MicroRNA-212 inhibits the proliferation, migration and invasion of renal cell carcinoma by targeting X-linked inhibitor of apoptosis protein (XIAP)

MicroRNAs have been found to be critical regulator of cancer cell biology. MicroRNA-212 (miR-212) was identified to be a critical cancer-associated microRNA playing either oncogenic functions or tumor suppressive roles in different types of human cancers. In this study, we found that the level of miR-212 in renal cell carcinoma (RCC) tissues was significantly lower than that in adjacent non-tumor tissues. Decreased level of miR-212 was associated with advanced T stage and TNM stage of RCC. The expression of miR-212 was decreased in RCC cell lines as compared with the HK-2 cell line. Overexpression of miR-212 inhibited cell viability, proliferation, migration and invasion of CAKI-2 cells. Knockdown of miR-212 increased cell viability and proliferation, migration and invasion of ACHN cells. In vivo experiments showed that miR-212 inhibited the proliferation and promoted the apoptosis of ACHN cells in nude mice and thus inhibited the in vivo tumor growth of CAKI-2 cells. Furthermore, we confirmed that X-linked inhibitor of apoptosis protein (XIAP) was the downstream target of miR-212. The expression level of miR-212 was negatively correlated with XIAP expression in RCC tissues. Moreover, XIAP mediated the tumor suppressive roles of miR-212 in RCC. Finally, we demonstrated that the aberrant expression of miR-212 and XIAP was evidently correlated with poor prognosis of RCC patients. In all, miR-212 can act as a prognostic biomarker for RCC patients and inhibits the growth and metastasis of RCC cells by inhibiting XIAP.


INTRODUCTION
Renal cell carcinoma (RCC), accounting for 2% to 3% of adults malignant tumors, is the most common type of cancer in adult kidney [1]. Clear cell renal cell carcinoma (ccRCC), compared with other three histologic subtypes including papillary RCC, chromophobe RCC, and oncocytomas, is the most aggressive and frequent RCC histologic subtype, accounting for around 80% of RCC cases [2]. RCC in early stage is curable by surgical resection, but around 20% RCC patients have the occurrence of metastasis at the time of diagnosis [3]. For RCC patients with metastasis, the 5-year survival rate was even lower than 10%. Additionally, 30% of RCC patients with localized RCC eventually developed distant metastases after receiving curative surgical resection [4]. However, it remains largely unknown regarding the molecular mechanisms of the progression of RCC. Therefore, it is of great importance to investigate the mechanisms of RCC growth and metastasis to identify novel early-diagnosis biomarkers and therapeutic targets for RCC patients.
MicroRNAs (miRNAs), a cluster of 20-24 nucleotide noncoding RNAs, post-transcriptionally inhibit the expression of targeted gene by preventing the translation of mRNAs or promoting the degradation of mRNAs [5,6]. Abnormal expression and function of miRNAs have been demonstrated to play important roles in the tumorigenesis and progression of many human cancers including RCC [7]. MiRNAs have been demonstrated to be critical regulators of the growth and metastasis of RCC, and are regarded as promising biomarkers and therapeutic targets of RCC [8,9]. Investigating the expression and function of miRNAs in RCC will contribute to identification of novel biomarkers and therapeutic targets for RCC patients.
MiR-212 is a novel cancer-associated microRNA and has been found to play critical roles in different types of human cancers. It was found to be down-regulated and play tumor suppressive roles in hepatocellular carcinoma [10], ovarian cancer [11], gastric cancer [12] and lung cancer [13,14]. On the other hand, miR-212 was also found to be overexpressed and play oncogenic roles in pancreatic cancer [15,16]. However, the expression and function of miR-212 in RCC remain unknown.
In this study, we identified that miR-212 was downregulated in RCC tissues and cells. MiR-212 inhibited the proliferation, migration and invasion of RCC cells.
In vivo studies also confirmed that miR-212 inhibited the growth of RCC cells in nude mice. X-linked inhibitor of apoptosis protein (XIAP) was found to be the direct downstream target of miR-212. The expression of XIAP in RCC tissues was negatively correlated with miR-212 level. MiR-212 exerted its tumor suppressive roles in RCC by targeting XIAP. Furthermore, Kaplen-Merier analysis demonstrated that both miR-212 down-regulation and XIAP overexpression predicted the poor prognosis of RCC patients.

