MicroRNA‐302c modulates peritoneal dialysis‐associated fibrosis by targeting connective tissue growth factor

Abstract Long‐term peritoneal dialysis (PD) can lead to the induction of mesothelial/epithelial‐mesenchymal transition (MMT/EMT) and fibrosis; these effects eventually result in ultrafiltration failure and the discontinuation of PD. MicroRNA‐302c (miR‐302c) is believed to be involved in regulating tumour cell growth and metastasis by suppressing MMT, but the effect of miR‐302c on MMT in the context of PD is unknown. MiR‐302c levels were measured in mesothelial cells isolated from the PD effluents of continuous ambulatory peritoneal dialysis patients. After miR‐302c overexpression using lentivirus, human peritoneal mesothelial cell line (HMrSV5) and PD mouse peritoneum were treated with TGF‐β1 or high glucose peritoneal dialysate respectively. MiR‐302c expression level and MMT‐related factors alteration were observed. In addition, fibrosis of PD mouse peritoneum was alleviated by miR‐302c overexpression. Furthermore, the expression of connective tissue growth factor (CTGF) was negatively related by miR‐302c, and LV‐miR‐302c reversed the up‐regulation of CTGF induced by TGF‐β1. These data suggest that there is a novel TGF‐β1/miR‐302c/CTGF pathway that plays a significant role in the process of MMT and fibrosis during PD. MiR‐302c might be a potential biomarker for peritoneal fibrosis and a novel therapeutic target for protection against peritoneal fibrosis in PD patients.


| INTRODUCTION
Peritoneal dialysis (PD) is one of the most important renal replacement therapies for patients with end-stage renal failure. However, continual exposure to non-biocompatible PD fluid (PDF), the mechanical stress of the dwelling solution, and catheter complications (including peritonitis and haemoperitoneum) may cause acute and chronic inflammation and injury of the peritoneum. In these conditions, the peritoneum undergoes structural and functional alterations, which ultimately lead to peritoneal fibrosis and ultrafiltration failure; however, these consequences subsequently limit the longterm clinical application of PD. 1 Mesothelial-to-mesenchymal transition (MMT) is the initial and reversible process that leads to fibrosis in some organs; because MMT is a source of peritoneal fibroblasts in PD and plays a central role in the peritoneal alterations leading to fibrosis during PD, an increasing number of studies have been carried out to analyse the characteristics of MMT in PD. [2][3][4][5] However, therapy has yet been found. Therefore, it is urgent to find an effective method to investigate the mechanism and an applicable treatment for peritoneal fibrosis.
MicroRNAs are non-coding RNAs that post-transcriptionally regulate gene expression by inducing target mRNA degradation or inhibiting its translation. Recent studies have confirmed that miR-NAs, such as those in the miRNA-200 family, miRNA-205, miRNA 30b, miRNA-21-5p and miRNA-129-5p, are important regulators of TGF-β1-induced MMT in PD. [6][7][8] In our previous study, we employed a miRNA array analysis of the PMCs from the effluent of PD patients and identified aberrant miRNA levels involved in the MMT of PMCs. The results indicated that the expression of 269 miRNAs was dysregulated; of these, 124 miRNAs were up-regulated and 145 miRNAs were down-regulated. MiR-302c was found to be downregulated nearly threefold in the PMCs from the effluents of patients undergoing PD for more than 1 year when compared with those from patients undergoing PD for less than 6 months. However, the role of miR-302c in the MMT process of PMCs remains unknown.
MiR-302c belongs to the miR-302 family, which is highly conserved and plays important roles in regulating cellular proliferation, differentiation and reprogramming. 9,10 MiR-302c was found to take part in multiple physiological and pathological processes. MiR-302c can regulate the differentiation of human embryonic stem cells, 11 and its effect on the formation and development of tumours has also been studied. MiR-302c may directly target the oestrogen receptor in human breast cancer, 12,13 and it has also been reported to be dysregulated in biliary tract cancer 14 and thyroid cancer. 15 Interestingly, it was delineated that miR-302c was involved in idiopathic pulmonary fibrosis via regulating the transition of epithelialto-mesenchymal (EMT). 16 More recently, Kai Zhu et al 17 revealed that miR-302c suppressed the tumour growth of hepatocellular carcinoma through the metadherin-mediated inhibition of EMT. These studies suggested a potential role of miR-302c in EMT and fibrosis.
Considering mesothelial cell is a kind of epithelial cell and MMT is actually a subtype of EMT, thus it aroused our interest to investigate the effect and mechanism of miR-302c in MMT and fibrosis during PD.
Connective tissue growth factor (CTGF), a downstream mediator of transforming growth factor-β1 (TGF-β1), was thought to take part in TGF-β1-induced fibrosis and to be related to the occurrence and development of fibrosis in many organs because it is a pro-fibrotic factor. 18,19 CTGF was also detected in the peritoneal fluid of patients undergoing PD. Some studies have revealed that peritoneal mesothelial cells can produce CTGF, which subsequently drives peritoneal fibrosis by inducing fibroblast proliferation and collagen synthesis; moreover, CTGF expressions are significantly increased in the thickened peritoneal membrane of PD patients with ultrafiltration failure (UFF). However, the exact mechanism has not been well described, especially in peritoneal fibrosis during PD. Consistent with the findings above, our previous studies have showed that the CTGF expression is up-regulated in both PD rats and the effluent liquid from PD patients with peritoneal fibrosis; these data indicate a positive correlation between CTGF and TGF-β1. 20,21 The goal of this study was to identify the role of miR-302c in peritoneal fibrosis. We analysed miR-302c expression in mesothelial cells isolated from the PD effluent from patients on PD and evaluated its relationship with markers of MMT and peritoneal fibrosis. Furthermore, we employed lentiviral miR-302c to up-regulate the expression of miR-302c and then observed whether the overexpression of miR-302c can alleviate the MMT and peritoneal fibrosis during PD and the relationship between it and CTGF. We have been suggested that that miR-302c may inhibit TGF-β1-induced MMT and fibrosis by suppressing the expression of CTGF.

