miR-200 family promotes podocyte differentiation through repression of RSAD2

Mature podocytes are highly differentiated cells with several characteristic phenotypic features that are involved in the glomerular filtration function. During kidney development, a series of changes of the morphological characteristics and cellular functions may happen in podocytes. The miR-200 family functions in various biological and pathological processes. But the underlying molecular mechanisms of miR-200 family that functions in podocyte differentiation remain poorly understood. Herein is shown that miR-200a, miR-200b and miR-429 are significantly upregulated during the differentiation of podocytes, with highest upregulation of miR-200a. In these cells, restraint of miR-200 family by RNA interference assay revealed a prominent inhibition of cell differentiation. More intriguingly, miR-200 family directly inhibited the radical S-adenosyl methionine domain-containing protein 2 (RASD2) expression. Moreover, further upregulation of RSAD2 combining with restraint of miR-200 family revealed a promotion of podocyte dedifferentiation and proliferation. In addition, the expression of RSAD2 is consistent with that of in vitro podocyte differentiation in prenatal and postnatal mouse kidney, and significantly down-regulated during the kidney development. Together, these findings indicate that miR-200 family may potentially promote podocyte differentiation through repression of RSAD2 expression. Our data also demonstrate a novel role of the antiviral protein RSAD2 as a regulator in cell differentiation.

that miR-200 family was one of the most abundant miRNAs in kidney 14 . miR-200 was expressed in the developing pronephros, the skin and part of the somites 15 . However, the accurate role of miR-200 family in podocyte differentiation is not clear. Therefore, a study in our laboratory has identified the miRNAs and mRNA expression profiles in undifferentiated and differentiated podocytes using miRNA and mRNA microarray 16 . These works lay the groundwork for our further molecular mechanism study in podocyte differentiation.
RSAD2, also known as Viperin or Cig5, plays a key role in the innate immune response system. RSAD2 is rapidly induced by both type I and II interferons and has a wide variety of antiviral activity 17 . RSAD2 subsequently localizes to the endoplasmic reticulum (ER) via its N-terminal amphipathic α -helix 18 , the process of which appears to result in the disruption of lipid raft microdomains and the prevention of influenza virus from budding of the plasma membrane 19 . Although increasing amounts of researchers have reported on the antiviral effect of RSAD2 in various organs and cells, very few studies have been conducted on the expression or function of system during cell development. One recent study from Steinbusch et al. has shown that RSAD2 knockdown induces ER stress and skews the chondrocyte phenotype towards hypertrophy in developing chondrocytes in vivo 20 . However, whether and how the RSAD2 is regulated by miRNA, especially by miR200 family during podocyte differentiation is still unclear.
Herein, we hypothesize that miR-200 family promotes podocyte differentiation through repression of RSAD2 expression. Our results indicate that the expression of miR-200 family is significantly up-regulated in differentiated podocytes comparing with the undifferentiated one, and the inhibition of miR-200 family can arrest cell differentiation. Furthermore, bioinformatic analysis and Luciferase reporter assay revealed that RASD2 was directly negatively regulated by miR-200 family. Together, we show that miR-200 family and RASD2 cooperate to assure a normal differentiation of podocytes.

Upregulation of miR-200a, miR-200b and miR-429 during the differentiation of podocytes.
To investigate the expression level of miR-200 family during the differentiation of podocytes, a recent study in our laboratory has identified the miRNAs and mRNA expression levels in undifferentiated and differentiated podocytes using miRNA and mRNA microarray 16 . The miRNA microarray showed that miR-200a, miR-200b and miR-429 expressions were significantly increased in differentiated podocytes (Fig. 1a). Notably, the three members are all located on chromosome 4, which indicated a potential key role of the three members of miR-200 family in podocyte differentiation. Further real-time quantitative PCR (qPCR) assay showed that miR-200a (1996 ± 470, P < 0.01), miR-200b (543 ± 92, P < 0.01) and miR-429 (463 ± 74, P < 0.01) expressions were significantly increased in differentiated podocytes (Fig. 1b).