XIAP is the downstream target of miR-212 in RCC
To clarify the molecular mechanisms underlying the tumor suppressive roles of miR-212 in RCC, we used the public database to search for the potentially downstream targets of miR-212. X-linked inhibitor of apoptosis protein (XIAP), a critical regulator of cell proliferation, apoptosis and motility [17][18][19], was predicted to be a potentially downstream target of miR-212. As shown in Figure 5A, XIAP 3'-UTR contained the complementary sequences for miR-212 binding. To validate miR-212 could indeed bind with the 3'-UTR of XIAP, we performed luciferase activity assay for wild type XIAP 3'-UTR and mutated XIAP 3'-UTR. Overexpression of miR-212 significantly decreased while miR-212 knockdown significantly increased the luciferase activity of wild type XIAP 3'-UTR (P<0.05, Figure 6B). In contrast, miR-212 overexpression or knockdown did not have any effect on the luciferase activity of mutated XIAP 3'-UTR ( Figure 6B). Furthermore, qRT-PCR and western blot confirmed that miR-212 overexpression decreased the mRNA (P<0.05, Figure 5C) and protein (P<0.05, Figure  5D) level of XIAP in CAKI-2 cells. MiR-212 knockdown increased the mRNA (P<0.05, Figure 5E) and protein (P<0.05, Figure 5F) level of XIAP in CAKI-2 cells. These data demonstrate that miR-212 could inhibit the expression of XIAP in RCC cells by interacting with the 3'-UTR of XIAP.

IAP expression is negatively correlated with miR-212 level in RCC tissues
To further confirm the regulatory effect of miR-212 on XIAP, we measured the expression level of XIAP in RCC tissues. qRT-PCR demonstrated that XIAP mRNA level was significantly increased in RCC tissues (P<0.05, Figure 6A). Correlation analysis for XIAP and miR-212 showed that the level of miR-212 was negatively correlated with the XIAP level in RCC tissues (P<0.05, Figure 6B). Western blot further demonstrated that in RCC tissues with high miR-212 level, the expression of XIAP was significantly decreased, compared with the tissues of low miR-212 level (P<0.05, Figure 6C). Taken together, these data demonstrate that miR-212 can inhibit XIAP expression in RCC.

MiR-212 downregulation and XIAP overexpression predict poor prognosis of RCC
After confirming the expression and function of miR-212 and XIAP in RCC, we lastly examined whether aberrant expression of miR-212 and its downstream target XIAP could predict the prognosis of RCC patients. The mean expression level of miR-212 and XIAP was defined as cutoff value to divide RCC patients into: miR-212 low group and high group, and, XIAP low group and high group. As shown in Figure 9A and 9B, decreased expression of miR-212 was correlated with decreased   rate of overall survival (OS) (P<0.05) and recurrent-free survival (RFS) (P<0.05). Furthermore, Kaplen-Merier analysis also demonstrated that patients with increased expression of XIAP had significantly decreased rate of OS (P<0.05, Figure 9C) and RFS (P<0.05, Figure 9D). These indicate that miR-212 and XIAP can both serve as prognosis predictor for RCC patients.

