miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in renal cell carcinoma

Renal cell carcinoma (RCC) is the most prevalent malignancy of kidney and accounts for approximately 4% of all cancer diagnoses in adults. Previous studies demonstrated microRNA-195-5p (miR-195-5p) as a tumor suppressor which is deregulated in many human cancers. However, the role of miR-195-5p in RCC is largely unknown. In the present study, we demonstrated that miR-195-5p was downregulated and negatively correlated with advanced clinical stage in RCC. Overexpression of miR-195-5p significantly suppressed RCC cells growth in vitro and in vivo, induced apoptosis and enhanced chemosensitivity to sorafenib. Conversely, suppression of miR-195-5p exhibited a reverse effect. REGγ, a proteasome activator, was identified as a novel downstream target of miR-195-5p in RCC. Knockdown of REGγ inhibited proliferation, induced apoptosis, increased sorafenib chemosensitivity and suppressed the wnt/β-catenin pathway in RCC cells. Moreover, restoration of REGγ markedly abolished the effects of miR-195-5p in RCC, and the wnt/β-catenin pathway was suppressed by miR-195-5p overexpression while activated by miR-195-5p inhibition in RCC cells. Our findings suggest that miR-195-5p is critical in REGγ-mediated regulation of wnt/β-catenin pathway in RCC development and may serve as a novel target for RCC treatment.


INTRODUCTION
Renal cell carcinoma (RCC) is the most prevalent malignancy of kidney and accounts for approximately 4% of all cancer diagnoses in adults [1]. At present, surgical resection remains the most effective treatment method for RCC, due to the high resistance of the cancer to conventional chemotherapy and radiotherapy; however, approximately 20-30% of post-surgery treatment patients eventually develop local recurrence or distant metastasis [2,3]. It is estimated that one third of patients present with metastatic disease when first diagnosed with RCC, www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 38), pp: 63986-64000 Research Paper and the 5-year survival rate of metastatic RCC is less than 10% [4,5]. Therefore, the identification of novel therapeutic molecules for RCC may help understanding the pathogenesis of the disease and improving the outcome of the patients.
MicroRNAs (miRNAs) are a class of endogenous, approximately 22 nucleotides in length, single strand noncoding RNAs that regulate gene expression by binding to the 3'untranslated region (UTR) of the corresponding target genes [6,7]. miRNAs have been shown to be deregulated in various human cancers and may function as oncogenes or tumor suppressors in cancer progression [8]. microRNA-195-5p (miR-195-5p) is a member of the microRNA-15a/b/16/195/497 family that is located on chromosome 17p13.1 [9]. Recently, the aberrant expression of miR-195-5p and its tumor suppressive role were demonstrated in a majority of human cancers namely, breast cancer [10], prostate cancer [11] and hepatocellular carcinoma [12]. However, the function of miR-195-5p in RCC is largely unknown.
REGgamma (REGγ), also known as PSME3, or PA28gamma, was first discovered in the sera of systemic lupus erythematosus patients and identified as the Ki antigen [13]. It is a member of the 11S proteasome activator family that can stimulate the proteolytic activity of the 20S core proteasome, independent of ubiquitination and ATP consumption [14,15]. REGγ is overexpressed in multiple human cancers, such as colorectal cancer [16], breast cancer [17], thyroid cancer [18] and hepatocellular carcinoma [19], suggesting the potential roles of this protein in cancer development.
In the present study, we found that miR-195-5p was downregulated and negatively correlated with advanced clinical stage in RCC. miR-195-5p suppressed cell growth, induced apoptosis and enhanced chemosensitivity to sorafenib via REGγ-mediated regulation of the Wnt/βcatenin pathway in renal cell carcinoma. Our findings indicated that miR-195-5p may act as a tumor suppressor in RCC and may serve as a novel therapeutic target in RCC treatment.

