Int J Clin Exp Pathol 2016;9(9):9283-9289 www.ijcep.com /ISSN:1936-2625/IJCEP0030816 Original Article miR-302b suppresses the high-sucrose induced epithelial-mesenchymal transition in diabetic nephropathy Chengjie Zhang1*, Pingling Yang2*, Aimei Li3, Qing Li4 1Department of Emergency, Binzhou People’s Hospital, Binzhou 256610, Shandong, China; 2Clinical Laboratory, People’s Hospital of Donggang District, Rizhao 276800, Shandong, China; 3Department of Infectious Diseases, Anqiu People’s Hospital, Anqiu 262100, Shandong, China; 4Clinical Laboratory, The First Hospital of Zibo City, Zibo 255200, Shandong, China. *Equal contributors. Received April 19, 2016; Accepted July 22, 2016; Epub September 1, 2016; Published September 15, 2016 Abstract: Objective: Diabetic nephropathy (DN) is a kind of vascular complication for diabetes, and its pathogenesis remains complicate. This study aimed to investigate the potential role of miR-302b in the high-sucrose induced epithelial-mesenchymal transition (EMT) during the development of DN and to reveal its possible molecular mecha- nism. Methods: The podocytes and epithelial cells were treated with the high-sucrose (30 mM) to construct the DN model. The overexpressed vector of miR-302b was packaged by the virus packaging method and then was trans- fected into the podocytes and epithelial cells. Results: The EMT-related protein including Slug, snail, vimentin, and fibronectin was significantly increased, whereas E-cadherin, β-catenin and α-catenin were significantly decreased by the overexpression of miR-302b. Conclusion: Taken together, our study revealed that the overexpression of miR- 302b functioned as a protector in the development of DN by suppressing the high-sucrose induced EMT process. Keywords: Diabeticnephropathy, miR-302b, podocytes, epithelial cells, epithelial-mesenchymal transition Introduction that there are many miRNAs involving in the biology of DN [6]. For example, serum miR-93 Diabetic nephropathy (DN) remains to be one of was down-regulated in DN cells and can regu- the vascular complications of diabetes, which late the progression of DN by regulating the is the main cause for the deaths caused by dia- downstream genes [7]. miR-192 is up-regulated betes [1]. Previous studies have reported that in DN, and suppression for miR-192 promotes the pathological changes for DN are mainly pre- the improvement of renal fibrosis [8]. In addi- sented as the glomerular mesanggial cell prolif- tion, it has been reported that miR-302b is eration and the glomerular basement mem- often down-regulated in variety kinds of tumors, brane thickness [2, 3]. The pathological chang- such as hepatocellular cancer, ovarian cancer, es for DN are mainly presented as the glomeru- and esophagus cancer [9, 10], and can sup- lar mesanggial cell proliferation and the glo- press the inflammation during infection [11]. merular basement membrane thickness [4]. Therefore, to explore the potential pathogene- Recent evidence revealed that the tubular EMT sis for DN will be of great significance for the remains to be one of the most pathogenesis for clinical treatment of DN. DN [12]. A various miRNAs are involved in the tubular epithelial-mesenchymal transition [13, microRNAs (miRNA) are some 20-22 nt in 14]. It has been reported that miR-302b is length that play pivotal roles in various biologi- involved in the apoptosis biological process in cal processes at the transcriptional and post- the EMT of embryonic stem cells [15]. However, transcriptional level by targeting the 3’-UTR of few have demonstrated the correlation between genes [5]. Increasing evidence has reported miR-302 expression in the tubular EMT of DN. miR-302b in protecting diabetes nephropathy In the present study, we constructed the DN were packaged using the 3rd generation lentivi- model by high-sucrose treatment using the ral packaging system (purchased from Addgene; podocytes and epithelial cells in vitro and ana- http://www.addgene.org/). Followed by the lyzed the expression of miR-302b to assess the transfection into 293FT cells using the effects of miR-302b in protecting DN. Further Lipofectamine 2000 (Life Technologies, Invi- experimental studies were conducted to ana- trogen, USA). After 48 h of incubation, the lyze the ETM-related protein expression. This supernatant containing virus was collected, study aimed to investigate the possible roles of cleared by centrifugation, filtered by 0.45 miR-302b in protecting DN and to elucidate its μmmillipore filter, concentrated with PEG-it™ potential molecular mechanism. Virus Precipitation Solution, and then proceed- ed to titeration. For infection, a final concentra- Materials and methods tion of 8 μg/mL polybrene (Millipore, TR-1003-G) was added together with the viral supernatant Cell preparation and treatment into MSCs in passage 4 at 30%-40% conflu- ence. The cells were infected with MOI=30. The The mice glomerular podocytes and the epithe- efficiency of gene transduction was evaluated lial cells were purchased from the FuDan IBS by immunofluorescence, PCR and Flow-cyto- Cell Central (Shanghai, China), and