LncRNA UCA1 promotes renal cell carcinoma proliferation through epigenetically repressing p21 expression and negatively regulating miR-495

Abstract

Long non-coding RNAs have recently emerged as important regulators in the pathogenesis and progression of cancers. The long non-coding RNA urothelial carcinoma–associated 1 is reportedly upregulated and functions as an oncogene in some tumors. However, the role of urothelial carcinoma–associated 1 in renal cell carcinoma is not well elucidated so far. In this study, we found that urothelial carcinoma–associated 1 was overexpressed in renal cell carcinoma tissues compared with the adjacent normal tissues, and higher urothelial carcinoma–associated 1 expression levels were positively associated with advanced tumor stage and poor survival time in renal cell carcinoma patients. Further studies showed that knockdown of urothelial carcinoma–associated 1 suppressed renal cell carcinoma cell proliferation and S-phase cell number in vitro. Moreover, urothelial carcinoma–associated 1 was found to be associated with enhancer of zeste homolog 2, which suppressed p21 expression through histone methylation (H3K27me3) on p21 promoter. We also showed that knockdown of urothelial carcinoma–associated 1 increased the p21 protein expression through regulating enhancer of zeste homolog 2. In addition, bioinformatics analysis and dual-luciferase reporter assays confirmed that miR-495 was a target of urothelial carcinoma–associated 1 in renal cell carcinoma, and urothelial carcinoma–associated 1 promoted cell proliferation by negatively regulating miR-495. These findings illuminated that urothelial carcinoma–associated 1 promoted renal cell carcinoma progression through enhancer of zeste homolog 2 and interacted with miR-495. Overall, overexpression of urothelial carcinoma–associated 1 functions as an oncogene in renal cell carcinoma that may offer a novel therapeutic target for renal cell carcinoma patients.

Introduction

Renal cell carcinoma (RCC), accounting for 3% of all human malignancies, is one of the 10 most common cancers and ranks the third most prevalent genitourinary cancers. ¹ Localized RCC remains a surgical disease and about 20%–30% patients who present with limited disease at the time of nephrectomy develop metastasis. The median survival time to relapse after nephrectomy is about 15–18 months; patients with advanced RCC have a poor 5-year survival rate. ² Although there are a number of new therapeutic options for RCC, for example, tyrosine kinase inhibitors and angiogenesis inhibitors, only an unsatisfactory response rate can be achieved so far. ³ Compared with other tumors, there are very few tumor biomarkers for renal cancer. ⁴ Therefore, novel biomarkers and therapeutic targets are urgently required to guide clinical decisions.

Long non-coding RNAs (lncRNAs), that are more than 200 bases in length, consist of exons and introns in structure, without ORFs, and are not highly conserved. Recent reports suggest that lncRNAs play an important role in regulation of diverse cellular processes such as cell growth and apoptosis and cancer metastasis. ⁵ Several lncRNAs have been reported to be participated in renal cancer, such as, Qiao et al. ⁶ found that a decrease in lncRNA growth arrest–specific 5 (GAS5) expression was associated with RCC genesis and progression and overexpression of GAS5 can inhibit the RCC progression. A recent study indicated that LncRNA metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) could function as a competing endogenous RNA to regulate zinc finger e-box binding homeobox 2 (ZEB2) expression by sponging miR-200s in clear cell kidney carcinoma. ⁷ HOX transcript antisense RNA (HOTAIR) was overexpressed in RCC, and knockdown of HOTAIR led to the weakening of the recruitment and binding abilities of enhancer of zeste homolog 2 (EZH2) and H3K27me3 locus with lncRNA HOTAIR and inhibited cell proliferation. ⁸

Human urothelial carcinoma–associated 1 (UCA1) was first reported to be overexpressed in bladder cancer and was suggested to serve as a biomarker for the diagnosis of bladder cancer. ⁹ UCA1 was also highly expressed in some cancer including breast tumor, ¹⁰ colorectal cancer (CRC), ¹¹ and esophageal squamous cell carcinoma, ¹² suggesting that UCA1 may play a common important role in human cancers. A previous report showed that UCA1 expression levels were significantly increased in RCC tissues; ¹³ however, the role of UCA1 in RCC remains to be well elucidated.

