MiR-3940-3p Regulates The Inflammatory Response Of Ischemic Stroke Wind-Phlegm Collateral Obstruction Syndrome By Targeting IRAK4

Object: The purpose of this study was to explore the association of miR-3940-3p and ischemic stroke wind-phlegm collateral obstruction syndrome(IS-WP),to confirm whether miR-3940-3p can be regard as a potential biomarker for IS-WP and to investigate the underlying mechanism by which miR-3940-3p affected IS-WP.Methods:1.50 IS-WP patients and 50 healthy controls were enrolled in this study,the relative expression level of miR-3940-3p between IS-WP patients and control subjects were detected by using quantitative real-time polymerase chain reaction(qRT-PCR).2.Receiver operating characteristic(ROC)curve was performed to evaluate the the diagnostic value of miR-3940-3p on IS-WP.3.Luciferase reporter gene assay was applied to validated that miR-3940-3p binds to IRAK4 gene.4.The lentiviral vectors were added into the HBMEC and these cells were classified into three groups: overexpression miR-3940-3p group(miR-3940-3p-mimics),knockdown miR-3940-3p group(miR-3940-3p-inhibitor)and empty vector control group(miR-3940-3p-NC).The expression of miR-3940-3p at the transcriptional level was detected by qRT-PCR to determine the transfection efficiency of overexpression and knockdown miR-3940-3p.5.QRT-PCR was used to detect the expression level of IRAK4 m RNA in the overexpression miR-3940-3p group,knockdown miR-3940-3p group,empty vector control group and control group after oxygen-glucose deprivation treatment.6.Western Blot(WB)was utilized to detect the expression level of IRAK4 protein in the four groups,including overexpression miR-3940-3p group,knockdown miR-3940-3p group,empty vector control group and control group,after oxygen-glucose deprivation treatment.7.The levels of IL-6,IL-8 and TNF-α inflammatory cytokines were detected by Enzyme-linked immunosorbent assay(ELISA)among the overexpression miR-3940-3p group,knockdown miR-3940-3p group,empty vector control group and control group after oxygen-glucose deprivation treatment.Results:1.QRT-PCR showed that the expression level of miR-3940-3p in the IS-WP patients was statistically significantly lower than that in the healthy control subjects(P<0.05).2.Receiver operating characteristic(ROC)curve analysis revealed that the area under the curve(AUC),sensitivity and specificity of miR-3940-3p for the diagnosis of IS-WP was 0.924,88% and 84%,respectively.3.Double luciferase reporter gene assay showed that the fluorescence activity of the 3’UTR binding site wild-type group of IRAK4 gene(IRAK43’UTR-WT)was significantly decreased compared with the 3’UTR binding site blank control group of IRAK4 gene(IRAK4 3’-UTR-NC)(P < 0.001),moreover,the fluorescence activity of the 3’UTR binding site wild-type group of IRAK4 gene was significantly lower than that of the 3’UTR binding site mutant group of IRAK4 gene(IRAK4 3’-UTR-MU)(P<0.001).4.Excellent transfection efficiency of overexpression and knockdown miR-3940-3p of HBMEC was established according to the qRT-PCR results that among the lentivirus-transfected HBMEC,the expression level of miR-3940-3p was significantly increased in the overexpression miR-3940-3p group compared with the empty vector control group(P<0.05),and the expression level of miR-3940-3p in the knockdown miR-3940-3p group was significantly decreased than the empty vector control group(P<0.05).5.QRT-PCR results showed that compared with empty vector control group,the IRAK4 m RNA expression in the overexpression miR-3940-3p and knockdown miR-3940-3p groups was not significantly different(P > 0.05,respectively).6.WB results showed that the expression level of IRAK4 protein in the overexpression miR-3940-3p group was significantly lower than that in the empty vector control group(P<0.05),while the expression level of IRAK4 protein in the knockdown miR-3940-3p group was significantly higher than the empty vector control group(P<0.05).7.ELISA results revealed that the expression levels of inflammatory cytokines IL-6 and IL-8 in the overexpression miR-3940-3p group were significantly decreased compared with that in the empty vector control group(P<0.05),while the expression levels of IL-6 and IL-8 in the knockdown miR-3940-3p group were significantly increased compared with the empty vector control group(P<0.05).Conclusion:1.MiR-3940-3p was significantly associated with the occurrence of IS-WP.2.MiR-3940-3p is a potential biomarker of IS-WP.3.MiR-3940-3p negatively modulated the expression level of IRAK4 protein by targeting IRAK4 gene,thereby regulating the expression levels of IL-6 and IL-8 inflammatory cytokines.4.MiR-3940-3p may negatively regulate the expression of IRAK4 protein through targeting IRAK4 gene,to affect the inflammatory response of IS-WP,thus affecting the occurrence of IS-WP.