Downregulated miR-203 attenuates IL-β, IL-6, and TNF-α activation in TRAF6-treated human renal mesangial and tubular epithelial cells

Abstract

Circulating microRNAs (miRNAs) are attracting major interest as novel non-invasive biomarkers for human autoimmune diseases including lupus nephritis (LN). A previous study showed that altered miR-203 expression may provide highly diagnostic for systemic lupus erythematosus. However, whether miR-203 is a diagnostic biomarker for LN is still unknown. In the present research, serum samples from 35 cases of active LN patients, 58 cases of inactive LN patients, and 74 cases of healthy volunteers were collected to analyze the expression profiles of miR-203 by qRT-PCR. The serum concentration of complement component 3 (C3) and complement component 4 (C4) was detected using nephelometry method. The expression of inflammatory cytokines, including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α), were analyzed using enzyme-linked immunosorbent assay (ELISA). The effect of miR-203 overexpression on the TNF receptor associated factor 6 (TRAF6)-induced inflammation of human renal mesangial cells (HRMCs) and human renal tubular epithelial cell line (HK-2) were evaluated. Results showed that miR-203 in serum of active LN patients was significantly down-regulated when compared with serum from inactive LN patients and healthy volunteers. Receiver operating curve (ROC) showed that decreased circulating miR-203 was a significant diagnostic biomarker for active LN patients, with an area under curve (AUC) of 0.974; sensitivity was 85.79%, and specificity was 89.40%. Significant downregulation of C3 and C4, and obvious upregulation of IL-β, IL-6, and TNF-α, was observed in serum of active LN patients. Furthermore, circulating miR-203 expression was positively correlated with the serum concentrations of C3 and C4, and negatively correlated with the serum expression of IL-1β, IL-6, and TNF-α in active LN patients. In addition, transfection of HRMCs and HK-2 cells with miR-203 mimics could suppress TRAF6-induced IL-β, IL-6, or TNF-α expression compared to cells treated with the mimics control group. In summary, decreased circulating miR-203 might be a candidate diagnostic biomarker for human active LN, and it attenuated IL-β, IL-6, and TNF-α activation in TRAF6-treated HRMCs and HK-2 cells.

Keywords: Circulating; active LN; biomarker; inflammation; miR-203.

Figure 1

Circulating miR-203 expression levels in LN patients and healthy volunteers. A. qRT-PCR analysis of miR-203 expression in serum from 93 LN patients and 74 healthy volunteers. U6 was chosen as a housekeeping internal control for normalization of miR-203 expression. B. miR-203 expression was lower in serum from 35 active LN patients compared with that in serum from 58 inactive LN patients. Data are mean ± SD from triplicate experiments, *P<0.05. miR: microRNA, LN: lupus nephritis, U6: RNU6B.

Figure 2

ROC curve analysis of circulating miR-203 for ability to discriminate LN patients from healthy volunteers and active LN patients from inactive LN patients. A. Diagnostic value differentiating LN patients from healthy volunteers; an AUC value was 0.897 and the corresponding sensitivity was 76.82% and specificity was 81.56%. B. Diagnostic value differentiating active LN patients from inactive LN patients; an AUC value was 0.974 and the corresponding sensitivity was 85.79% and specificity was 89.40%. Data are mean ± SD from triplicate experiments. ROC: receiver operating characteristic, AUC: area under the curve.

Figure 3

Serum concentration of C3 and C4 and expression of IL-1β, IL-6, and TNF-α in serum of LN patients. A. The concentration of C3 and C4 was lower in serum from active LN patients compared with that in serum from inactive LN patients. B. ELISA showed that serum expression of IL-1β, IL-6, and TNF-α was markedly upregulated in active LN patients compared to inactive LN patients. Data are mean ± SD from triplicate experiments, *P<0.05. C3: complement component 3, C4: complement component 4, IL-1β: interleukin 1 beta, IL-6: interleukin 10, TNF-α: tumor necrosis factor α, ELISA: enzyme-linked immunosorbent assay.

Figure 4

The effect of miR-203 overexpression on the TRAF6-induced IL-β, IL-6, TNF-α activation of HRMCs and HK-2 cells. A. qRT-PCR results revealed that miR-203 was efficiently overexpressed by transfection of miR-203 mimic in HRMCs and HK-2 cells, with U6 as an internal control. B. TRAF6 treatment significantly elevated the concentration of IL-1β, IL-6, and TNF-α compared to the control group. C. HRMC cells transfected with miR-203 mimics showed lower IL-1β, IL-6, and TNF-α expression than that in the mimic control group. D. Transfection of HK-2 cells with miR-203 mimics resulted in significantly decreased expression of IL-1β, IL-6, and TNF-α compared to the control group. Data are mean ± SD from triplicate experiments, *P<0.05. TRAF6: TNF receptor associated factor 6, HRMCs: human renal mesangial cell line, HK-2: human renal tubular epithelial cell line.