Increased HDAC3 and decreased miRNA-130a expression in PBMCs through recruitment HDAC3 in patients with spinal cord injuries

Abstract

The study was performed to investigate the molecular mechanism for SCI patients. The interaction between miRNA-130a and HDAC was demonstrated in PBMCs from SCI patients. Increased HDAC3 and decreased miRNA-130a were observed in PBMCs from AS patients. Next, HDAC3 loss-of-function or HAAC3 inhibition promoted the expression of miRNA-130a, and HDAC3 could be recruited to the promoter region of the gene, miRNA-130a, in PBMCs. In addition, linear regression analysis indicated that mRNA expression results were highly negative correlated between HDAC3 and miRNA-130a in PBMCs from SCI patients. Furthermore, miRNA-130a down expression increased the expression of HDAC3 in PBMCs. Loss-of-function of miRNA-130a promoted PBMCs apoptosis, but HDAC3 loss-of-function had no significant effect on the apoptotic cell. In addition, miR-130a overexpression decreased, whereas miR-130a inhibition increased, the expression of TNF-α in PBMCs. Furthermore, HDAC3 loss-of-function or HAAC3 inhibition associated with simultaneous up-regulation the expression of miR-130a and down-regulation the expression of TNF-α in PBMCs. In conclusion, HDAC3 regulated a distinct underlying molecular and pathogenic mechanism of SCI by forming a negative feedback loop with miR-130a and enhanced TNF-1α expression.

Keywords: HDAC3; PBMCs; Spinal cord injury; miRNA-130a.

Figure 1

The expression of HDAC3 and miRNA-130a in PBMCs from SCI patients. The mRNA (A) and protein (C) expression of HDAC3 are respectively measured by Quantitative real-time PCR and western blotting in PBMCs from SCI patients. Densitometric quantification for western blotting (D). miRNA-130a expression is measured by Quantitative real-time PCR (B). Correlation between HDAC3 and miRNA-130a (E). Values are expressed as mean ± SEM, n = 3 in each group. R ≤ -0.8 means highly negative correlation between cervical scrapings and tumor tissue PAX1 methylation levels.

Figure 2

Regulation of miRNA-130a by HDAC3. The PBMCs are treated with vehicle solvent, DMSO, HDAC-siRNA and trichostatin A (TSA), and the expression of miRNA-130a is measured by Quantitative real-time PCR (A). The protein expression of HDAC3 is measured by western blotting in PBMCs from SCI patients (B). ChIP assay is performed using HDAC3 antibody to detect binding at the miRNA-130a promoter region. Percentage input was calculated with 2^{(Ct [1% of input]–Ct [ChIP])} (C). The apoptotic cells was detected by flow cytometric analysis of annexin V/PI double staining (D) and the percentage of apoptotic cells (E). Values are expressed as mean ± SEM, n = 3 in each group.

Figure 3

Regulation of PBMCs apoptosis via miRNA-130a. The protein expression of HDAC3 is measured by western blotting in PBMCs from SCI patients (A) and densitometric quantification for western blotting (B). The apoptotic cells was detected by flow cytometric analysis of annexin V/PI double staining (C) and the percentage of apoptotic cells (D). Values are expressed as mean ± SEM, n = 3 in each group.

Figure 4

Regulation of TNF-α1 by HDAC3 via miRNA-130a. The mRNA (A) and protein (B) of TNF-1α are respectively measured by Quantitative real-time PCR and western blotting in PBMCs with miRNA-130a loss-of-function or gain-of-function. The mRNA expression of TNF-1α and miRNA-130a are measured by Quantitative real-time PCR in PBMCs with HDAC3 loss-of-function or inhibition (C). The protein expression of TNF-1α is measured by western in PBMCs with HDAC3 loss-of-function or inhibition (D). Values are expressed as mean ± SEM, n = 3 in each group.