| Objective Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel Phlebovirus in family Bunyaviridae recently found in China. It’s an urgent need to clarify the mechanism of infection and the pathogenesis, which has important implications for the development of effective prevention and treatment measures. However, research in this area is currently lacking. We try to acquire expression levels of miRNAs in serum samples and establish THP-1 cell infection model in order to identify relationship between miRNAs with viral infection. The research will facilitate in-depth understanding and exploration of SFTS virus pathogenesis, disease progression and immune function and provide a reference for clinical prevention, the development of new antiviral therapies.Methods 1) We first identified differentially expressed microRNAs in serum of infected patients obtained by TaqMan Low Density Array(TLDA) serum miRNA expression profiling, and validated by quantitative RT-PCR (qRT-PCR) followed by analysis and identification of viral infections and related miRNAs host immune response.2) THP-1 cells were established in vitro SFTSV infection model. We examined the expression of significant changes in the expression level of miRNAs and mRNAs. Combined with bioinformatics, we predict differentially expressed miRNAs’functions and try to explore the pathogenesis of viral infection from the changed genes of the virus itself and the roles of viral proteins, host-virus interactions and state of the host ifself. Real-time quantitative PCR was also used for verification of miRNA expression in order to further confirm the virus infection associated micro RNAs.Results 1) Serum TLDA revealed 117 miRNAs were up-regulated and 30 were down-regulated (fold-change>2) compared with healthy controls. There were 22 miRNAs fold change between 2-4,25 between 4-10,27 between 10-40,14 between 40-200 and 32 more than 300 times. There were 22 out of 117 differentially expressed miRNAs in terms of viral infection and immune reponse.20 miRNAs were up-regulated and 2 were down-regulated. Then we use qRT-PCR technique to validate expression levels of 22 selected candidate miRNA in TLDA gene microarray expression profiling. Nine miRNAs were confirmed by qRT-PCR (miR-146a、 miR-15b、miR-24、miR-628-5p、miR-101、miR-126、miR-155、miR-221、miR-34a)2) By means of Affymetrix miRNA array, THP-1 cells as vitro SFTSV infection model, we analyzed significantly altered expression levels of miRNA and mRNA.548 target genes were finally found and 16 differentially expressed miRNAs can regulate target genes. There were 13 miRNAs up-regulated, hsa-miR-149,, hsa-miR-155, hsa-miR-1915, hsa-miR-28-3p, hsa-miR-221, hsa-miR-193b, hsa-miR-146a, hsa-miR-628-5p, hsa-miR-29a, hsa-miR-132, hsa-miR-1908, hsa-miR-638, hsa-miR-1469; 3 were down-regulated, hsa-miR-1307, hsa-miR-345, hsa-miR-320a. These miRNAs target important regulatory factors in TOLL signaling pathway, like TNF、CAT、IL1B、STAT1、CCL5、MAP3K11、 CD81、LITAF、CARD9 and reponse to drug factors SLC1A3、CENPF. Through qRT-PCR, differential expression of miR-146a、miR-155 were examined from virus-infected THP-1 cells. MAS3.0 software was used to analyze the GO/KEGG functional annotation of up/down regulation of miRNA target genes respectively. We found these genes were involved in the regulation of signaling pathways like antigen processing, the host against cytokine interactions, signal transduction and so on. The results prompted these differentially expressed miRNAs may play a significant role in regulating genes of major signaling pathways associated with SFTSV infection.Conclusion Our study showed microRNAs associated with SFTSV infection viral pathogenesis and host-pathogen interactions at serum and cellular level. The study provide a novel insight on host miRNAs in dissecting viral infectious pathogenesis, host response and immune systems in humans and a reference on the development of prevention and control and therapeutic strategies in SFTS and other viral diseases. |
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