| Animal model
Male ICR mice weighing 28-30 g were purchased from the Experi-

| Peritoneal equilibrium test (PET)
PET measurements were carried out as previously reported. 22 Briefly, a 2.5% dextrose dialysate exchange was performed overnight using a PD patient's standard dwell volume. The peritoneal cavity was drained for over 20 minutes, then instilled 2.5% dextrose dialysate into the peritoneal cavity. After 4 hours, remove 10 mL effluent to determine the urea, creatinine and glucose levels. Peritoneal membrane transport function was designated as high, high average, low average or low transporter.

| Human peritoneal mesothelial cells (HPMCs)
To determine the expression of miR-302c in PD patients, the overnight (8 hours) peritoneal lavage of continuous ambulatory peritoneal LI ET AL. | 2373 dialysis (CAPD) patients were collected and centrifuged to isolate peritoneal mesothelial cells as previously reported. 23 All patients involved were >18 years of age. Patients were excluded if they were >65 years old or had history of abdominal infection or malignancy.
Patients with a history of kidney transplantation, haemodialysis for >3 months before PD or immunosuppressive agent use for a long time were also excluded. Ultimately, 20 PD patients were included.
Newly enrolled patients receiving CAPD less than 3 months were used as controls (PD start, n = 10) and patients who were receiving CAPD for more than 1 year were used as the PD >1 year group (n = 10). MiR-302c expression in the PMCs was detected by Taq

| Cell culture
HMrSV5 (a human peritoneal mesothelial cell line, HPMCs) was obtained from Dr Pierre Ronco (Tenon Hospital, Paris, France). Cells were grown in Dulbecco's modified Eagle's medium/F12 medium containing 10% foetal bovine serum and incubated at 37 in a humidified incubator with 5% CO 2 . In in vitro experiment, cells were incubated with 5 ng/mL TGF-β1 (Prospecbio, Inc., East Brunswick, NJ, USA) in serum-free medium for 0-48 hours to investigate the effect of TGF-β1 in the MMT change and expression of CTGF in HPMCs.
In addition, with the treatment of 5 ng/mL TGF-β1, HPMCs were transfected with LV-hsa-miR-302c (LV-pGIPZ served as a control), and with or without 10 ng/mL recombinant CTGF protein (R&D System, Minneapolis, MN, USA) to explore the precise role of miR-302c and CTGF in TGF-β1 induced MMT in HPMCs. MiR-302c and U6 primer sets were purchased from Applied Biosystems.

| Real-time PCR
Total RNA was extracted from visceral peritoneum or cells using  Table   S1.
The real-time PCR analyses were performed with the ABI Prism 7900 sequence detection system (Applied Biosystems).
Expression was calculated using the 2 −ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase. Three independent experiments were performed, and the results were given as means ± SD.

| Statistical analysis
Data obtained from this study were expressed as means ± SD, and the statistical significance was analysed using a one-way ANOVA and an unpaired t test (GraphPad Prism, version 5.01, San Diego, CA, USA).
Differences were considered statistically significant at P < 0.05.

| MiR-302c was down-regulated in PMCs from the effluent of PD patients; miR-302c expression negatively correlated with MMT and CTGF expression
To confirm our previous findings from a miRNA array analysis, we measured miR-302c and MMT-related factors in the peritoneal mesothelial cells from the effluents of PD patients. The average duration of dialysis in the PD >1 year group (23.80 ± 13.26 months) was significantly longer than that in the PD start group (1.70 ± 0.82 months). Other clinical characteristics, including sex, age, BMI and D/PCr (4 hours) in the peritoneal equilibrium test, were not significantly different (Table 1). We found that there was a marked reduction in miR-302c levels with prolonged dialysis ( Figure 1A), as well as increased vimentin levels ( Figure 1B2 and C) and reduced Zo-1 levels ( Figure 1B3 Figure 1G).