miR-195-5p was downregulated and negatively correlated with advanced clinical stage in RCC
The expression of miR-195-5p was determined in 67 paired ccRCC tumor tissues and matched normal tissues in order to elucidate the potential role of miR-195-5p in the development of RCC. The results showed that miR-195-5p was significantly downregulated in RCC tumor tissues compared with adjacent normal tissues ( Figure 1A). And, we found that the miR-195-5p expression levels in 4 RCC cell lines (786-O, ACHN, caki-1 and A498) were markedly decreased compared to the immortalized primary human proximal tubular cell line HK-2 ( Figure 1B). In addition, TCGA database also indicated a significant lower miR-195-5p expression in RCC compared with non-tumor tissues ( Figure 1C). Next we investigated the realtionship between miR-195-5p expression level and clinicopathologic factors in the 67 ccRCC patients. We found that miR-195-5p expression was significantly correlated with clinical stage, histological grade, tumor stage and lymph node metastasis (P<0.05), but was not significant associated with patients' gender, age and tumor size (Table 1). Moreover, Kaplan-Meier analysis revealed that ccRCC patients with low miR-195-5p expression levels had a significantly shorter overall survival time than those with high miR-195-5p levels ( Figure 1D). These results indicated that miR-195-5p may play a tumor suppressive role in renal cell carcinoma.
To further define the suppressive role of miR-195-5p in renal cell carcinoma. 786-O cells were infected with a miR-195-5p lentiviral expression vector (LV-miR-195-5p) to overexpress miR-195-5p or the negative control lentivirus (LV-miR-NC). The infection efficiency was examined by fluorescent microscopy and miR-195-5p expression was evaluated by qRT-PCR ( Figure 2G). The cells were subsequently collected and implanted subcutaneously in 6-week-old nude mice. The xenograft tumor growth was estimated following 3 weeks of tumor implantation. A significant decrease in the size and weight of tumors was observed in the miR-195-5p overexpressed group compared with that in the control group ( Figure  2H, 2I). The relative miR-195-5p levels in xenograft tumors were confirmed to be upregulated in LV-miR-195-5p groups ( Figure 2J). The number of Ki-67-positive cells in tumors from miR-195-5p overexpression group was lower than that in the control group as shown by immunohistochemistry ( Figure 2K), indicating a tumor suppressor potential. www.impactjournals.com/oncotarget

miR-195-5p induced apoptosis and cell cycle arrest in RCC cells
The effects of miR-195-5p on RCC cells apoptosis and cell cycle distribution were determined by flow cytometry. Our results revealed an obvious increase of cell apoptosis in 786-O and caki-1 cells after overexpression of miR-195-5p, whereas suppression of miR-195-5p significantly inhibited RCC cells apoptosis ( Figure 3A, 3B). In addition, cell cycle analysis indicated that the percentage of cells in G0/G1 phase was markedly higher in the miR-195-5p mimics group and lower in the miR-195-5p inhibitors group compared with the control groups ( Figure 3C, 3D). These results suggested that miR-195-5p may suppress cell growth by inducing apoptosis and cell cycle arrest in RCC cells.

miR-195-5p enhanced RCC cells chemosensitivity to sorafenib
The standard first-line treatment for advanced RCC is the targeted therapy and sorafenib is widely used for targeted therapy in RCC patients. As miR-195-5p exhibited

REGγ is a direct downstream target of miR-195-5p in RCC
A bioinformatics approach was used to understand the mechanism of miR-195-5p in the suppression of RCC progression. A total of 3 bioinformatic databases including TargetScan, PicTar and miRanda were used in combination to predict the putative targets of miR-195-5p ( Figure 5A). REGγ, which was reported as an oncogene in many human cancers, was selected as a potential target of miR-195-5p among the screened candidate genes ( Figure  5B). Dual-luciferase reporter assay, qRT-PCR and western blot analysis were carried out to confirm the relationship between REGγ and miR-195-5p. Dual-luciferase reporter assay showed that the luciferase activity in 786-O cells was significantly reduced when co-transfected with miR-195-5p mimics and the wild type (wt) but not the mutant (mut) 3'-UTR of REGγ ( Figure 5C). This finding indicated that miR-195-5p could directly bind to the 3'-UTR of REGγ in RCC. In addition, the qRT-PCR results indicated that the mRNA level of REGγ is down-regulated after transfection with miR-195-5p mimics while up-regulated following transfection with miR-195-5p inhibitors in RCC cells ( Figure 5D, 5E). In addition, western blot analysis indicated that the REGγ protein level was downregulated following overexpression of miR-195-5p while upregulated following suppression of miR-195-5p ( Figure 5F, 5G). Furthermore, the immunohistochemical analysis of the xenograft tumor tissues generated from LV-miR-195-5p and LV-miR-NC 786-O cells revealed a marked reduction of REGγ expression when miR-195-5p was overexpressed ( Figure  5H). These findings collectively suggest that REGγ is a direct downstream target of miR-195-5p in RCC.

