The Study On MiRNAs’ Regulation Function And Diagnostic Efficiency During Virus Infectious Diseases

Infectious diseases can be caused by a variety of pathogens, including viruses, bacteria, fungi, protozoa, and worms. They are easy to be widely and rapidly spread in a large geographical area, and it is difficult to prevent and treat for them, especially viral diseases, such as the Ebola virus, the influenza virus H7N9, hepatitis B virus (HBV) and hepatitis C virus (HCV), causing serious consequences for the society and the country. Therefore, rapid and accurate screening for virus infectious diseases have been urgently needed and also become a hot topic for the prevention and treatment due to their easy spread and the lack of effective therapeutic measures. So far, a lot of diagnostic techniques for virus infection have been developed based on viruses, nucleic acid and proteins, mainly including virus separation and the detection for viral nucleic acid and some biomarkers, respectively. However, the current biomarkers for virus detection cannot be satisfying and it is significantly meaningful to seek for new biomarkers.MicroRNAs (miRNAs) are a class of small noncoding RNAs (about 22 nucleotides, nt), commonly found in eukaryotic cells. At present, as the experiment technologies are developed and detailed researches of miRNAs are carried out, more than 28000 miRNAs have been found, of which there are more than 2500 human encoded mature miRNAs. miRNAs play critical roles in a variety of physiological and pathological processes including cell growth, development and apoptosis, tumorigenesis and virus infection by regulating gene expression or post-transcriptional translation, which work by perfect or imperfect base pairing between short seed regions of miRNAs and 3’UTR of target mRNAs. In recent years, it has been well documented that miRNAs can directly target virus genome to regulate the replication and pathogenesis of viruses. At the same time, their own expression has been confirmed to have strain specificity and certain time tropism, which makes them possible to serve as biomarkers and to be applied for the diagnosis and treatment of virus infection diseases.We aim to make a study on the specific expression of miRNAs to find out new biomarkers during H7N9 and HCV infection, and then make a pilot analysis of their regulation function and diagnostic efficiency. Ultimately, we hope to achieve rapid screening for infectious diseases by establishing a miRNA panel based on the SPR(Surface Plasmon Resonance). As so far, it is still not very clear about the pathogenesis and related regulation during H7N9 infection. We studied the pathogenicity of hemagglutinin (HA) for its vital roles in Influenza A virus infection, further observed the Let-7e expression at a cellular level and analyzed its possible regulation function. In addition, according to the principle that miRNAs can directly target the virus genome to regulate the virus infection, the HA gene segment was taken for an example and the optical human encoded miRNAs were predicted by bioinformatics in specific organization. These candidate miRNAs may be more reliable targets for laboratory researches. Besides, the expression profile of serum miRNAs in patients with HCV viremia was analyzed and the diagnostic efficiency was evaluated, which may provide a basic reference for studies on the process of HCV infection.This study consists of the following three chapters:First Chapter The pathological response induced by the avian influenza virus A/Anhui/(H7N9) hemagglutinin protein and miRNA-mediated homeostasis responseObjective To study the pathological process induced by the functional protein HA/H7N9, and further analyze the Let-7e expression and it’s regulation function at a cellular level.Methods On one hand, at animal level, the pathology of HA was explored. Female Balb/c mice with the age of six weeks were purchased from the laboratory animal center of Guangdong, China, and then were randomly divided into three groups (6 animals/group):BSA, HA, and HA combined with incomplete adjuvant. Mice were subcutaneously injected at week 0 and week 3 with BSA, HA 100ng, and HA 10ng combined with adjuvant 0.2ml. The weight of the mice body, spleen and thymus were collected 2 weeks after the second immunization. In addition, lymphocyte proliferation assay was carried out with CCK8 to further explore the immune response induced by HA.On the other hand, at cellular level, the inflammatory response induced by HA was studied and the regulation function of Let-7e was analyzed. THP-1 is a kind of immunocytes that mainly play roles in chemotactic and phagocytic process. First of all, HA, at a final concentration of 100ng/ml, was added into THP-1 cells which were seeded in six well plates and cells and the medium were collected at Oh,2h,4h, 8h,12h,24h. Secondly, different concentration gradient of HA (Ong/ml, lOng/ml, 100ng/ml,200ng/ml) was added similarly and cells and the medium were also collected after 4h. Furthermore, the synergistic effect of HA and lipopolysaccharide (LPS) on inflammatory response was explored in THP-1. Finally, the roles of toll-like receptors in the inflammatory response induced by HA were analyzed through transfection THP-1 with siRNA to silence the expression of TLR4. All RNA samples were stored at -70℃. RT-qPCR was performed to detect the expression of inflammatory cytokines including IL1a, IL1b, IL6 and TNFa and the level of Let-7e in cells and the medium. Statistical analysis was conducted by using SPSS 13.0.Result HA significantly increased the thymus coefficient and