Figure 4

Anti-miR-146a rescues TRAF6, IRAK1, IRAK2 genes. CHME3 cells were transfected with 100 pmol anti-miR-146a. Cy3-labeled negative control and mock transfected cells were used as control. After 24 h of transfection, JEV infection was given (MOI 5). For better comparison, we also used a JEV-infected group untransfected with miR-146a mimic harvested after 24 h post infection. (A) Western blots showing upregulation of TRAF6, IRAK1, and IRAK2 genes upon silencing miR-146a. Anti-miR-146a also upregulated TRAF6, IRAK1, and IRAK2 genes after JEV infection. (B) Densitometry plot showing upregulation of TRAF6, IRAK1, and IRAK2 genes by anti-miR-146a w.r.t. JEV infection. The image density was normalized by β-tubulin. For statistical analysis, the protein levels of JEV-infected samples were compared to anti-miR-146a and anti-miR-146a + JEV samples. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, ***denotes P < 0.001. (C) Taqman RT-PCR analysis of anti-miR-146a-transfected samples to confirm silencing of miR-146a. No silencing was observed in Cy3-labeled negative control. Cy3-labeled control was used as control for t-test. (D) Graph bars showing reduced luciferase activity upon miR-146a overexpression. JEV infection after 24 h post miR-146a mimic transfection further reduced NF-κB activity. β-galactosidase activity was used to normalize luciferase activity. miR-146a was overexpressed, JEV was infected, and miR-146a + JEV samples were compared to scrambled control for statistical analysis. (E) Graph showing increased luciferase activity of NF-κB luciferase vector upon silencing of miR-146a by anti-miR-146a. Anti-miR-146a treatment 24 h prior to JEV infection also increased NF-κB luciferase activity. JEV-infected group was used for comparison for statistical analysis. All experiments were repeated thrice and are represented as mean ± SE. The fold change is significant where *denotes P < 0.05, **denotes P < 0.005, and ***denotes P < 0.001.