Knockdown of REGγ inhibited proliferation and increased chemosensitivity to sorafenib by suppressing the wnt/β-catenin pathway in RCC cells
REGγ expression was knocked down by siRNA and the efficiency was confirmed by qRT-PCR and western blot analysis ( Figure 6A, 6B). The results of the MTT assay demonstrated that knockdown of REGγ significantly inhibited cell proliferation of both 786-O and caki-1 cell lines ( Figure 6C). Flow cytometry results indicated that knockdown of REGγ obviously induced apoptosis ( Figure 6D) and cell cycle arrest ( Figure 6E) in RCC cells. Moreover, we estimated chemosensitivity of 786-O and caki cells to sorafenib following REGγ knockdown as described. After sorafenib treatment, cell viability and apoptosis were determined by the MTT assay ( Figure 6F) and flow cytometry ( Figure 6G, 6H), respectively. The results indicated that REGγ knockdown increased RCC cells chemosensitivity to sorafenib.
We have reported before that REGγ is involved in skin carcinogenesis via direct degradation of GSK-3β and modulation of the Wnt/β-catenin pathway in our previous study [20]. And the significant role of the Wnt/ β-catenin signaling in RCC initiation and development has also been well addressed by previous studies [21].
Moreover, previous studies have showed that Wnt/βcatenin pathway is involved with sorafenib sensitivity [22,23]. We presumed that REGγ may regulate Wnt/ β-catenin pathway to exert its effects in RCC as well and conducted western blot analysis to verify that. We observed an increased GSK-3β level, while β-catenin, c-Myc and CyclinD1 were decreased in RCC cells following knockdown of REGγ ( Figure 6I). These results suggested that knockdown of REGγ inhibited cell proliferation and increased chemosensitivity to sorafenib by suppressing the wnt/β-catenin pathway in RCC.

Restoration of REGγ markedly abolished the effects of miR-195-5p in RCC
To further confirm whether the roles of miR-195-5p in RCC was mediated by REGγ, we transfected pcDNA5-REGγ plasmid to restoring REGγ expression when miR-195-5p was overexpressed in 786-O cells ( Figure 7A). Data showed that restoration of REGγ significantly abolished the effects of miR-195-5p on RCC cells proliferation, apoptosis and cell cycle distribution ( Figure 7B, 7C and 7D). Consistently, the increase of chemosensitivity to sorafenib by miR-195-5p was also abolished by REGγ restoration in 786-O cells ( Figure 7E, 7F). In addition, we determined the effect of miR-195-5p on Wnt/β-catenin pathway. Our results revealed that overexpression of miR-195-5p significantly suppressed Wnt/β-catenin pathway, while suppression of miR-195-5p exhibited an opposite results in RCC cells ( Figure 7G, 7H). These results collectively suggested that miR-195-5p may exert its roles via REGγ-mediated regulation of Wnt/β-catenin pathway in RCC.