were cul- metry. tured in the RPMI 1640 medium containing 10% fetal bovine serum (FBS, Sigma-Aldrich, Western blotting USA). Cells were lapped with the radioimmunoprecipi- For cell treatment, cells were treated with high- tation (RIPA) assay (Sangon, Shanghai, China) sucrose at the concentration of 30 mM with dif- containing phenylmethanesufonyl fluoride ferent time points at 6 h, 12 h, 24 h, 48 h, and (PMSF, Sigma), and then were centrifuged at 72 h. Cells treated with the low-sucrose at con- 12,000 rpm for 10 min at 4°C. Supertanant centration of 5.5 mM were considered as the was collected for the measurement of protein control. concentrations using BCA protein assay kit qRT-PCR analysis (Pierce, Rochford, IL). For Western blotting, pro- tein sample was subjected onto a 12% sodium Total RNA was isolated from the cells according dodecylsulfate-polyacrylamide gel electropho- to the manufacturer’s instructions. Comple- resis (SDS-PAGE), followed by transferred onto mentary DNA (cDNA) was produced using the the polyvinylidencefluoride (PVDF) membranes reverse transcriptase (iScript™ cDNA Synthesis (Mippore). Then the PVDF membraneswere Kit; Bio-Rad Laboratories). Expression for the blocked in Tris-Buffered Saline Tween (TBST) targets was detected by the SYBR green-based containing 5% non-fat milk for 1 h at room tem- quantitative RT-PCR (SYBR Green Master mix; perature. Consequently, the membrane was Thermo Scientific, Waltham, MA, USA). U6 incubated with rabbit anti-human antibodies served as the internal control. The primers (Slug, snail, vimentin, fibronectin, E-cadherin, used for the target gene amplification were as β-catenin and α-catenin, 1:100 dilution, follows: miR-302b: 5’-GGGUCUCCCAACCCUUG- Invitrogen, USA) and overnight at 4°C. Then UA-3’, and U6: 5’-CGCAAGGATGACACGCAAA- membrane was incubated with horseradish- TTC-3’, and the universal reverse primer was peroxidaselabeled goat anti-rat secondary anti- 5’-5’-CAGTGCGTGTCGTGGAGT-3’. body (1:1000 dilution) at room temperature for 1 h. Finally, the PVDF membranes were washed Cell transfection 3 times with 1× TBST buffer for 10 min each. The signals were detected after incubation with The coding sequence of miR-302b was trans- a chromogenic substrate using the enhanced fected into the pEGFP to produce the vector of chemiluminescence (ECL) method. Additionally, pEGFP-miR-302b (GENEchem, Shanghai, Chi- GAPDH served as the internal control. na). The pEGFP vectors are served as the con- trol. Vectors were transfected into the 293FT Statistical analysis cells using the virus packaging method. Briefly, 293FT cells were cultured in the 96 well plates All the experiments were conducted 3 times till they were 80% confluent. Then the vectors independently in this study. Data were expre- 9284 Int J Clin Exp Pathol 2016;9(9):9283-9289 miR-302b in protecting diabetes nephropathy Figure 1. Influence of high-sucrose treatment on the miR-302b expression in the diabetic nephropathy (DN)-related cells. A: High-sucrose (30 mM) treatment resulted in a significant low expression of miR-302b in podocytes at 24 h till to 48 h; B: High-sucrose (30 mM) treatment resulted in a significant low expression of miR-302b in epithelial cells. No significant difference for the miR-302b expression in the two kinds of cells was observed between the 48 h and the 72 h. *: P<0.05 and **: P<0.01, compared to the control, and @: P<0.05 compared to cells treated with sucrose at 48 h. ssed as the mean ± standard deviation (SD). cells (Figure 2). The relative mRNA expression Student’s t test was used to compare the differ- of miR-302b in the podocytes and in the epithe- ence between two groups. Data were analysed lial cells was both significantly increased com- by the SPSS 19.0 (Chicago, USA). The P<0.05 pared to the control (P<0.05). In addition, the were considered as the statistically significant. flow cytometry and immunofluorescence assay revealed that the virus vector was successfully Results transfected into the two kinds of cells. High-sucrose treatment on the miR-302b ex- miR-302b overexpression promotes the trans- pression formation of epithelial cells Both the two kinds of cells including podocytes We further analyzed the epithelial cell transfor- and epithelial cells were treated with high- mation-related protein expression to investi- sucrose at a concentration of 30 mM to assess gate the influence of miR-302b expression on the effects of high-sucrose treatment on the podocytes and epithelial cells transformation miR-302b expression (Figure 1). Until 24 h, no (Figure 3). In the podocytes, the expression of significant difference was observed for the proteins including Slug, snail, vimentin, and expression of miR-302b expression in the two fibronectin was significantly decreased com- kinds of cells. However, the relative expression pared its control (P<0.05), while E-cadherin, of miR-302b in the two kinds of cells was sig- β-catenin and α-catenin were all significantly nificantly decreased from 12 h till 48 h (P<0.05), increased with time increasing (P<0.05, Figure which indicated the miR-302b expression was 3A).