Up-regulation of Serum MiR-130b-3p Level is Associated with Renal Damage in Early Lupus Nephritis

Abstract

Systemic lupus erythematosus (SLE) is a common but severe autoimmune systemic inflammatory disease. Lupus nephritis (LN) is a serious complication of SLE,affecting up to 70% of SLE patients. Circulating microRNAs (miRNA) are emerging as biomarkers for pathological conditions and play significant roles in intercellular communication. In present research, serum samples from healthy control, early and late stage LN patients were used to analyze the expression profile of miRNAs by microarray. Subsequent study demonstrated that miR-130b-3p in serum of patients with early stage LN were significantly up-regulated when compared with healthy controls. In addition,we have also observed that the expression of a large amount of circulating microRNAs significantly decreased in patients with late stage LN. The further analysis found that the expression of serum miR-130b-3p was positively correlated with 24-hour proteinuria and renal chronicity index in patients with early stage LN.Transfection of renal tubular cellline(HK-2)with miR-130b-3p mimics can promote epithelial-mesenchymal transition (EMT). The opposite effects were observed when transfected with miR-130b-3p inhibitors. MiR-130b-3p negatively regulated ERBB2IP expression by directly targeting the 3'-UTR of ERBB2IP The circulating miR-130b-3p might serve as a biomarker and play an important role in renal damage in early stage LN patients.

Figure 1. erum miRNA expression in patients with lupus nephritis (LN).

(A) Serum miRNAs profile from 4 early stage LN patients (eGFR > 30 mL/min/1.73 cm²) versus 4 healthy controls. (B) The fold of serum miRNA changes of early stage LN versus healthy controls. (C) SerummiRNAsprofile from 4 late stage LN patients (eGFR < 30 mL/min/1.73 cm², CKD 4–5 stage) versus 4 healthy controls. (D) SerummiRNAs profile from 4 late stage versus 4 early stage LN patients. (E) The concentration of total RNAs present in 12 μL Rnase water from 200 μL serum of the validation group. The vertical line represented the changing fold equal to –2 (down-regulation), 1, 2 (up-regulation and invisible in Fig. 1D). The plots above the horizontal line represent the miRNAs with P values less than 0.05. *P < 0.05 **P < 0.01.

Figure 2. Expression of miR-130b-3p and miR-1233-3p in validation group.

(A) Serum miR-130b-3p indifferent stage of LN patients. (B) Receiver operating characteristic (ROC) curves with corresponding area under the ROC curve (AUC) for miR-130b-3p in discriminating LN patients with early stage CKD (stage1–3) from healthy control. (C) Serum miR-1233-3p indifferent stage of LN patients. The line indicates the median value per group. Fold regulation is expressed as RQ based on 2^−∆∆Ct *P < 0.05 **P < 0.01

Figure 3. The association of serum MiR-130b-3p and SLE activity and renal damage.

(A) Altered expression of serum miR-130b-3p in early stage LN patients with or without SLE activity. The line indicates the median value per group. Fold regulation is expressed as RQ based on 2^−∆∆Ct.; The correlation between serum miR-130b-3p and (B) anti-dsDNA, (C) C3, (D) C4, (E) 24-hour urine proteinor, (F) renal chronicity index in early stage LN.

Figure 4. The role of miR-130b-3p in renal tubular cells.

(A) The expression of miR-130b-3p was significantly upregulated in HK-2 cells post transfecting with miR-130b-3p mimics than with miR-control at a 30nM concentration. (B) MiR-130b-3p expression in HK-2 cells was determined by quantitative RT-PCR 24 h post transfection of miR-130b inhibitors. (C,D) Transfection of HK-2 cells with miR-130b-3p mimics resulted in significantly increased mRNA expression of α-smooth muscle actin (α-SMA) and decreased mRNA expression of E-cadherin (E-cad) compared to NC mimics in the presence of TGF-β1 (10 ng/ml) for 72 h; Inhibition of miR-130b-3p partially reversed TGF-β1-induced EMT. (G-F) HK2 cell’s expression of E-cadherin or α-SMA protein after transfection of miR-130b-3p mimics or inhibitors and correspondent controls when in the absence or presence of TGF-β1 (10 ng/ml). All results are expressed as mean ± SD. for three independent experiments. *p < 0.05, **p < 0.01

Figure 5. miR-130b-3p promotes TGF-β1-induced EMT in renal tubular epithelial cells by targeting ERBB2IP.

(A,B) Transfection of HK-2 cells with miR-130b-3p mimics resulted in significantly decreased protein expression of ERBB2IP compared to NC mimics both in the absence and presence of TGF-β1 (10 ng/ml) for 72 h. (C). Transfection of HK-2 cells with miR-130b-3P mimics resulted in significantly decreased mRNA expression of ERBB2IP in the absence and presence of TGF-β1 (10 ng/ml) for 72 h (D) The effects of mimics negative controls (NC-mimics), wild-type miR-130b-3p mimics(miR-130b-wt) and miR-130b-mut on luciferase activity in 293T cells after transfection with 500 ng psiCHECK-2 reporter plasmids. All results are expressed as mean ± SD. for three independent experiments. *p < 0.05, **p < 0.01.