DISCUSSION
An increasing number of studies have shown that dysregulation of miRNAs is a common event in human cancers including renal cell carcinoma. miR-195-5p was reported as a tumor suppressor in various human cancers.
In our study, we found that miR-195-5p was significantly downregulated and negatively correlated with advanced clinical stage in RCC. That was consist with a previous study which reported that miR-195-5p expression is deceased in RCC by using TaqMan Low Density Arrays [24]. In addition, patients with low miR-195-5p expression level had a significantly shorter overall survival time than those with high miR-195-5p level, indicating the potential value of miR-195-5p as a prognosis predictor in RCC patients. The overexpression of miR-195-5p significantly inhibited cellular proliferation, in accordance with induction of apoptosis and cell cycle arrest in 786-O and caki-1 RCC cells. In addition, inhibition of miR-195-5p had a converse effect on RCC cellular proliferation, apoptosis and cell cycle distribution. Furthermore, overexpression of miR-195-5p significantly suppressed RCC tumorigenesis in a xenograft nude mice model. Our results indicated that miR-195-5p acted as a tumor suppressor in RCC development.
The resistance to chemotherapy drugs remains the major impediment towards successful cancer treatment. Emerging evidence suggests that miRNAs can modulate chemosensitivity via regulation of multiple target genes. For example, miRNA-185-5p was shown to modulate chemosensitivity of human non-small cell lung cancer to cisplatin via targeting ABCC1 [25]. While miRNA-155 can regulate cell survival, growth and chemosensitivity by targeting FOXO3a in breast cancer [26]. In addition, miR-146a was reported to enhance chemosensitivity in epithelial ovarian cancer via reduction of SOD2 expression [27]. In accordance with the aforementioned findings, miR-195-5p was also demonstrated to modulate chemosensitivity in various human cancers. It was reported that miRNA-195-5p sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w [28]. In breast cancer, miR-195-5p can increase the sensitivity of the cells to adriamycin treatment via Raf-1 inhibition [29]. Qu et al. also demonstrated that miRNA-195-5p chemosensitizes colon cancer cells to doxorubicin by targeting BCL2L2 [30]. As miR-195-5p acted as a tumor suppressor in RCC development, the effect of miR-195-5p in the modulation of chemosensitivity to sorafenib in RCC cells was investigated. In our study, miR-195-5p was shown to enhance the chemosensitivity of RCC cells to sorafenib by measuring cell viability and apoptosis following sorafenib treatment.
To explore the mechanism by which miR-195-5p exerts its effect in RCC, the potential target genes of miR-195-5p were predicted by 3 bioinformatic databases and REGγ was selected as a candidate gene. By using Dualluciferase reporter assay, qRT-PCR, western blot analysis and immunohistochemical staining of the xenograft tumor tissues, we demonstrated that miR-195-5p could directly bind to the 3'-UTR of REGγ and suppress its expression in RCC. In addition, knockdown of REGγ significantly inhibited proliferation, induced apoptosis and increased sorafenib chemosensitivity in RCC cells, and restoration of REGγ markedly abolished the effects of miR-195-5p in RCC. These results suggest that miR-195-5p may exert its function by targeting REGγ in RCC.
In skin carcinogenesis, we have showed that REGγ could modulate the Wnt/β-catenin pathway [20], which is also crucial in RCC development. The regulation of Wnt/β-catenin pathway by REGγ in RCC was estimated by western blot analysis. We observed that knockdown of REGγ markedly suppressed the wnt/β-catenin pathway in RCC cells. Given that REGγ could regulate the Wnt/βcatenin pathway and that REGγ was a direct target of miR-195-5p in RCC, we presumed that miR-195-5p may have effect on wnt/β-catenin pathway in RCC. Results of western blot showed that the wnt/β-catenin pathway was suppressed by miR-195-5p overexpression while activated by miR-195-5p inhibition in RCC cells. To the best of our knowledge, miRNA-195-5p was reported to suppress colorectal cancer cell proliferation via regulation of FGF2 and the Wnt/β-catenin pathway [31], indicating that the latter pathway may be involved in the tumor suppressive function of miR-195-5p. Our results suggest that miR-195-5p is critical in REGγ-mediated regulation of wnt/βcatenin pathway in renal cell carcinoma.
In summary, our study demonstrated that miR-195-5p was downregulated in RCC tissue samples compared with matched normal tissues. miR-195-5p suppressed cell growth, induced apoptosis and enhanced chemosensitivity to sorafenib via REGγ-mediated regulation of the Wnt/βcatenin pathway in renal cell carcinoma, which was summarized by the schematic model ( Figure 8). Our findings suggested that miR-195-5p acted as a tumor suppressor in RCC and may serve as a new target for RCC therapy.

Tissue samples
All tumor tissues and adjacent normal tissues were obtained from 67 patients with primary clear cell RCC (ccRCC) who underwent radical nephrectomy at the Department of Urology, Shanghai Tenth People's Hospital, Tongji University from 2007 to 2010. None of the patients received preoperative treatment. All samples were immediately snap-frozen in liquid nitrogen following surgery, and stored in liquid nitrogen until further use. Our work has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). The study was approved by the Ethics Committees of Shanghai Tenth People's Hospital and informed consent was obtained from each patient prior to his/her participation in the study.

Cell culture
Human RCC cell lines (786-O, ACHN, caki-1 and A498) were obtained from the Institute of Cell Research of the Chinese Academy of Sciences (Shanghai, China). The immortalized primary human proximal tubular cell line (HK-2) was obtained from the American Type Culture Collection (ATCC, Rockville, USA). The cell lines 786-O and caki-1 were cultured in RPMI-1640 medium (Gibco, USA), ACHN and A498 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) and HK-2 was cultured in F-12K medium (Gibco). All media were supplemented with 10% fetal bovine serum (Gibco), 50 U/ml of penicillin and 50 μg/ml of streptomycin (Invitrogen, USA). Cells were all incubated in a humidified incubator containing 5% CO 2 at 37 °C.

EdU proliferation assay
Cell proliferation was measured by the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) during DNA synthesis using the EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China). The assay was carried out as determined by the manufacture's instruction. Briefly, the transfected cells were seeded in 96-well plates and incubated with 50 μM EdU for 2 h prior to the fixation process. The cell nuclei were stained with Hoechst. Finally, the EdU positive cells were captured and quantified by fluorescence microscopy. Three independent experiments were conducted for each sample.