In the study, we found that UCA1 was overexpressed in RCC tissues compared with the adjacent normal tissues and negatively associated with the over survival time. Further studies showed that UCA1 silencing decreased RCC cell proliferation and S-phase cell number. We also showed that knockdown of UCA1 increased the p21 expression through regulating EZH2 and promoted cell proliferation by negatively regulating miR-495. Overall, UCA1 functioned as an oncogene in RCC that may offer a novel therapeutic target for RCC patients.

Methods

Patient samples

A total of 50 cases of RCC tissues and the adjacent normal tissues were obtained from March 2006 to January 2010 at Department of Urology at the Affiliated Hospital of Hebei Engineering University. The tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C for RNA detection after surgery. None of the patients had undergone chemotherapy or radiotherapy prior to surgery. Informed consent was obtained from the all patients, and the research procedure was approved by the Medical Ethics Committee of the Affiliated Hospital of Hebei Engineering University. All of the RCC cases were clinically and pathologically identified to be of the clear cell type by two senior pathologists. Clinical stages and T stages were determined according to the 2011 Union for International Cancer Control (UICC) tumor–node–metastasis (TNM) classification guidelines.

Cell culture

Human RCC cell lines (786-O, ACHN, Caki-1, and Caki-2) were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (CCCAS, Shanghai, China). Immortalized normal human proximal tubule epithelial cell line HK-2 was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). The RCC cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). HK-2 cells were cultured in keratinocyte-SFM (Gibco/Invitrogen, Vienna, Austria) with 100 U/mL of penicillin and 100 µg/mL of streptomycin. Cells were cultured in a sterile incubator maintained at 37°C in 5% CO₂.

RNA isolation and quantitative reverse transcription polymerase chain reaction assay

Total RNA was extracted from cells or tissue specimens using RNAiso Plus (TaKaRa, Dalian, China), RNA (1 µg) was reverse transcribed in a final volume of 20 µL using random primers under standard conditions for the PrimeScript RT Reagent Kit (TaKaRa). The SYBR Premix Ex Taq (TaKaRa) was used to detect the expression of UCA1 and p21, according to the manufacturer’s instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was normalized to messenger RNA (mRNA) expression. The mRNA expression was detected using an Applied Biosystems 7500 Detection System, and the 2^−ΔΔCt method was chosen to calculate the relative expression levels (7500 Fast; Applied Biosystems, Foster City, CA, USA). The primer sequences used are given in Supplement Table 1. All results were repeated three times. All results are shown as the mean ± standard deviation (SD) of three independent experiments.

Cell transfection and luciferase reporter vector construct

RNA interference was conducted using synthetic small-interfering RNA (siRNA) oligo. Two synthetic siRNA oligos against UCA1 and a negative control sequence are given in Supplement Table 1. The siRNA was transfected into RCC cells in a six-well plate using the Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). After 48 h of transfection, the efficiency of knockdown was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). To construct luciferase reporter vectors, UCA1 complementary DNA (cDNA) fragment containing the predicted potential miR-495 binding sites or mutant sites was amplified by PCR and then cloned to pMIR-Report Luciferase Vector in MluI/HindIII sites (Ambion, Austin, TX, USA).

Cell proliferation assays

The CCK-8 assay (TransGen, Beijing, China) was used to examine cell proliferation according to the manufacturer’s instructions; 5 × 10³ cells were seeded at 96-well plates at 37°C for 24 h and then were transfected with si-NC, si-UCA1, or si-UCA1 + miR-495. After 24, 48, 72, and 96 h, 10 µL CCK-8 was added to the plates and incubated at 37°C for 2 h. The absorbance at 450 nm was detected by automatic microplate reader (Bio-Rad, Hercules, CA, USA).

Cell cycle analysis

ACHN cells transfected with si-NC, si-UCA1, and si-UCA1 + miR-495 were stained with propidium iodide (PI) using the CycletEST PLUS DNA Reagent Kit (BD Biosciences, Becton Dickinson, Franklin Lakes, NJ, USA) following the protocol and analyzed by FACScan. The percentage of the cells in G0/G1, S, and G2/M phases was counted and compared.

Western blotting analysis

The western blotting assays were performed according to the previous report. ¹⁴ The protein levels of p21 were analyzed by western blotting using a rabbit polyclonal anti-human p21 (1:1000; Cell Signaling Technology, USA). Normalization was performed by blotting the same samples with an antibody against β-actin (1:1000; Cell Signaling Technology, Danvers, MA, USA).