Restraint of miR-200 family revealed inhibition of cell differentiation and apoptosis.
To confirm the biological function of miR-200 family in podocyte differentiation, we examined cell cycle distribution after a simultaneous inhibition of the expression of miR-200a, miR-200b and miR-429. Compared with DMPC group (76.3 ± 0.5%), a significant decrease of cells in the G0/G1 phase of cell cycle was observed following miR-200a, miR-200b and miR-429 co-inhibition (miR-200a/200b/429 inhibitors-DMPC group; 63.3 ± 4.5%, P < 0.001) (Fig. 2a). Triplicate assays were done for each RNA sample and the relative amount of each miRNA was normalized to GAPDH. The expression of miRNAs in MPC groups was standardized as percentile change of numerical one hundred. The data are the average of three independent experiments and are shown as the mean ± S.D. Statistically significant differences between two groups was indicated by ** P < 0.01. In addition, research has revealed a rearrangement of cytoskeleton during differentiation of MPCs 1,21 . An ordered array of actin fibers and microtubules extends into the forming cellular processes during differentiation. These cytoskeletal rearrangements and process formation are accompanied by the onset of Nephrin, CD2AP and WT1 synthesis, the process-associated proteins marking specifically differentiated podocytes. Consequently, we studied the effect of miR-200 family inhibition on the rearrangement of cytoskeleton and expression of biomarkers, which would reveal the effect of miR-200 family on podocyte differentiation. Indeed, comparative analysis of F-actin distribution revealed that the process extension was prominently inhibited in miR-200a/200b/429 inhibitors-DMPC (Fig. 3a). Semiquantification of F-actin expression showed that, compared with the DPMC group (166.7 ± 13.6%), the mean relative fluorescent intensity in each cell of the miR-200a/200b/429 inhibitors-DPMC group is significantly decreased (118.7 ± 11.6%, P < 0.001, Fig. 3b).  (Fig. S1). Statistical result of TUNEL-positive apoptotic cell rate was shown in (c). The data are the average of three independent experiments and are shown as the mean ± S.D. Statistically significant differences between two groups was indicated by ***P < 0.001. Besides, Nestin, the intermediate filaments-associated protein, was significantly down-regulated in miR-200a/200b/429 inhibitors-DMPC group (147.6 ± 9.3%) compared with the DMPC group (246.4 ± 21.1%, P < 0.001, Fig. 3c,d). Moreover, The expressions of podocyte-specific markers, Nephrin, CD2AP and WT1 significantly down-regulated in miR-200a/200b/429 inhibitors-DMPC group (148.3 ± 18.1% of Nephrin, P < 0.001; 139.8 ± 18.6% of CD2AP, P < 0.001; 119.2 ± 12.5% of WT1, P < 0.001, Fig. 3c,d) compared with the DMPC group (256.0 ± 15.8% of Nephrin, P < 0.001; 226.8 ± 25.3% of CD2AP, P < 0.001; 180.2 ± 9.9% of WT1, P < 0.001, Fig. 3c,d).
Since restraint of miR-200 family inhibited podocyte differentiation, we further explored the effect of restraint of miR-200 family on podocyte apoptosis. Indeed, the TUNEL assay showed that restraint of miR-200a, miR200b and miR-429 evidently decreased the apoptotic cell numbers (Fig. 2c,e). Subsequent flow cytometry analysis showed that restraint of miR-200a, miR200b and miR-429 significantly decreased the rate of Annexin V-positive apoptotic cells in DMPC group (9.19 ± 2.2%) compared with the DMPC group (13.56 ± 1.7%, P < 0.001, Fig. 2b,d).
