Functional Mechanism Of Long Non-coding RNA MSTRG.15570.6 In Dengue Virus Infection

Dengue fever(DF)is a life-threatening mosquito-borne viral infection caused by dengue virus(DENV).Non-coding RNAs(ncRNAs)are involved in the process of DENV infection.As an important part of ncRNAs,the research on long non-coding RNAs(LncRNAs)in DENV infection is still in its infancy.The aim of this study was to investigate the mechanism of action of LncRNAs in dengue infection and to discover potential biomarkers or therapeutic targets for dengue infection.In order to obtain relevant data on the involvement of LncRNAs in viral infection and disease progression.The clinical features and epidemiological studies were carried out in this study.The clinical information,serum and peripheral blood mononuclear cells(PBMC)of dengue hospitalized patients were collected in Xishuangbanna.The results showed that in severe dengue fever(SD),the proportion of male and Han nationality was high.The majority DENV serotype in SD is DENV-1,and there are 6 Zika virus(ZIKV)positive cases in SD.secondary infection in SD accounted for 76.47%,and DF accounted for only 20%.The analysis of the three-dimensional structure of the protein found that the C/PrM/E of DENV-1 and DENV-2 were more stable than the previous strains in the same region.6 strains of DENV-2 were found to have no common mutation compared with DENV strains from previous years.SD may be associated with cross-infection of heterotypic dengue virus and ZIKV co-infection.After high-throughput sequencing of PBMC,a large number of differential mRNAs and LncRNAs were found between healthy group and dengue patient group.The same result also appears between SD and DF.7 LncRNAs and 8 mRNAs were selected to real-time quantitative PCR(qPCR)detection,it was found that the expression trend was basically consistent with the transcriptome results.DENV 118 strains infected PBMC in vitro,and the expression of 11 unknown LncRNAs was detected by qPCR.The results shown that DENV infection could induce the expression of MSTRG.15570.6.The same results also appeared in the process of DENV infection of THP-1,HUVEC,Huh-7 cells.The full length of MSTRG.15570.6 was obtained by 5’ cDNA ends rapid amplification technology(RACE)and 3’ RACE,and its length was measured to be 2202bp,located on human chromosome 12.MSTRG.15570.6 is an antisense Lnc RNA.After prediction ofsecondary structure by bioinformatics,it was found that there are a lot of loop structures at its 5’ end.Between 992 and 1211 have the potential to encode amino acids.The results of nucleocytoplasmic separation and probe in situ hybridization(FISH)were shown that MSTRG.15570.6 existed in both nucleus and cytoplasm.In the study of theMSTRG.15570.6 biological function of Huh-7 and HUVEC cells,it was found that the expression of MSTRG.15570.6 could promote the replication of DENV through siRNA interference or lentiviral vector-mediated overexpression experiments.Through RNA pull-down experiments and protein profiling,11 potential interacting proteins were screened.After siRNA interfered the expression of MSTRG.15570.6,the mRNA expressions of autophagy related proteins CSNK2B,CSNK2A1,SQSTM1 and SLC3 A2 were decreased by qPCR detection.In Huh-7 cells,DENV induced autophagy by electron microscopy and detection of autophagy-related genes.The mRNA expressions of autophagy-related proteins ATG5 and ATG12 were decreased in Huh-7 after siRNA interfered with the expression of MSTRG.15570.6.This indicated that autophagy induced by MSTRG.15570.6 and DENV was positively regulated in Huh-7 cells,that is,decreased expression of MSTRG.15570.6 inhibited autophagy.Bioinformatics predicted that MSTRG.15570.6 could regulate the expression of IFIT3.In Huh-7 cells,the mRNA expression of IFIT3 was inhibited after interfering with the expression of MSTRG.15570.6.Therefore,we speculate that the expression of MSTRG.15570.6 and IFIT3 is positively correlated.Studies have shown that DENV infection can induce the expression of MSTRG.15570.6 both in vivo and in vitro.In vitro experiments show that the presence of MSTRG.15570.6 can promote virus replication.It is speculated that autophagy and IFIT3 play a key role,but the specific mechanism needs further study.This study is helpful for understanding DENV infection and pathogenic mechanism,and provides a reference for exploring the role of LncRNAs in virus infection and screening related therapeutic target molecules.