Luciferase reporter assay

The ACHN cells seeded in the 96-well plate were transfected with wild-type UCA1 or wild-type UCA1 + miR-495 mimic or mutant UCA1 or mutant + miR-495 mimic fragment using Lipofectamine 3000 (Invitrogen). After 48 h of transfection, cells were harvested and luciferase activity was measured by chemiluminescence in a luminometer (PerkinElmer Life Sciences, Boston, MA, USA) using the dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.

RNA immunoprecipitation assay

RNA immunoprecipitation was performed in 786-O and ACHN cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA), and the EZH2, lysin-specific demethylase 1 (LSD1), and SUZ12 (Millipore) antibodies were used according to the manufacturer’s protocol; normal mouse IgG (Millipore) was used as a negative control and anti-snRNP70 was used as a positive control (Millipore). RNA immunoprecipitation assay (RIP) was performed to investigate whether ribonucleoprotein complex contained UCA1 and its potential binding protein (EZH2) in renal cancer cells. ¹⁵ Samples were incubated with Proteinase K with shaking to digest the protein, and then, immunoprecipitated RNA was isolated. Final analysis was performed using qRT-PCR and shown as fold enrichment of UCA1.

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed using an EZ-ChIP Kit (Millipore) according to the manufacturer’s instructions. The RCC 786-O and ACHN cells were treated with formaldehyde and incubated for 10 min to generate DNA–protein cross-links. Cell lysates were then sonicated to generate chromatin fragments and immunoprecipitated with EZH2-, H3K27me3-, and LSD1-specific antibody (Millipore) or IgG as control. Precipitated chromatin DNA was recovered and analyzed by qRT-PCR.

Statistics analysis

The statistical analysis was performed by SPSS 17.0 (IBM SPSS Statistics Software, Somers, NY, USA). Expression of UCA1 and miR-495 was calculated and measured by parametric (Student’s t-test) and non-parametric tests for paired samples (Wilcoxon test). In all cases, p values <0.05 were considered as statistically significant.

Results

The UCA1 expression levels are upregulated in RCC tissues and cells

Initially, we detected the UCA1 expression levels in 50 RCC tissues and matched adjacent normal tissues using qRT-PCR analysis. The results showed that UCA1 expression was significantly upregulated in renal cancer tissues compared with adjacent normal tissues (Figure 1(a), p < 0.05). Next, we investigated the correlation between UCA1 expression and clinical factors in RCC patients. The UCA1 expression was classified as higher expression and lower expression groups according to UCA1 median expression levels. Clinicopathological analysis showed that higher UCA1 expression was significantly correlated with advanced TNM stage (p < 0.05; Table1), whereas there was no significant correlation between UCA1 and other clinicopathological characteristics such as gender, age, and vein invasion (Table 1). Moreover, we demonstrated that the overall survival time was also significantly shorter in the higher UCA1 expression group compared with the lower UCA1 group (Figure 1(b)). Furthermore, UCA1 expression was detected in four different RCC cell lines (786-O, ACHN, Caki-1, and Caki-2) and normal human proximal tubule epithelial cell line HK-2. The results showed that UCA1 expression level was significantly higher than that in HK-2 cell (Figure 1(c)). The knockdown of UCA1 in 786-O and ACHN was used by two si-UCA1-1 and si-UCA1-2. The siRNA-UCA-2 had a higher suppression of UCA1 and then was used in the following experiment for knockdown of UCA (Figure 1(d) and (e)). We also observed significant upregulation of UCA1 by transfection of pcDNA3.1-UCA1 plasmid into 786-O and ACHN cells (Figure 1(f)).

Figure 1.