RSAD2 was a predicted target gene of miR-200 family. We then looked for miR-200 family putative targets. On the basis of previous data, In general, the predicted miR-200 family target genes were downregulated in DMPCs because miR-200 family expression was upregulated in DMPCs. Then, we further predicted the miR-200 family target genes by comparing the upregulated miRNAs and the downregulated mRNAs on two web sites: http://www.microrna.org/ microrna/getGeneForm.do and http://mirdb.org/miRDB/. Both of the web sites showed the same sequences of RSAD2 targeting by miR-200 family (Fig. 4a). In accordance with our hypothesis, our previous mRNA microarray study showed a significant down-regulation of RSAD2 in DMPCs (Fig. 4b) 16 . Therefore, we confirmed a prominent down-regulation of RSAD2 mRNA (P < 0.05, Fig. 4c) and protein expression in DMPCs (P < 0.01, Fig. 4d).
To investigate whether miR-200 family can indeed target RSAD2, a dual luciferase reporter assay was conducted. The miR-200a, miR-200b and miR-429 mimics and vectors containing the 3′ -UTR of RSAD2 were transiently transfected into HEK293 cells. To test the RSAD2 putative binding site, we generated a mutant RSAD2 construct, RSAD2-mut, in which the 3′ -UTR of RSAD2 was altered using a site-directed mutagenesis kit. Results showed that miR-200a (P < 0.01), miR-200b (P < 0.05) and miR-429 (P < 0.05) significantly down-regulated the luciferase activity of RSAD2 construct (Fig. 5a), whereas luciferase activity was not generated from the mutant  (Fig. S5). Data are the average of three independent experiments and are shown as the mean ± S.D. Statistically significant differences between two groups was indicated by *P < 0.05. construct (Fig. 5b). In addition, Western blot assay showed that miR-200 family (miR-200a, miR-200b and miR-429) evidently affected protein levels of RSAD2 (Fig. 5c). All together, these results suggested that miR-200 family directly regulated the expression ofRSAD2 by interacting with its 3′ -UTR.
The expression of RSAD2 in mouse renal cortex. Next, we analyzed the appearance of RSAD2 in developing renal cortex by examining its expression in the prenatal and postnatal mouse kidney that displays glomeruli at different stages of development. We used immunofluorescence assay to detect RSAD2 expression in the mouse kidney at prenatal day (E) 18.5 and postnatal day (P) 5, 7, 14 and 49. As shown in Fig. 7a, RSAD2 expression was found at all stages of renal glomerulus and tubule formation during prenatal and postnatal development. Importantly, the expression of RSAD2 was major localized in the glomerulus, but not the tubule at P14 and P49. The result indicates a positively correlative of RSAD2 with podocyte during its differentiation. Moreover, there was a decrease in the expression of RSAD2 in glomeruli between P5 and P7. Further analysis of RSAD2 expression in renal cortex was conducted by using western blot assay. Consistently, compared with the E18.5 group, the expression of RSAD2 significantly down-regulated at P7 (P < 0.01, Fig. 7b), P14 (P < 0.001, Fig. 7b) and P49 (P < 0.001, Fig. 7b). These results are consistent with results in vitro study in podocytes.

Discussion
In the present study, we show a relationship between miR-200 family and RSAD2 in podocyte differentiation. The results showed that miR-200 family modulated cell differentiation through the inhibition of RSAD2. Furthermore, the blockade of miR-200 family by RNA interference inhibited the expression of RASD2 and arrested cellular differentiation. Our studies suggested the hypothetic miR-200s/RSAD2 signal is crucial for podocyte differentiation. Since functional defects of differentiating podocytes may lead to structural damage of glomerulus and failure of  Western blot assay was used to quantitatively evaluate the RSAD2 expression. A cropped blot was shown above the histogram, and the corresponding full-length blot was shown in the supplementary data (Fig. S7). Data are means ± S.D. Statistically significant differences are indicated (**P < 0.01, ***P < 0.001). the glomerular filtration barrier, which in turn triggers a cascade of events that can finally lead to renal disease. Thus, our findings emphasize that the miR-200s/RSAD2 signal may represent a promising pathway in exploring molecule mechanisms of podocyte differentiation.
