, lung viral load below Yorkshire.And the use of 5-week-old PRRSV, PCV, PRV three viruses are negative healthy weaned Tongcheng 22 and Large White 12, according to sibling uniformly and randomly divided into infection group and control group, after the pre-test manual infection, test pigs intramuscularly PRRSV-Wu H3 strains, the control group pig intramuscular DMEM; necropsy on day 7 after infection, take the lungs and other tissues and organs, one side of each lung PBS bronchial lavage techniques to collect use PAMs, total RNA was extracted for the high-throughput sequencing of small RNA, the other side of the lung tissue samples after collection with 4% paraformaldehyde, used to make pathological and immunohistochemical analysis. Clinical observations, six pigs within 2-5 days of infection has appeared depression, feeding, vomiting, breathing, high temperature, fever and other symptoms, Yorkshire infection than Tongcheng body temperature more illness and more obvious; observed gross anatomy lung disease, the group found to be infected lung edema Yorkshire, the surface a lot of bleeding, lung consolidation appears dark red area, extensive lymph nodes, lungs swelling Tongcheng, consolidation degree than Large white light pink elastic, spongy. After collecting samples of lung tissue with paraffin sections stained by HE after inverted microscope and found through the city and Large White pigs infected with PRRSV, lung lesions appear both interstitial pneumonia, bronchioles and perivascular mononuclear macrophage infiltration, most significant thickening of the alveolar wall, alveolar narrow cavity mainly necrotic cells, there is a homogeneous slurry of red dye and macrophages, compare the severity of the histological lesions, compared with Large White Tongcheng more serious. The control group through the city and Large White pigs lung tissue structure clear, no abnormal changes. With horseradish peroxidase-labeled goat anti-mouse Ig G antibody PRRSV to observe the distribution of PRRSV antigen in a lung by immunohistochemistry, in exactly the same under the microscope image acquisition conditions, each slice in 40 randomly selected magnification At least five horizons, software IPP6.0 quantitative analysis, observed that the reaction product of PRRSV antibodies in lung infection in pigs mainly in alveolar macrophages, alveolar type II epithelial cells, lymphocytes and exfoliated cells, in the same species inside, relative to the control group, the group of pigs infected with PRRSV lungs clear positive signal distribution, while in the infected group, Tongcheng pig lungs PRRSV distribution is less than the Large White.(2) Tongcheng pigs and Large White 12 PAM samples infected with PRRSV RNA before and after the small group of high-throughput sequencing, each sample to obtain at least 0.5G of data, to obtain at least 10,327,597 of the original sequence alignments have to give 391 mature mi RNA known pig, 67 differentially expressed in different combinations.PAM select 12 pigs as sequencing sample, total RNA was extracted after 12 c DNA library was constructed, small group of high-throughput RNA sequencing on Illumina Hi Seq2000 platform, the raw data were obtained for each sample in more than 0.5G, at least get 10327597 original sequence, the number of pure filtered sequence reads obtained account for more than 95% of the original sequence of the total number of reads, about 20 bp nucleotide error rate are less than 0.02%. Mi RDeep2 will compare the use of sequencing and Gene Bank sequences in the pig genome sequence and mi RBase mature mi RNA sequences found in 12 samples averaged 391 have been reported to mature mi RNA pigs. Combinations of two T test screened out 67 differentially expressed mi RNAs, screening criteria for the T-test P value ≤0.05, fold change> 1 is raised, fold change <1 is lowered, which Tongcheng pigs infected with the control group( TC-INJ-CON) mi RNA expression significantly down compared with four(mi R-101, mi R-146 b, mi R-296-3p and mi R-664-5p), and increase the expression of mi RNA has five(mi R-22-3p, mi R-24-2-5p, mi R-27b-5p, mi R-423-5p and mi R-7137-5p); and Large White infection group and control group(LW-INJ-CON) compared to significantly downregulated There are 36 of mi RNA, mi RNA upregulated 20; Tongcheng Large White pigs infected and infected group(TC-INF-LW) compared with 5 and 3 downregulated mi RNA upregulated mi RNA; Tongcheng in the control group and the control group Large White(TC-CON-LW) compared with four down and three up-regulated mi RNA mi RNA. Among them, 12 mi RNAs appear in multiple combinations, such as mi R-101 down-regulated in the TC-INF-CON, and regulated in the LW-INF-CON and the TC-CON-LW, mi R-296-3p and mi R146 b in TC-INF-CON two portfolios and LW-INF-CON both down, and mi R-423-5p and mi R7137-5p in TC-INF-CON two portfolios and LW-INF-CON is regulated.(3) forecast to 67 differentially expressed mi RNAs target host genes 22,234 on binding targets PRRSV genome has 119, host to a number of target genes enriched GO Terms and KEGG pathway, and the formation of mi RNA-m RNA regulatory networks.Prediction using software Target Scan Human6.2 differentially expressed mi RNAs target gene in the pig genome, obtained 22243 m RNA-mi RNA interaction sites using software mi Randa predicted mi RNA differentially expressed in the PRRSV genome has 119 combined targets. Tongcheng infection control group was expressing mi RNAs target group has 4474, enriched to 801 GO Terms and 50 KEGG pathway, Yorkshire infection group and control group of differentially expressed mi RNAs target group has 12,856, the rich Set to 901 GO Terms and 57 KEGG pathway. A total of 42 in the two paths, there are 12 and the signal transduction pathways and 18-related diseases such as cancer pathways, as well as macrophage receptor FcγR-mediated phagocytosis pathway, suggesting that this pathway may host antiviral innate immunity or PRRSV invasion mechanisms. Combined with the group transcriptome sequencing data analysis results(see separate article Liang Wan doctoral thesis), the differential expression of mi RNA Tongcheng pigs and m RNA analysis, we found that eight differentially expressed mi RNAs can target 143 differentially expressed genes, four downregulated mi RNA upregulated genes corresponding to 37, four up-regulated mi RNA corresponding to 38 down-regulated genes, the 143 differentially expressed genes using online software DAVID as GO and KEGG analysis, enriched to 51 biological processes(BP), 17 types of cell components(CC) and 10 kinds of biological function(MF) classification, to participate in the p53 signaling pathway, JAK-STAT signaling pathway, the cancer pathways, cytokine and cytokine receptor interaction in using Cytoscape software production mi RNA- m RNA regulatory protein networks, we found mi R-101 and mi R-25b-5p, etc. for multiple target groups have regulation.