The UCA1 expression levels are upregulated in RCC tissues and cells. (a) The UCA1 expression levels were measured by qRT-PCR assays in 50 cases of RCC tissues compared to adjacent non-tumor tissues. UCA1 expression was normalized to GAPDH (*p < 0.05). (b) Kaplan–Meier curves and log-rank test were used for the overall survival (OS) time of 50 cases of RCC patients, divided according to UCA1 median expression levels. (c) The four RCC cell lines (786-O, ACHN, Caki-1, and Caki-2) and normal human proximal tubule epithelial cell line HK-2 were detected by qRT-PCR assays, and UCA1 expression was normalized to GAPDH (*p < 0.05). (d and e) The UCA1 expression was evaluated by qRT-PCR after transfecting si-NC, si-UCA1-1, and si-UCA1 into 786-O or ACHN cells, and UCA1 expression was normalized to GAPDH (*p < 0.05). (f) The UCA1 expression was evaluated by qRT-PCR assays by transfecting pcDNA3.1-NC or pcDNA3.1-UCA1 into 786-O and ACHN cells, and UCA1 expression was normalized to GAPDH (*p < 0.05). Error bars represent the mean ± SD of triplicate experiments.

Table 1.

Correlation between the LncRNA UCA1 expression and clinicopathological characteristics in RCC patients.

Characteristics	Patients number	UCA1 expression (N = 50)		p value
		Lower (n = 26)	High (n = 24)
Gender				0.832
Female	18	9	9
Male	32	17	15
Age (years)				0.924
⩽60	33	17	16
>60	17	9	8
Grade				0.420
G1, 2	36	20	16
G3, 4	14	6	8
Vein invasion				0.817
Yes	20	10	10
No	30	16	14
TNM stage				0.030**
T1–T2	35	22	13
T3–T4	15	4	11

TNM: tumor–node–metastasis.

**

p < 0.05.

UCA1 binds and interacts with EZH2 in RCC cells

Previous reports have shown that HOTAIR could bind to polycomb-repressive complex 2 (PRC2; EZH2, SUZ12, and EED) enhancing methylation of H3K27me3, resulting in gene silencing. ¹⁶ Furthermore, we detected the distribution of UCA1 in RCC cells. According to the distribution of GAPDH and U1, the nucleus or cytoplasm was separated. The results showed that UCA1 existed in both the nucleus and the cytoplasm in ACHN cells (Figure 2(a)). To investigate potential interaction between UCA1 and EZH2, RIPs were performed in 786-O and ACHN cells. We showed that UCA1 was significantly enriched with the EZH2, LSD1, and SUZ12 antibodies compared with IgG (control antibody) in 786-O and ACHN cells (Figure 2(b) and (c)), while U1 binding with SNRNP70 was used as positive control (Figure 2(d)). These results suggested that UCA1 could epigenetically repress underlying targets’ expression at transcriptional level.

Figure 2.

UCA1 binds and interacts with EZH2 in RCC cells. (a) Subcellular localization of UCA was detected using fractionation. After nuclear and cytoplasm separation in ACHN cells, RNA was extracted from fractions and UCA1 expression was measured by qRT-PCR. GAPDH was used as a cytoplasm marker and U6 was used as a nucleus marker. (b and c) RNA levels in immunoprecipitates with EZH2, LSD1, and SUZ12 were determined by qRT-PCR in 786-O and ACHN cells. Expression levels of UCA1 were presented as fold enrichment relative to IgG immunoprecipitates in 786-O and ACHN cells. (d) The U1 binding with SNRNP70 was used as positive control and anti-IgG as a negative control. (e and f) The levels of p21 were detected by qRT-PCR during knockdown of UCA1 in 786-O and ACHN cells, and the results are represented as the average ± SD based on three independent experiments (*p < 0.05).

UCA1 inhibits the expression of p21 via regulating EZH2 in RCC cells

The underlying target genes of UCA1 in RCC cells are further explored. P21 had been reported to be inhibited by EZH2, ¹⁷ which could be a target of UCA1. After knockdown of UCA1, the qRT-PCR results showed that expression of p21 was upregulated in 786-O and ACHN cells (Figure 2(e) and (f)). Furthermore, the western blot assays showed the same results after knockdown of UCA1 in 786-O and ACHN cells (Figure 3(a)). Moreover, we demonstrated that knockdown of EZH2 and LSD1 expression led to increased p21 expression in 786-O and ACHN cells (Figure 3(b)). To further detect whether UCA1 inhibited p21 expression through interacting with EZH2 and LSD1, the ChIP analysis was carried out. The results demonstrated that EZH2, H3k27me3, and LSD1 could directly bind to p21 promoter region (Figure 3(c)). However, knockdown of UCA1 reduced their binding ability and H3K27 trimethylation and demethylation modifications (Figure 3(d)). The protein expression of p21 was inhibited by overexpression of UCA1, but cotransfection of pcDNA-UCA1 and si-EZH2 reversed the effects in 786-O and ACHN cells (Figure 3(e)). Therefore, these data indicated that UCA1 repressed p21 expression via EZH2 in RCC cells.