Several studies have indicated that miRNAs play crucial roles in kidney development. The first functional studies addressing roles for miRNAs in podocytes in kidney have used a conditional approach to knock down Dicer, which is required for the production of mature miRNAs [22][23][24] .
Our recent study also indicated the miRNA-mRNA regulatory networks during podocyte differentiation 16 . In this study, we found that miR-200a, miR-200b and miR-429 were upregulated in DMPCs, whereas the expression of miR-200c and miR-141 were not affected (Fig. 1). Notably, the three miR-200a/200b/429 are all located on chromosome 4, which indicates a potential key role of the three members on chromosome 4 in podocyte differentiation, but not the other two members (miR-200c and miR-141) on chromosome 6. Thus, a negative control or miR-200a, miR-200b and miR-429 inhibitors were all co-transfected together into DMPCs for 48 h. We found that restraint of miR-200 family significantly inhibited podocyte differentiation (Figs 2 and 3). Though the members of the miR-200 family (the miR-200b/200c/429 group and the miR-200a/141 group) share the similarities of their seed sequences 25 , the biological function of miR-200a/200b/429 subfamily in our study may undergo a chromosome-dependent molecular mechanism.
As regulators of gene expression, miRNAs can work by either targeting the mRNA for destruction or by inhibiting protein synthesis 26,27 . Because miR-200a/200b/429 were up-regulated in DMPCs, we examined the down-regulated genes in DMPCs to predict the target genes using the microRNA and miRDB databases. Finally, we selected the RSAD2 gene as a putative target gene. The RSAD2 (Viperin/Cig5) is known to be an antiviral protein 17,28 . However, the RSAD2 is recently reported to be a novel chondrogenic regulator in developing chondrocytes in vivo 20 .
We have reported a downregulation of RSAD2 in DMPCs using a mRNA microarray study (Fig. 4b) 16 . We further confirmed a prominent down-regulation of RSAD2 protein expression in DMPCs (Fig. 4c).We then constructed a vector containing the mutated 3′ -UTR of the RSAD2 gene, and confirmed that the RSAD2 gene was regulated by miR-200a/200b/429 subfamily using a dual luciferase reporter assay (Fig. 5). These data indicated that miR-200a/200b/429 negatively regulated RSAD2 expression by binding to the 3′ -UTR of the RSAD2 gene.
Several genes have been reported to be the targets of the miR-200 family, including ZEB1 29 , ZEB2 30 , Foxf2 31 and SIP1 32 . However, most of them were reported to be involved in cancer. Our further assay by restraint of miR-200 family expression combining with higher expression of RSAD2 in podocytes showed a restriction of podocyte differentiation (Fig. 6), which further confirmed the pivotal role of miR-200s/RSAD2 signaling in podocyte differentiation. Besides, we also revealed the role of miR-200s/RSAD2 signaling in podocyte proliferation. Inhibition of the miR-200s/RSAD2 signaling significantly increased the podocyte proliferative rate (Fig. 6). Since report has revealed the critical role of mir-200 family in cell proliferation 33 , These results indicated that the novel role of the miR-200s/RSAD2 signaling in podocyte proliferation.
The RSAD2 is generally considered as an antiviral protein. The RSAD2 would be induced by interferons to resist the infection of virus 34 . We initially doubted the RSAD2 signaling may result from the treatment of interferon in podocyte culture. However, in our study, the RSAD2 maintained at a low level in interferon-induced MPCs (Fig. 4b,c), which indicated an entirely different role of RSAD2 may play in podocytes. One recent study from Steinbusch et al. have shown a novel role of the RSAD2 in developing chondrocytes in vivo 20 . Here we bring direct new evidences that miR-200s-mediated RSAD2 plays a crucial role in podocyte differentiation.