Flow cytometry
Cell apoptosis was determined using Annexin V-FITC apoptosis detection kit (BD Biosciences, Erembodegem, Belgium) in accordance with the manufacturer's instructions. Cells were collected, washed twice with cold PBS and resuspended in Annexin V-binding buffer. Subsequently, they were incubated with 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) in the dark at room temperature for 15 min. The apoptosis rate was analyzed by flow cytometry using BD FACS Calibur (Beckman Coulter, CA, USA). Whereas as regards the cell cycle distribution analysis, the cells were collected and resuspended in PBS containing PI and 50 μg/ml RNase A (Sigma-Aldrich) in the dark at 37 °C for 30 min. Flow cytometry was conducted to analyze cell cycle distribution. Each experiment was independently repeated 3 times.

Xenograft assays in nude mice
The hsa-miR-195-5p sequence was cloned into the lentiviral vector PHY-502 carrying a green fluorescent protein (GFP) sequence by Hanyin Co. (Shanghai, China). The recombinant has-miR-195-5p expression lentivirus (LV-miR-195-5p) and the negative control lentivirus (LV-miR-NC; Hanyin Co. Shanghai, China) were prepared and titered to 10 9 TU/ml (transfection unit). 786-O cells were infected with LV-miR-195-5p or LV-NC and then selected by puromycin (Sigma-Aldrich) in order to generate the www.impactjournals.com/oncotarget stably transfected miR-195-5p overexpressing cell lines. Subsequently, the cells were implanted in the dorsal flanking sites of male BALB/c nude mice (6 weeks) at a density of 2×10 6 cells in 100 μl of PBS. Following 3 weeks of tumor implantation, mice bearing tumors were sacrificed for the assessment of tumor size, weight and immunohistological examination. Athymic nude mice were provided by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China). The animal care and animal experiments were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Dual-Luciferase reporter assay
The 3'-UTR sequences of human REGγ mRNA, containing the putative miR-195-5p binding site, were amplified by PCR and cloned into the XhoI /BglII site of the pGL3 vector (Promega, Madison, WI, USA) in order to construct the wild-type REGγ 3'-UTR plasmid (wt 3'-UTR). The mutant REGγ 3'-UTR plasmid (mut 3'-UTR) was generated using the Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China). The cultured 786-O cells were cotransfected with wt or mut REGγ 3'-UTR plasmid, miR-195-5p mimics (miR-195-5p), negative control miR (miR-NC) and a control Renilla luciferase pRL-TK vector (Promega) using Lipofectamine 2000 reagent (Invitrogen). Following 48 h of incubation, the cells were harvested and lysed. The luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. The firefly luciferase fluorescence was normalized to Renilla luminescence from the same vector. Experiments were independently repeated 3 times.

Western blot
Equal amounts of total protein extracted from cells were loaded on a polyacrylamide gel (10%) and subjected to electrophoresis. The separated proteins were transferred to pure nitrocellulose (NC) membranes and blocked with 5% fat free dry milk in PBS for 1 h at room temperature. The membrane was subsequently incubated with primary antibody at 4 °C overnight. After washed three times with PBS-T, membranes were incubated with a fluorescentlabelled secondary antibody (1:5,000 dilutions) for 1 h at 4 °C. The specific signals that corresponded to the protein expression were visualized by a LI-COR Odyssey Infrared Imaging System. A total of 3 independent experiments were carried out.

Immunohistochemistry
Tumors excised from nude mice were fixed in 4% paraformaldehyde, dehydrated through a graded series of ethanol solution and embedded in paraffin. The sections were cut at 4 mm and stained with hematoxylin and eosin (H&E). The sections were incubated with primary antibody versus REGγor Ki-67 overnight at 4 °C for immunostaining experiments. Subsequently, the sections were incubated with biotinylated goat anti-rabbit antibody IgG for 20 min at room temperature and then for 30 min with Streptavidin-HRP peroxidase. Diaminobenzidine (DAB)-H 2 O 2 was used as a substrate for the peroxidase enzyme.

Statistical analysis
All statistical analyses were conducted using SPSS software (version 17.0, SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (Version 5.0, GraphPad Prism Software Inc., San Diego, CA). The data are presented as the mean ± standard deviation (SD). Statistical significance between normal and tumor tissues was determined using the non-parametric Mann-Whitney U-test. Statistical analysis between two groups was conducted using Twotailed unpaired Student's t-test. The relationship between miR-195-5p expression level and clinicopathologic factors was evaluated using Pearson's Chi-square test. Patient survival was evaluated using the Kaplan-Meier method and compared using logrank test. Significant differences were obtained for a p value less than 0.05 (*p < 0.05).