| Background and Aim Systemic lupus erythematosus(SLE) is a common but severe autoimmune disease. Lupus nephritis(LN) is a common and serious complication that is often associated with a poor long-term prognosis; up to 70% of SLE patients are affected of LN and about 10-30% of which will progress to end-stage renal failure(ESRD) circulating mi RNAs significantly altered in disease conditions than in healthy individuals. Therefore, we attempted to 1) reveal the changes of mi RNAs in serum during different stages of CKD caused by LN utilizing microarray technology, 2) investigated these disrugulated circulating mi RNAs correlation with renal damage and SLE activity 3)investigated the possible molecular mechanism of the disrugulated mi RNAs involved in early stage LN.Methods: 1. A total of 96 serum samples, including 60 LN patients with different stages of CKD and 36 healthy controls, collected from May to October 2013,were included in this study. First, serum samples(n=4 from each group) from healthy control, early stage LN patients(CKD 1-3 stage) and late stage(CKD 4-5 stage) LN patients were used to analyze the expression profile of human mi RNAs by Human Serum & Plasma mi Scriptmi RNA PCR Array, which profiles the detectable expression of 372 mi RNAs. 2. Another 52 LN patients(including 40 patients with early stage LN and 12 patients with late stage LN and 32 age- and sex-matched healthy control were recruited for the validation group. Total RNA was isolated from 200μL of serum by mi RNeasy Serum/Plasma Kit, Nanodrop 2000 was used for Detecting RNA concentration. Two mi RNAs, mi R-130b-3p, mi R-1233-3p, which increased the most and have the lowest p value in early stage LN in the screening group were selected to be validated.The area under the ROC curve(AUC) for disregulated circulating mi RNA was used to evaluate the vavlue in predicting early stage LN from healthy control. 3. Human renal tubular epithelial cell line HK-2 was transfected with mi R-130b-3p mimics or inhibitors or correspondent controls Then, HK-2 cells were stimulated with 10ng/m L recombinant TGF-β1 for 72 hours. Aft er 72 h, the expression m RNA and protein levels of E-cadherin(Ecad) and α-smooth muscle Actin(α-SMA) m RNA were quantified by real-time PCR and westen blot. The PCR fragment of ERBB2 IP 3’-UTR of HK-2 cells was digested with Xho I and Not I and cloned into psi Check-2.. For the reporter assays, human embryonic kidney 293 T cell line(293T) was transiently transfected with reporter plasmids and negative control mimics(NC mimics), wild-type mi R-130b-3p mimics or mutant mi R-130b-3p mimics using Lipofectamine 2000 At 48 h post transfection, cells were harvested for analysis. Luciferase activity was determined by using a Promega luciferase assay system..Results 1. We found that the expressions of seven micro RNAs, including mi R-130b-3p, mi R-1233-3p, mi R-18a-3p, mi R-628-3p, mi R-1260 b, mi R-1539 and mi R-378 e were increased in early stage LN patients compared to healthy controls.On the other hand, the expression level of 75 circulating mi RNAs were found decreased more than 2 times, and 21 mi RNAs down-regulated significantly(P<0.05) in late stage LN patients compared to healthy controls; the expression level of 100 circulating mi RNAs in detected were found decreased more than 2 times, and 53 mi RNAs downregulated significantly(P<0.05) in late stage LN patients compared to early stage LN group. There was no mi RNAs were found up-regulated more than 2 times or increased significantly(P<0.05) in late stage LN group compared to healthy controls or early stage LN patients. The results have a implication that circulating mi RNA may reduced in patients with severe chronic renal failure. 2. the level of mi R-130b-3p were significantly higher in early stage LN patients compared to healthy controls [IQR 16.2(8.7, 42.7)vs9.6(4.8, 17.4), p<0.01)], and decreased significantly in late stage LN group. The mi R-1233-3p was not significantly changed between controls and early stage LN patients [IQR 10.4(6.4, 16.9) vs 8.7(3.8, 16.9) p>0.05], but significantly down-regulated in late stage LN group.The area under the ROC curve(AUC) for mi R-130b-3p in predicting early stage LN from healthy control is 0.683±0.062(95%CI=0.561-0.805, P<0.01). The association of serum mi R-130b-3p and various disease activity parameters were found not significant correlated such as anti-ds DNA, C3, C4.that the expression of serum mi R-130b-3p was significantly and positively correlated with 24-hour proteinuria and chronicity index. Our data also showed that serum mi R-130b-3p was well correlated with serum triglyceride level. 3.Transfection of HK-2 cells with mi R-130b-3p mimics resulted in significantly increased expression of α-smooth muscle actin(α-SMA), PPAR-γ,COL-1,COL-4 and decreased expression of E-cadherin(E-cad) in the presence of TGF-β1(10ng/ml) both on RNA and protein level. Furthermore, inhibition of mi R-130b-3p partially reversed TGF-β1-induced EMT and fibrosis. Western blotting showed that after transfected by mi R-130b-3p mimics both in the absence and presence of TGF-β1 showed significantly decreasing of ERBB2 IP and PPAR-γ protein. Following co-transfection of human 293 T with the constructs and one of the mimics: negative control, wild-type mi R-130b-3p, mutant mi R-130b-3p, luciferase activity was measured in each case. The results showed that mi R-130b-3p induced a decrease of ERBB2 IP 3’-UTR luciferase activity compared to negative control and mutant mi R-130b-3p, but mutant mi R-130b-3p had similar effect compared to negative controland; the wt-mi R-130b-3p induced a decrease of wtPPARγ 3’-UTR luciferase activity compared to negative control and mutant-PPARγ 3’-UTR.Conclusions: The expression level of circulating mi RNA disregulated in pateients with deferent CKD stage.Circulating mi RNA may reduced in patients with severe chronic renal failure. Mi R-130b-3p up-regulated in early stage LN patients and negatively regulated ERBB2 IP expression by directly targeting the 3’-UTR of ERBB2 IP. The circulating mi R-130b-3p might serve as a biomarker and play an important role in renal damage in early stage LN patients. |
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