Figure 3.

UCA1 inhibited the expression of p21 via regulating EZH2 in RCC cells. (a) The p21 protein expression in 786-O and ACHN cells was detected by western blotting analysis after knockdown of UCA1. (b) The p21 expression levels were detected by qRT-PCR after knockdown of EZH2 and LSD1 in 786-O and ACHN cells. GAPDH was normalized for mRNA expression. (c and d) Chromatin immunoprecipitation–qPCR was used to analyze the EZH2 and LSD1 occupancy, H3K27me3 binding to the p21 promoter regions in 786-O and ACHN cells, and IgG as a negative control. (e) The p21 protein expression in 786-O and ACHN cells was detected by western blotting analysis after transfecting pcDNA 3.1-NC, pcDNA3.1-UCA1, or pcDNA3.1-UCA + si-EZH2. Results are represented as the average ± SD based on three independent experiments (*p < 0.05).

UCA1 promotes RCC cell proliferation by negatively regulating miR-495

Recent studies have indicated that lncRNAs may act as endogenous sponge RNA to interact with miRNAs.^7,15 To investigate the underlying mechanism of UCA1 in RCC cell, we found that there is a binding site for miR-495, according to the predicted online software by miRanda (http://www.microrna.org/) (Figure 4(a)). MiR-495 is a tumor suppressor gene reported in RCC. ¹ Our results found that miR-495 was downregulated in RCC tissues compared to matched normal tissues (Figure 4(b)). Interestingly, UCA expression was negatively correlated with miR-495 (r = −0.355, p < 0.05; Figure 4(c)). Furthermore, we evaluated the expression of miR-495 by overexpression or knockdown of lncRNA UCA1. When the UCA1 was knocked down, the expression level of miR-495 was significantly increased in 786-O and ACHN cells (Figure 4(d)). However, the expression level of miR-495 was significantly decreased after overexpression of UCA1 (Figure 4(e)). We also detected the expression of UCA1 by transfecting miR-495 mimic or miR-495 inhibitor, and the results showed that miR-495 mimic or miR-495 inhibitor had less effect on UCA1 expression (Figure 4(f)). The results indicated that UCA1 was the upstream targets of miR-495. The luciferase assays were then used to evaluate the possibility of bioinformatical prediction in ACHN cells. The results revealed that the miR-495 mimic significantly suppressed the luciferase activity of UCA-wt, but had less effect on the UCA-mut (Figure 5(a)). Taken together, these results suggested that lncRNA UCA downregulated the RNA levels of miR-495 through directly binding to it.

Figure 4.

MiR-495 was a target of UCA1 in RCC cells. (a) Alignment of potential UCA1 sequences with miR-495 as identified by miRanda (http://microrna.org). UCA1 mutated at the putative binding site. (b) MiR-495 is downregulated in RCC tissues compared to levels in adjacent normal tissues as determined by qRT-PCR. The expression of miR-495 was normalized to U6. (c) The correlation between UCA1 and miR-495 expression was detected in 50 RCC tissues (r = −0.355, p < 0.05). The ΔCt values were subjected to Pearson’s correlation analysis. (d) The expression of miR-495 was increased after knockdown of UCA1 and (e) was decreased after overexpression of UCA1 in 786-O and ACHN cells. (f) The expression of UCA1 was not been affected after transfecting miR-495 mimic or miR-495 inhibitor into 786-O and ACHN cells. Results are represented as the average ± SD based on three independent experiments (*p < 0.05; n.s.: not statistically significant).

Figure 5.

UCA1 promoted RCC cell proliferation by negatively regulating miR-495. (a) Luciferase reporter activity was detected after transfected with pmirGLo-UCA-wt, pmirGlo-UCA1-wt + miR-495 mimic, pmirGLo-UCA-mut, or pmirGlo-UCA1-murt + miR-495 mimic into ACHN cells. Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. Error bars represent the mean ± SD of triplicate experiments (*p < 0.05). (b) CCK-8 cell proliferation was used to detect the cell proliferation ability by transfecting si-NC, si-UCA1, and si-UCA1 + miR-495 inhibitor into ACHN cells. Absorbance at 450 nm is shown. (c and d) The cell-cycle assays and analysis were performed by flow cytometry analysis after transfecting si-NC, si-UCA1, and si-UCA1+miR-495 inhibitor into ACHN cells. Results are represented as the average ± SD based on three independent experiments (*p < 0.05; ‘#’ or n.s.: not statistically significant).

