MicroRNA-155 Ihibites EV71 Replication Through Targeting PICALM In Human Neuroblastoma Cells

Objective:The genus Enterovirus(EV), a member of the Picornavirus family, belongs to RNA viruse which may cause poliomyelitis, hand, food, mouth disease(HFMD). MicroRNAs(miRNAs) are a class of highly conserved, small noncoding RNAs molecules which contain 18- to 25- nucleotides(nts) in almost all eukaryotes. miRNAs regulate gene expression at the post-transcriptional level, influencing important physiological and pathological processes. In this study, on the basic of miRNA expression profiling in EV71 infected neuroblastoma cells, we selected and verified microRNA-155(mi R-155) can regulate EV71 replication by using miRNA mimic and miRNA inhibitor, and further clarified that miR-155 can inhibit the EV71 replication through targeting PICALM in human neuroblastoma cells(SK-N-SH cells). Methods:(1) SK-N-SH cells after EV71 infection are collected and extracted total RNA. The miRNA gene chip was used to obtain changed miRNA expression profile and chose the more remarkable miRNAs; In addition, qRT-PCR was used to verify the result of gene chip.(2) The miRNA mimic is transfected into SK-N-SH cells to over-regualate miRNA expression level, and CPE, virus TCID50, virus qRT-PCR, Western blotting(WB) were applied to detect the expression of EV71 replication compared to negative control and blank control, and verify the role of miRNA in regulating EV71 replication.(3) By using the database of miRNA target genes: MiRanda, TargetScan and MiRbase to find miRNA functional targets. QRT-PCR and WB are used to detect the relationship between mi RNA and target gene. Constructing over-expressional vector and synthesing small interfering RNA(si-RNA) to up-regulate and down-regulate gene expression, respectively.The role of miRNA on EV71 replication was detected and simultaneously explored miRNA regulates EV71 replication through the target gene in SK-N-SH cells. Results:(1) After EV71 infection, the results of both miR-155 gene chip and q RT-PCR showed that miR-155 expression was increased and the changes are positively related to infection time and EV71 titer.(2) SK-N-SH cells was transfected with miR-155 mimic, the expression of miR-155 obviously increased, EV71 replication was inhibited compared to negative control in SK-N-SH cells. Meanwhile, this change positively depends on miR-155 expression level.(3) Using biological software, such as miRBase and gene function to select the target gene of miR-155. The results showed that PICALM mRNA level and protein level were obviously decreased after EV71 infection in SK-N-SH cells. Using PICALM over-expression vector and PICALM siRNA upregulate and downregulate the gene expression respectively, the result showed that the over-expression of PICALM could reduce inhibition by miR-155 on EV71 replication while PICALM si-RNA could further inhibit EV71 replication. Conclusions:Our results found that miR-155 can inhibit EV71 replication through targeting PICALM in SK-N-SH cells.