DISCUSSION
Accumulating studies have demonstrated miRNAs actively participated in the development and progression of various human cancers [20]. Identifying cancerassociated miRNAs and their biological functions in cancer cells can promote the identification of biomarkers and therapeutic targets for human cancers. In this study, we used miRNAs screen assay to identify the differentially expressed miRNAs between RCC tissues and non-tumor tissues. Among numerous miRNAs, miR-212 was one of the significantly downregualted miRNAs in RCC tissues. qRT-PCR further confirmed that the expression of miR-212 was significantly decreased in RCC tissues. In RCC tissues of advanced T stage and TNM stage, the level of miR-212 was significantly decreased. Furthermore, we demonstrated that miR-212 was significantly decreased in RCC cell lines. These data demonstrate that miR-212 is a tumor suppressive microRNA in RCC. Functionally, miR-212 was found to inhibit the growth of hepatocellular carcinoma by targeting FOXA1 [10]. Study of gastric cancer also demonstrated that miR-212 inhibited the proliferation of gastric cancer cells by inhibiting [21]. However, study of pancreatic cancer cells found that miR-212 promoted cell proliferation by targeting patched-1 [16]. In this study we demonstrated that overexpression of miR-212 could inhibit the viability, proliferation, migration and invasion of RCC cells while miR-212 knockdown promoted these biological functions of RCC cells. Therefore, miR-212 exerted its tumor suppressive roles in RCC by inhibiting cell viability, proliferation, migration and invasion.
To clarify the detailed mechanisms by which miR-212 exerts tumor suppressive effects in RCC cells, we further investigated the downstream target of miR-212. XIAP, an important regulator of cell apoptosis pathway, has been found to be aberrantly expressed in various types of human cancers [22][23][24][25]. Poorly differentiated carcinomas showed significantly elevated levels of XIAP than well differentiated carcinomas. XIAP expression in metastatic tissues was also significantly higher than that in primary tumor sites [26]. XIAP could inhibit the function of caspase-3, 7 and 9, and thus prevent cell apoptosis [27][28][29]. Interestingly, XIAP was also found to interact with the Rho GDP dissociation inhibitor (RhoGDI) to promtoe the cell motility [19]. In this study, qRT-PCR, western blot and luciferase activity assay demonstrated that XIAP was the direct downstream target of miR-212 in RCC. More importantly, XIAP was found to mediate the functional influence of miR-212 on cell viability, proliferation, migration and invasion. Therefore, these data indicate that miR-212 inhibits the proliferation, migration and invasion of RCC cells by targeting XIAP protein, and can potentially serve as a promising therapeutic target for RCC. It is worth to mention here that one miRNA potentially has multiple downstream targets. Previous stduies showed FOXA1, HBEGF, MECP2 and PED were the downstream targets of miR-212 in different types of cells. It is worth to investigate in the future whether the regulatory effects of miR-212 on these protein can be observed in RCC cells.
Increasing studies showed that miRNAs could serve as promising prognostic indicators of human cancers [30]. Therefore, we evaluated the prognostic value of miR-212 in RCC. Decreased expression level of miR-212 was correlated with poor prognosis of RCC patients. Additionally, XIAP overexpression in RCC was correlated with poor prognosis of RCC patients. These indicate that miR-212 and XIAP are promising prognostic predictors for RCC patients. It will be of great importance in the future to investigate whether miR-212 and XIAP can be employed as used as biomarkers for RCC early diagnosis.
In summary, this study finds that miR-212 is down-regulated in RCC and its decreased expression is associated with RCC progression. In vitro and in vivo studies confirm that miR-212 inhibit the proliferation, migration and invasion of RCC cells. Furthermore, we identify that XIAP protein was the direct downstream target of miR-212. Lastly, miR-212 and XIAP are prognostic predictors for RCC patients.

Clinical tissues
60 RCC samples and adjacent non-tumor tissues were collected after getting informed consent from the RCC patients. All patients received partial or radical nephrectomy in the Department of Departments of Urology and Henan Institute of Urology during January 2005 to December 2010. The clinical tissues were stored at liquid nitrogen after surgical resection. All protocols involving clinical tissues were approved by the Ethics Committee of Zhengzhou University.

Screen for differentially expressed miRNAs between RCC tissues and non-tumor tissues
TaqMan LDA Human microRNA Panel v2.0 (Applied Biosystems, Foster City, CA, USA) was used to identify the differentiated miRNAs between RCC tissues and adjacent non-tumor tissues. Three pairs of RCC tissues and adjacent non-tumor tissues were used for miRNAs screen. After generating cDNAs by reverse transcription, the TaqMan real-time PCR assay for these cDNAs was performed. GeneSpring GX software (version 7.3.1, Agilent Technologies) was used to analyze the relative miRNA expression. P < 0.05 was used to define the differentially expressed miRNAs.