Furthermore, we investigated the roles of UCA1 in cell proliferation in ACHN cells by performing CCK-8 cell proliferation assays. As expected, knockdown of UCA1 inhibited the cell proliferation; however, cotransfection of si-UCA1 and miR-495 inhibitor reversed the effect in ACHN cells (Figure 5(b)). In addition, Cell cycle analysis further confirmed that knockdown of UCA1 inhibited the S-phase cell number, but cotransfection of si-UCA1 and miR-495 inhibitor reversed the effect in ACHN cells (Figure 5(c) and (d)). These results suggested that UCA1 promoted the RCC cell proliferation by negatively regulating the miR-495.

Discussion

In recent years, lncRNAs have gained massive attention as a potentially novel and crucial regulators of gene expression and cellular processes. An increasing number of evidences have shown that lncRNAs were closely related to many human diseases, including cancer progression. ¹⁸ Their expression profiling in various cancer types have been widely examined, and many of these lncRNAs were correlated with cancer diagnosis and prognosis. ¹⁹ A study was performed with a total of 1240 patients from 15 articles that were included, and the results indicated that a significantly shorter over survival time was observed in patients with high expression level of UCA1 in various cancers. ²⁰ Overexpression of lncRNA UCA1 promoted osteosarcoma progression and correlates with poor prognosis. ²¹ Increased UCA1 was associated with tumor proliferation and metastasis and predicted poor prognosis in CRC. ²² In this study, we found that UCA1 was overexpressed in RCC tissues compared with the adjacent normal tissues, and higher expression levels of UCA1 were associated with advanced tumor stage.

Numerous studies suggest that UCA1 play critical roles in some cancer development and progression. Wang et al. ²³ found that upregulation of UCA1 contributed to progression of hepatocellular carcinoma through inhibition of miR-216b and activation of fibroblast growth factor receptor 1 (FGFR1)/extracellular signal–regulated protein kinase (ERK) signaling pathway. In epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer, UCA1 induced non-T790M acquired resistance to EGFR-TKIs by activating the AKT/mammalian target of rapamycin (mTOR) pathway. ²⁴ Another study found that UCA1 exerted oncogenic functions in non-small-cell lung cancer by targeting miR-193a-3p. ²⁵ Detecting the expression of UCA1 in plasma for early gastric cancer may be a novel diagnostic and predictive biomarker. ²⁶ In addition, recent evidence also showed that UCA1 contributed to the progression of oral squamous cell carcinoma via regulating WNT/β-catenin signaling pathway. ²⁷ Our results showed that knockdown of UCA1 inhibited the RCC cell proliferation and found that UCA1 could bind with EZH2 and recruit it to p21 promoter regions to repress its transcription in RCC cells. Moreover, UCA1 could negatively regulate the miR-495 expression and promote the cell proliferation by binding to miR-495.

EZH2, a core subunit of PRC2, is a histone methyltransferase that specifically catalyzes the trimethylation of lysine residue 27 of histone 3 (H3K27me3) of target genes, and previous study reported that HOTAIR could promote tumor cell invasion and metastasis by recruiting EZH2 and repressing E-cadherin in oral squamous cell carcinoma. ²⁸ Plasmacytoma variant translocation 1 (PVT1) was associated with enhancer of zeste homolog 2 (EZH2) and promotes cell proliferation through epigenetically regulating p15 and p16 by EZH2. ²⁹ Here, we further demonstrated that p21 transcription could be repressed by EZH2 mediated by UCA1 in RCC cells. In addition, our results also show that the UCA1 interacted with miR-495. Taken together, our findings indicated that UCA1 may function as an oncogene and its increased expression contributed to RCC development and progression.

In conclusion, our results demonstrated that UCA1 promoted RCC through EZH2 and negatively regulated the miR-495. Further study about UCA1 involved in RCC may offer a novel therapeutic target for RCC patients.