We should point out that signals controlling podocyte differentiation may have cross-talk with each other, we cannot exclude the possibility that other signals may participate in the miR-200s/RSAD2 signal. Therefore, further study is needed to investigate a clear signaling network that may take part in the miR-200s/RSAD2 signal. In that regard, the anti-viral action of RSAD2 by localizing to the endoplasmic reticulum 28 , may be a new potential direction in miR-200s-mediated podocyte differentiation. In addition, other correlative cells of nephron, such as capillary endothelial cells and parietal epithelial cells, also play crucial roles in kidney development. Further investigation of the miR-200s/RSAD2 signal in these cell types will strengthen our understanding of the miR-200s/RSAD2 relationships.
In the present study, we demonstrated that miR-200a, miR-200b and miR-429 are significantly up-regulated during the podocyte differentiation. MiR-200 family restraint reveals a significant inhibition in podocyte differentiation. More intriguingly, miR-200 family directly inhibited the RASD2 expression, which can reverse miR-200a/200b/429-mediated promotion of cell differentiation (Fig. 8). It is the first report on a novel role of the antiviral protein RSAD2 as a regulator in cell differentiation. Hence, these findings indicate that miR-200 family may potentially promote podocyte differentiation through repression of RSAD2 expression.

Materials and Methods
Cell cultures. Mouse podocytes from a conditionally immortalized cell line (MPC) were cultured, as described previously 35  RNA extraction and real-time quantitative PCR (qPCR). Total RNA isolation and qPCR for detecting miR-200a, miR-200b, miR-429 and RSAD2 mRNA expression were carried out by the following procedures. Briefly, total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. For miRNAs expression, total RNAs smaller than 200 nt were isolated using the miRcute miRNA Isolation Kit (Tiangen Biotech, Beijing, China). One microgram of total miRNA per sample was copied into complementary DNA (cDNA) using the miRcute miRNA First-strand cDNA Synthesis Kit (Tiangen Biotech, Beijing, China). One microliter of reverse transcription reaction was used for each specific miRNA qPCR assay, which was amplified using miRcute miRNA qPCR Detection Kit (Tiangen Biotech, Beijing, China) following the manufacturer's instructions. MiRNAs expression levels were normalized to GAPDH level. For gene expression, RNAs were retro-transcribed with the PrimeScript TM RT reagent kit (Takara Biotechnology, Japan) and qPCR performed using SYBR ® Premix Ex Taq TM reagent kit (Takara Biotechnology, Japan). Gene expression levels were normalized to GAPDH level. All reactions were done in triplicate using Mastercycler ep realplex 2 (EppendorfAG, Hamburg, Germany) and expression levels calculated using the comparative CT method (2 −ΔΔCT ).
Western blot analysis. Preparation of cell lysates has been described in our previous studies. Equal amounts protein loadings were separated by SDS-PAGE on 8% or 10% gels and electrophoretically transferred to a PVDF membrane. Nonspecific binding sites were blocked with 5% nonfat milk powder in phosphate-buffered saline (PBS) and 0.05% Tween 20 for 1 h at room temperature. Membranes were incubated in the following primary antibodies at appropriate concentrations: RSAD2 (1:1000 dilution), β -actin (1:1500 dilution), Nestin (1:1000 dilution), Nephrin (1:1000 dilution), CD2AP (1:1000 dilution) and WT1 (1:1000 dilution). Secondary antibodies were used at 1:2500. Following additional washes before application of chemiluminescent substrate (Millipore, Billerica, MA, USA), the membranes were exposed on MXG film (Kodak, Rochester, NY) using varying exposure times. β -actin was used as an internal control. The quantification of bands was performed using the Image J software (NIH, Bethesda, MD).
