Fig 2: Recovery of tumor necrosis factor receptor-associated factor 3 (TRAF3) expression by anti-miR-32 transfection suppresses expression levels of total interferon regulatory factor (IRF)3 and IRF7.(a) CHME3 cells were transfected with Cy3-labeled control anti-miR,anti-miR-32 and anti-miR-32 plus Tat C treatment. Phosphorylated (p)IRF3 level increased after anti-miR-32 treatment, while the total IRF3 level was downregulated in anti-miR-32-transfected cells, showing a positive relationship between cellular TRAF3 level and activation of IRF3. (b) pIRF7 was increased after anti-miR-32 treatment and anti-miR-32 plus Tat C treatment, again showing a positive role of TRAF3 in IRF7 activation. Total IRF7 level was decreased in anti-miR-32-transfected cells showing that recovery of TRAF3 could modulate the transcription of IRF3and IRF7. (c,d) Densitometry analysis of pIRF3, pIRF7, total IRF3 and total IRF7 normalized to β-tubulin. Experiments were performed three times and data are presented as mean ± SE. Results were significant (*P ≤ 0.05).

Fig 3: Overexpresssion of miR-32 suppresses tumor necrosis factor receptor-associated factor 3(TRAF3) protein expression. (a) Western blot analysis for TRAF3 in CHME3 cells after miR-32 overexpression. Plasmid pCMV-miR-32 was transfected into CHME3 cells. The empty vector was used as the negative control. Cell lysates were prepared after 24 hours of transfection, and western blot analysis was performed using anti-TRAF3 antibody. miR-32 overexpression significantly reduced both mRNA and protein levels of TRAF3 (P ≤ 0.05) (indicated by * in the transfected group) compared with empty vector. (b) Quantitative (q)PCR analysis of miR-32 overexpression in CHME3 cells, using TaqMan miR-32 assay. miR-32 expression was found to be 7.5-fold higher in miR-32-overexpressed cells. (c) Densitometry quantification of TRAF3 normalized to β-tubulin. (d) qPCR analysis for detection of changes in transcript level of TRAF3 after miR-32 overexpression in CHME3 cells. All experiments were performed at least three times and data are presented as mean ± SE.

Fig 4: Anti-miR-32 transfection rescues tumor necrosis factor receptor-associated factor 3 (TRAF3) protein expression in CHME3 cells. (a) Transfection efficiency of anti-miR, by using Cy3-labeled anti-miR as negative control. (b) Quantitative (q)PCR analysis of cellular miR-32 level after anti-miR-32 transfection, to confirm the suppression of miR-32. The expression level of miR-32 decreased by 40% in cells transfected with anti-miR-32; compared to cells transfected with scrambled anti-miR negative control (*P ≤ 0.05). (c) Western blot analysis of TRAF3 in CHME3 cells after anti-miR-32 transfection, showing the recovery of TRAF3 expression level in cells treated with anti-miR-32 and anti-miR-32 plus Tat. Anti-miR-32 transfection was performed at a concentration of 100 pmol/l. After 24 hours of anti-miR-32 transfection, a set of transfected cells were treated with 500 ng/ml Tat C protein to augment the cellular expression level of miR-32. (d) Densitometry analysis of TRAF3 normalized to β-tubulin. There was a significant (**P ≤ 0.005) recovery of TRAF3 expression level.

Fig 5: Expression of miR-32 increases with Tat C treatment in a dose-dependent manner.(a) CHME3 cells were treated with an increasing dose of HIV-1 Tat C protein. After 24 hours, cells were harvested for RNA isolation and protein lysate preparation. miR-32 assays were performed by quantitative PCR with TaqMan probes and primers specific for human miR-32. Data was normalized to the expression level of the small RNA, RNU24, and results are shown as fold change compared with untreated control. Changes in miR-32 expression level were significant (P ≤ 0.05). (b), Western blot analysis for tumor necrosis factor receptor-associated factor 3(TRAF3) of the same samples treated with increasing concentrations of Tat C, showing a gradual reduction in TRAF3 protein expression. (c) Western blot image intensity was normalized to β-tubulin. All experiments were performed three times and are presented as mean ± SE. Changes in the level of expression of TRAF3 in response to increasing dose of Tat C were significant (**P ≤ 0.005, *P ≤ 0.05) compared with the untreated group.