The Discovery Of MiR-374a And The Role Of Its Target Genes In The Pathogenesis Of MERS-CoV Infection

Middle East Respiratory Syndrome Coronavirus(MERS-Co V)is one of the largest RNA viruses which has been found with strong pathogenicity and high mortality characteristics.The emergence of MERS,a new infectious disease,has brought new challenges to mankind.However,there are few studies on the pathogenesis of MERS-Co V.Mi RNA as a new type of regulatory factors plays a very important role in the pathogenesis of the virus.In this study,the pathogenesis of MERS-C o V was studied from the perspective of mi RNA.The study of pathogenesis of MERS-Co V was helpful to further elucidate the interaction between MERS-Co V and its host,to discover the new anti-virus drugs and to provide new targets and strategies.Objective To explore the role of important regulatory micro RNAs in the pathogenesis of MERS-Co V and the related molecular mechanisms.Methods1.MERS-Co V infection-related mi RNA screening: mi RNA expressionchips were used for screening MERS-Co V infection-related mi RNAand q RT-PCR was also used to verified the expression of mi RNA in the cells after infection of MERS-Co V.2.2.To investigate the effect of mi R-374 a on cell proliferation,construct the overexpression vector of mi R-374 a was constructed and was transfect transiently them into cells.MTT assay was used to detect the effect of mi R-374 a overexpression on cell proliferation.3.Predictive analysis of target genes of mi R-374 a.Prediction of mi R-374 a target gene was predicted by MTS-Co V-infected cell m RNA expression profiling and comprehensive on-line analysis(Targetscan,pichtar).q PCR validation assay validated the results.The targeting of Bol A33'UTR was detected by double luciferase reporter gene.And the effect of mi R-374 a on the m RNA and protein levels was detected by q PCR and Western blotting.4.Cloning and expression of the Bol A3 gene and antibody preparation: The Bol A3 gene was amplified by q RT-PCR and constructed into p QE30 prokaryotic expression vector for expression.The rabbits were immunized using the purified recombinant protein.5.The distribution of Bol A3 in the cells and the study of ATP production: a variety of cell lines including lung,liver,kidney,brain and other tissue were cultured for detection of Bol A3 expression by Western blotting methods.EGFP-Bol A3 fusion expression vector were constructed and transiently transfected into the cells to to detect the localization of Bola3 protein and test the effect of inhibition of Bol A3 on cell function..The Bol A3 inhibition model was established and the effective region of Bol A3 RNAi was screened for making Bol A3 RNAi adenovirus To test the effect of inhibition of intracellular expression of Bola3 on ATP production.Results1.This study found that MERS-Co V infection induced mi RNA differential expression profiling.We found at least seven mi RNAs with significant increase in signal density,Mi R-374 a,which may play an important role in the pathogenesis of MERS-Co V infection,was screened by q PCR.MERS-Co V could promote the expression of mi R-374 a in the cells.Functional test revealed that mi R-374 a overexpression in cells could inhibit cell proliferation.mi R-374 a may target the regulation of Bol A33'-UTR.The results showed that MERS-Co V could inhibit the expression of Bol A3.The results showed that mi R-374 a could inhibit the expression of Bol A3 at the m RNA and protein levels.2.Anti-Bol A3 polyclonal antibody preparation and identification: the construction of the Bola3 prokaryotic expression vector was transformed into the M15 competent state,protein expression was induced,the purified protein program was used to get immunized rabbit serum and Western blotting were used to identify the antibodies that were highly specific for the binding of prokaryotic and eukaryotic expression of Bol A3 protein.3.The distribution and localization of the Bola3 gene in the cell and its effect on ATP production were studied.The results showed that Bol A3 was expressed in various tissues such as liver,kidney,lung and brain.The expression of Bol A3 could be localized in mitochondria and nucleus.Our experimental study showed that the decrease in the expression of Bol A3 affected the formation of ATP,animportant substance in the cellular energy metabolism,which in turn affected cell survival.Conclusion This study explored the role of mi RNA in the pathogenesis of MERS-Co V infection.The results showed that MERS-Co V infection could cause the changes of mi RNAs in a variety of host cells.MERS-Co V infection could inhibit the expression of mi R-374 a and inhibit the expression of Bol A3.This suggest that MES-Co V infection may lead to a decrease in the expression of Bol A3 that may play a role in the pathogenesis of MERS-Co V infection.