Fluorescent staining. Cells cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at 4 °C, and incubated in blocking buffer (10% normal goat serum and 0.3% Triton X-100) for 30 min at 37 °C. Cells were labeled with Nephrin hAb (1:200 dilution) diluted in 5% of normal goat serum, 0.3% Triton X-100 in PBS for 2 h at 37 °C. After three times of gentle wash with PBS, Secondary antibodies coupled with green-fluorescent Alexa-488 were used at 1:500. For the observation of cytoskeletal assembly, cells were stained for 30 min in PBS containing 100 ng/ml rhodamine phalloidin (Cytoskeleton, Inc) and 5 μ g/ml DAPI (Sigma, St. Louis, MO, USA), rinsed in PBS for three times, and observed under a confocal laser scanning fluorescent microscope at FV1000 (Olympus, Tokyo, Japan). Images were then processed by Photoshop 7.0 (Adobe, San Jose, CA).
The tissue samples were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), sliced in 4 mm thick sections and incubated in blocking buffer (10% normal goat serum and 0.3% Triton X-100) for 30 min at 37 °C. Sections were labeled with RSAD2 hAb (1:200 dilution) diluted in 5% of normal goat serum, 0.3% Triton X-100 in PBS for 2 h at 37 °C. After three times of gentle wash with PBS, Secondary antibodies coupled with red-fluorescent Alexa-647 were used at 1:500. For the observation of nuclear, cells were stained for 2 min in PBS containing 5 μ g/ml DAPI (Sigma, St. Louis, MO, USA), rinsed in PBS for three times, and observed under a confocal laser scanning fluorescent microscope at FV1000 (Olympus, Tokyo, Japan). Images were then processed by Photoshop 7.0 (Adobe, San Jose, CA).
Annexin V/propidium iodide (PI) staining assay. To determine type and ratio of apoptotic and necrotic cell death, cells were detected with AnnexinV-FITC/PI Detection Kit (Tianjin Sungene Biotech, China) according to the manufacturer's protocol. Briefly, cells were harvested, washed in phosphate-buffered saline (PBS) and stained for 15 min at room temperature, with Annexin V-FITC (5 μ l) and PI (5 μ l) in binding buffer in the dark. The cells were then analyzed in a FACSCalibur flow cytometer (Beckman-Coulter, Fullerton, CA) equipped with CellQuest software (BD Biosciences, San Jose, CA). TUNEL staining. TUNEL staining was conducted using the in situ colorimetric TUNEL apoptosis assay kit (Beyotime, China), according to the manufacturer's manual. Briefly, the sections were washed one time in PBS and incubated in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. The sections were washed one time with PBS. Then sections were incubated in the cold 0.1% Triton X-100 for 2 min. The sections were washed two times with PBS. Then the sections were incubated with TdT enzyme solution for 60 min at 37 °C in darkness. Then sections were rinsed in PBS for three times, and observed under a confocal laser scanning fluorescent microscope at FV1000 (Olympus, Tokyo, Japan). Images were then processed by Photoshop 7.0 (Adobe, San Jose, CA).
Cell cycle analysis. Cells were harvested by centrifugation at 1500 rpm for 5 min. Supernatants were discarded and cells were suspended in ice-cold PBS and fixed in 70% ice-cold ethanol. Upon analysis, the cells were washed with PBS and then resuspended in DNase-free RNase A (100 μ g/ml) and left at 37 °C for 30 min. Cells were then incubated in PBS containing propidium iodide (10 μ g/ml) for 30 min at 4 °C. The cell cycle distribution was measured by a flow cytometer (BD FACS Calibur) equipped with CellQuest software. Statistical analysis. All data were expressed as mean ± SD. Independent-samples t-tests were applied where two groups of data were compared. The mean values are presented in bar charts, with T-bars referring to standard deviations. Multiple samples were statistically analysed for significance using one-way analysis of variances (One-Way ANOVA). Post hoc statistics were obtained using the LSD test (Fisher'sleast significant difference t-test) for multiple comparisons. All of the statistical analyses were performed using SPSS 11.5 software (IBM, Chicago, IL). P-values less than or equal to 0.05 were considered to be significant. All experiments were performed at least in triplicate.