| MiR-302c was down-regulated during peritoneal fibrosis in a mouse model of PD
We set up a PD mouse model according to our previous research and found that the submesothelial compact zone of the peritoneum was thickened with an extended time of PD (Figure 2A), and realtime PCR showed that miR-302c was down-regulated in the peritoneum of mice from the D30 group compared with the D0 group ( Figure 2B). Western blot analyses showed that MMT was present with increased vimentin ( Figure 2C2) and decreased Zo-1 levels (Figure 2C3), and the CTGF expression was up-regulated with time ( Figure 2C4). Similar results were also seen in their mRNA expression levels ( Figure 2D-F). These results suggested a correlation between miR-302c and MMT.

| MiR-302c overexpression protected the peritoneum from PD-induced peritoneal fibrosis in vivo
To determine the functional role of miR-302c in a mouse PD model of peritoneal fibrosis, we used LV-mmu-miR-302c to transfect mouse peritoneum and LV-pGIPZ as a control. It was found that obvious green fluorescence in the mesothelial cells and submesothelial compact zone of the peritoneum with the transfection of LV-mmu-miR-302c or LV-pGIPZ ( Figure 1A1). Meanwhile, miR-302c was obviously up-regulated in PD mice transfected with LV-mmu-miR-302c compared with those that received only PD fluid ( Figure 3B). This upregulation resulted in a significant attenuation of peritoneal fibrosis, as demonstrated by HE and Masson's trichrome staining (Figure 3A1). In the control group, the peritoneal tissues were almost normal and had no thickening of the submesothelial compact zone.
Compared with that in the control group, the submesothelial compact zone in the peritoneum of the PDF group was significantly thicker and enriched with numerous cells. However, the intraperitoneal injection of LV-mmu-miR-302c suppressed the thickening of the submesothelial compact zone ( Figure 3A2). Moreover, the downregulation of E-cadherin and the up-regulation of α-SMA and collagen I during PD were alleviated ( Figure 3C2-C4 and D-F). The upregulation of CTGF induced by PDF was also alleviated by miR-302c overexpression ( Figure 3C5 and G).

MMT in vitro
MMT has been proven to be an important mechanism in the pathogenesis of peritoneal fibrosis during PD. We next examined the

