Study On The Mechanism Of EV71 Inducing Human Neuroblastoma Cell SH-SY5Y Pyroptosis By Up-regulating MiR-146a

Objective: The results showed that EV71 infection could induce pyroptosis of SH-SY5 Y,and the possible molecular mechanism of EV71 induced pyroptosis of SH-SY5 Y was further discussed.Methods: In the previous research work,the RIP chip technology was used to detect the differentially expressed miRNAs in SH-SY5 Y after EV71 infection.Among the many differentially expressed miRNAs,some genes were verified by RT-q PCR,and miR-146 a that may be related to pyroptosis.Using bioinformatics technology to analyze and compare the five databases of Targetscan,miRNA,miRDB,Pictar,miRtar Base and consulting the literature to find three target genes TRAF6,CXCR4,BRCA1 downstream of miR-146 a that may be related to pyroptosis.RT-q PCR and Western blot were uesd to technology to determine TRAF6,CXCR4 as the research targets of downstream target genes.Western blot were used to determine the pyroptosis of SH-SY5 Y induced by EV71.Mi R-146a-inhibitor was used to adjust the expression of miR-146 a in cells,and then detect the changes in the level of pyroptosis after EV71 infection.After using Phace-TRAF6 and Phace-CXCR4 to overexpress TRAF6 and CXCR4 in cells,the changes of the level of pyroptosis after EV71 infection were detected.In addition,we used the TUNEL experiment to detect the pyroptosis of EV71 after SH-SY5 Y infectionResults: Western blot results showed that after cells were infected with EV71,the expression of intracellular pyroptosis-related protein caspase-1,GSDMD,NLRP3,and IL-1β increased,indicating that the virus can induce cells pyroptosis.RT-q PCR results showed that the expression level of miR-146 a in SH-SY5 Y infected with EV71 increased.After down-regulating the expression of miR-146 a in cells by transfection of miR-146a-inhibitor,the expression of pyroptosis-related proteins caspase-1,GSDMD,NLRP3,and IL-1β in SH-SY5 Y infected with EV71 was significantly reduced,indicating that miR-146a-inhibitor can reverse virus-induced pyroptosis.Combined with bioinformatics technology and literature review,we preliminarily identified TRAF6 and CXCR4 as the downstream target genes of miR-146 a.Western blot and RT-q PCR results showed that after EV71 infects cells,it can down-regulate the expression of TRAF6 and CXCR4 in cells.When miR-146a-inhibitor was transfected to down-regulate the expression of miR-146 a in cells,the expression of TRAF6 and CXCR4 increased,which suggested that miR-146 a had a targeting relationship with TRAF6 and CXCR4,and there was a negative correlation.After we overexpressed TRAF6 and CXCR4 in cells,we tested the expression of pyroptosis-related proteins in the cells infected by EV71,the results showed that the expression of caspase-1,GSDMD,NLRP3,and IL-1β decreased,which indicates that overexpression of TRAF6 and CXCR4 in cells can reverse virus-induced pyroptosis.TUNEL experiment results proved that EV71 can induce cell apoptosis after infecting SH-SY5 Y.Conclusion: The above experimental results suggest that EV71 can up-regulate the expression of miR-146 a and induce pyroptosis and apoptosis in SH-SY5 Y.When the expression level of miR-146 a in cells is down-regulated,the effect of EV71 on the pyroptosis of SH-SY5 Y can be reversed.When the expression levels of TRAF6 and CXCR4 in cells were up-regulated,the effect of EV71 on the pyroptosis of SH-SY5 Y could also be reversed.This shows that EV71 can induce pyroptosis and apoptosis in SH-SY5 Y,and this induction is achieved by the virus up-regulating the expression of miR-146 a in cells,and then regulating the expression of its downstream target genes TRAF6 and CXCR4.