MTS assay and BrdU assay
Cell viability was determined by MTS assay (Roche, USA) performed according to the manufacturer's instructions. The proliferative ability of RCC cells was determined by BrdU (5-bromodeoxyuridine) (chemiluminescent) (Roche, USA) assay based on manufacturer's instructions.
3D culture for cancer cells 300 μl of Matrigel was added to each well of 24well plates. After the gel was solidified, 5000 RCC cells suspended in 1mL RPMI 1640 medium were seeded into each well and 3D spheroids were formed after culturing the cells for 5 days. The spheroids were observed under the inverted microscope and pictures were captured. Relative sizes of these spheroids were compared using Image J software.

Migration and invasion assays
Cell migration and invasion were evaluated by Transwell assay. RCC cells were starved in basal medium for 24 hours before transwell assay. For invasion assay, matrigel diluted in basal medium (1:8 dilution) was used to coat the membrane of upper chamber. RCC cells suspended in basal medium were seeded into the upper chamber. 10% FBS used as chemoattractant was added into the bottom chamber. 12-24 hours later, RCC cells migrated or invaded through the chamber membrane were fixed with methanol and stained with crystal violet solution.

qRT-PCR
Total RNA was extracted from RCC tissues and cultured RCC cells using miRNeasy Mini Kit or RNeasy Mini Kit (Qiagen). Reverse Transcription for cDNA was performed using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) or iScript ™ cDNA Synthesis Kit (BIORAD). TaqMan Human MiRNA Assay Kit (Applied Biosystems) was used to evaluate the relative expression of miR-212 [31]. Platinum SYBR Green qPCR Supermix UDG kit (Invitrogen, Carlsbad, CA) was used to evaluate the relative level of XIAP. GAPDH and U6 were used as the internal control for XIAP and miR-212, respectively.

Western blotting analysis
Cellular protein were extracted from RCC tissues and cells using RIPA buffer (Thermo Scientifc, Rockford, IL, USA) supplemented with protease inhibitor (Beyotime Institute of Biotechnology, Haimen, China) and PMSF (Beyotime Institute of Biotechnology). Thirty micrograms of protein lysate were separated in 4-20% SDS-PAGE gels and transferred onto PVDF membranes (Beyotime Institute of Biotechnology). The membranes were incubated with primary antibodies including XIAP and GAPDH at 4°C overnight. Secondary antibodies conjugated with horseradish peroxidase (Boster Bio-engineering Limited Company, Wuhan, China) were incubated with the membranes for 2 hrs at room temperature. The protein signals were dectected using ECL kit (Beyotime Institute of Biotechnology).

In vivo experiments
BALB/c nude mice were used to establish the nude mouse xenograft model. CAKI-2 cells transfected with miR-212 overexpressing vector or control vectors were suspended in 100 uL PBS and were injected subcutaneously into the flank of nude mouse. The width and length of the tumor nodules were measured every three days. The volume of subcutaneous tumors was calculated as tumor volume = length × width × width/2. 3 weeks later, the xenograft tumor tissues were explanted for Ki67 staining (Cell Signaling, Danvers, MA, USA) and Tunel staining assay. In vivo protocols were approved by the Institutional Animal Care and Use Committee of Zhengzhou University.

Statistical analysis
The data in this study are presented as mean ± S.E.M. Statistical analysis including Pearson chi-squared test, a two-tailed Student's t test, a Kaplen-Merier plot and the ANOVA test were performed using SPSS software (Version 13, SPSS, Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, Inc, San Diego, CA, USA). P < 0.05 was regarded to be statistically significant

Author contributions
Chaohui Gu and Zhiyu Wang performed the in vitro experiments and wrote the manuscript; Zhibo Jin and Guanru Li performed the in vivo study; Yiping Kou, Zhankui Jia and Jinjian Jia analyzed the data; Fengyan Tian designed this study.