spleen coefficient of the mice in vivo and also promoted lymphocyte proliferation in vitro through direct stimulation. Besides, the expression of ILla and IL6 was significantly up-regulated by HA in THP-1 at various concentration and different time points. A weak trend of up-regulation was observed for ILlb, but no obvious change for TNFa. Moreover, HA and LPS showed synergy effect on the expression of IL1a, IL1b and IL6 to some extent. Interestingly, the abundance of Let-7e was increased in THP-1 and decreased in medium during the inflammatory process. At the same time, the expression of ILla, IL6 and TNFa compared with NC group was down-regulated and the level of let-7e was reversed inside and outside of THP-1 by silencing TLR4.Conclusion HA can cause the mice to produce significant pathological reaction, promoting the body’s immune response. Furthermore, HA mainly promotes inflammation through TLR4 pathway and Let-7e may play a critical role in homeostasis response to the inflammatory stress in THP-1.Second Chapter Analysis of human encoded miRNAs targeting the gene segments of H7N9 by bioinformaticsObjective To predict human encoded miRNAs targeting the gene segments of influenza A virus (H7N9) in specific tissue by bioinformatics.Methods According to the principle at miRNAs can directly target virus genome to regulate the infection process, the human encoded miRNAs that may regulate H7N9 gene segments were predicted in A549. In detail, the miRBase were screened for all human encoded miRNAs that had targets in these regions by miRNA prediction software (FindTar3) and their expression enrichment in A549 of lung was analyzed subsequently through online database (microRNA.org). The optimal human encoded miRNAs that may regulate the gene segments were picked out via comprehensive evaluation of the thermodynamic free energy of target regions and the expression level in A549.Result The number of miRNAs whose matching genes of seed regions were located in the conserved regions of gene segments were as follows:51 (HA),37 (NA),29 (PB2),19 (PA) and 26 (PB1-F2). After the analysis of miRNA expression level in A549, four miRNAs that have relative high expression enrichment and small thermodynamic free energy were regarded as the optimal human encoded miRNAs regulating the gene segments of H7N9:hsa-miR-16 (HA and PB2), hsa-miR-21 (NA), hsa-miR-9 (PA) and hsa-miR-193b (PB1-F2).Conclusion Predicting miRNAs targeting the gene segments of viruses in specific tissues or cells through rational utilization of bioinformatics provides a new way to find more reliable aimed miRNAs for laboratorial basic researches and clinical treatment although the results need to be further verified through experiments.Third Chapter Dysregulated serum miRNA expression profile and the evaluation of the diagnostic potential in hepatitis C virus infected patientsObjective To screen the dysregulated miRNA expression profile in HCV infected serum by using miRNA arrays, then verify the results and estimate the diagnostic potential to lay the foundation for further studies on miRNAs associated with HCV infection.Methods The serum samples from HCV patients without treatment was selected and collected from January 2011 to September 2013 in Zhujiang Hospital. All samples were separated and stored at -70℃ as soon as possible.768 miRNAs in serum pools with high abundance of HCV-RNA (HCV-RNA≥105) were screened with miRNA PCR arrays, and then the expression of hsa-miR-122, hsa-miR-134, hsa-miR-424-3p and hsa-miR-629-5p were verified in 68 serum samples by individual PCR including 39 cases with positive HCV-RNA (HCV-RNA≥5×102) and 29 healthy controls. On that basis, ROC curve was performed to analyze the diagnostic potential. Finally, preliminary analysis of the possible targets and function was carried out through miRNA prediction software and the literature.Result After screening,106 miRNAs met the basic conditions, including 51 up-and 55 down-regulated miRNAs, which may be associated with HCV infection. Hsa-miR-122, hsa-miR-134, hsa-miR-424-3p and hsa-miR-629-5p were verified to be up-regulated in HCV infected serum by individual PCR. The ROC curves showed that hsa-miR-134 and hsa-miR-424-3p could distinguish HCV patients with moderate sensitivity and specificity, whereas hsa-miR-122 had highest diagnostic efficiency and hsa-miR-629-5p showed low diagnostic potential. Furthermore, functional analysis of target proteins of these miRNAs indicated the involvement of viral replication, inflammation response, tumorigenesis and so on.Conclusion The abnormal expression spectrum of miRNAs in serum may be significant for the diagnosis and pathogenesis of virus infectious diseases, such as HCV.Summarization:1. HA can induce the body to produce obvious pathological reaction and promote the local inflammation in mice, which may play a vital role in the pathogenesis of H7N9.2. HA can induce the up-regulated expression of Let-7e in THP-1 and down-regulated expression in medium, which may give an explain for the increased level of Let-7e in H7N9 infected serum to some degree.3. Let-7e may act as an inhibiting factor to mediate the homeostasis response to inflammation stress, which is closely associated with H7N9 infection.4. The miRNAs targeting the virus gene segments predicted by bioinformatics in specific tissue may be more reliable targets for the laboratorial basic researches and clinical treatment.5. The dysregulated expression of miRNAs in HCV infected serum shows virus strain specificity, which may have certain clinical application value for the diagnosis and treatment of HCV infection.