| MiR-302c overexpression attenuates TGF-β1induced MMT and fibrogenesis in HPMCs through down-regulation of CTGF
To determine the functional role of miR-302c in the process of MMT and peritoneal fibrosis, we transfected HMrSV5 cells with LVhsa-miR-302c and then incubated them with 5 ng/mL TGF-β1 for 48 hours. We analysed miR-302c gene expression by detecting the green fluorescent protein (GFP) under a fluorescence microscope; we found that miR-302c was stably transfected in HMrSV5 cells We searched biological analysis software (www.microrna.org) and found that miR-302c has a biding site in the 3′UTR of CTGF genes ( Figure 5D), suggesting that miR-302c could regulate the expression of CTGF.
To clarify whether miR-302c modulates TGF-β1-induced MMT Masson's trichrome staining showed that the overexpression of miR-302c alleviates PD-induced peritoneal fibrosis (100× magnification). Under a fluorescence microscope, it was found that obvious green fluorescence in the mesothelial cells and submesothelial compact zone of the peritoneum with the transfection of LV-mmu-miR-302c or LV-pGIPZ. (A2) The graph indicates the quantification of the peritoneal thickness in the four groups. (B) Real-time PCR shows that the peritoneum transfected with LV-mmu-miR-302c overexpressed miR-302c compared to other groups. Western blot analysis (C1) of the overexpression of miR-302c inhibiting the PD-induced up-regulation of α-smooth muscle actin (α-SMA) and collagen I and the down-regulation of E-cadherin in the peritoneal membrane at the 30th day. In addition, the up-regulation of CTGF during PD was reversed by the overexpression of miR-302c, and the bar graphs (C2-C5) represent the α-SMA, collagen I, E-cadherin and CTGF protein band densities relative to β-actin. The values are the mean ± SE (n = 5). Similar results were shown by real-time PCR (D-G), *P < 0.05 vs Control group. # P < 0.05 vs PDF group. Col I: collagen I; E-cad: E-cadherin up-regulating miR-192 in the mesangial cells of diabetic mice. 32 Moreover, a study of the MMT process in tubular epithelial cells showed that the level of let-7d miRNA was significantly reduced by TGF-β stimulation, and the overexpression of let-7d could suppress TGF-β-induced MMT and renal fibrogenesis. 33  In clinical settings, PD effluent markers were used to assess peritoneal functionality and morphology. Established effluent biomarkers include cancer antigen 125 (CA125), VEGF, hyaluronan (HA), interleukin-6 (IL-6) and tumour necrosis factor (TNF-α). [36][37][38] In various studies, PD effluent was also used as a source of biomarkers for the early detection of long-term alterations in the peritoneum and peritoneal fibrosis. In our previous study, aberrant miRNA levels associated with PD were observed using a miRNA array analysis of cultured PMCs from the effluent of PD patients. MiR-302c was found to be down-regulated nearly threefold in patients undergoing PD for more than 1 year when compared with patients who had recently started PD. We further demonstrate that the expression levels of miR-302c are decreased in both PD patients and a cultured human peritoneal mesothelial cell line treated with TGF-β1; however, miR-302c expression is negatively correlated with the development of MMT and fibrosis during PD (Figures 1 and 2).
Lentivirus is a subtype of retrovirus and is originally developed There are many transcription factors and pathways involved in the TGF-β1-induced MMT process; these include ZEB1, SIP1, 40  Our previous study demonstrated the positive correlations between the levels of CTGF, TGF-β1 and FN proteins and the collagen thickness in PD rats. These studies indicated that CTGF is an important mediator implicated in the pathogenesis of peritoneal fibrosis.
In contrast to the diverse effects of TGF-β1, CTGF specifically targets the fibrosis pathway and is thus an attractive candidate for inhibiting the damage to the membrane. 48 Furthermore, increasing F I G U R E 5 Overexpression of miR-302c by lentivirus transfection inhibited TGF-β1-induced MMT and peritoneal fibrosis in HMrSV5 cells through down-regulation of CTGF. (A) LV-hsa-miR-302c was used to stably transfect HMrSV5 cells, and GFP was examined under a fluorescence microscope; the overexpression of miR-302c alleviated the fibroblast-like morphological changes in HPMCs. (B) Real-time PCR indicated that LV-hsa-miR-302c-transfected HMrSV5 cells overexpressed miR-302c compared to the other groups. Western blot analysis (C1) showed that the overexpression of miR-302c alleviated TGF-β1-induced MMT and fibrosis and prevented the down-regulation of E-cadherin and the up-regulation of α-smooth muscle actin (α-SMA) and collagen I in HMrSV5 cells; moreover, the up-regulation of CTGF induced by TGF-β1 was reversed by the overexpression of miR-302c, the bar graphs (C2-C5) represent the α-SMA, collagen I, E-cadherin and CTGF protein band densities relative to β-actin. The values are the means ± SE (n = 3). Similar results were shown by real-time PCR (E-H); (D) Location of the predicted miR-302c target site in the CTGF 3′UTR, as assessed by biological analysis (www. microrna.org). (I) Vimentin and Ecadherin protein expression in HPMCs treated with TGF-β1, LV-miR-302c and recombinant CTGF by western blot analysis. *P < 0.05 vs Control group. # P < 0.05 vs TGF-β1 group. Col I: collagen I; Ecad: E-cadherin the expression levels of CTGF can induce partial MMT in renal tubular cells. This process cannot be blocked by neutralizing anti-TGF-β1 antibodies, suggesting that this action is TGF-β1 independent. From these studies we can infer that CTGF may be a better therapeutic target for peritoneal fibrosis than TGF-β1.
Our study indicated that the levels of CTGF were significantly increased in both a cultured human peritoneal mesothelial cell line treated with TGF-β1 and the peritoneal tissue of a PD mouse model. The upregulation of CTGF is tightly correlated with the expression of MMTrelated genes and proteins and is negatively correlated with the expression of miR-302c. In addition, we observed that the overexpression of miR-302c decreased the CTGF mRNA and protein levels. These data indicate that miR-302c may exert a protective role in the MMT during PD by regulating the expression of CTGF, and the TGF-β1/miR-302c/CTGF pathway may take part in modulating the process of MMT during PD.
However, one study has demonstrated that TGF-β regulates CTGF through increasing the expression of the miR-302 cluster. 49 MiR-302c decreased the TGF-β-induced MMT in renal epithelial cells and the TGF-β-induced mesangial production of fibronectin and thrombospondin. These results indicate a feedback loop between TGF-β and miR-302c that needs further study in the process of MMT and fibrosis in PD.
In summary, our study suggests that miR-302c is a vital factor protecting MCs from undergoing TGF-β1-induced MMT during PD.
We also demonstrated that miR-302c may inhibit TGF-β1-induced MMT by suppressing the expression of CTGF. Therefore, our data provide new information regarding the mechanism of peritoneal fibrosis during PD and may offer a new therapeutic target.