Long noncoding RNA PVT1 inhibits renal cancer cell apoptosis by up-regulating Mcl-1

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) is up-regulated in various human cancers, and our results indicated that PVT1 was up-regulated in clear cell renal cell carcinoma tissues. The Cancer Genome Atlas cohort analysis revealed that in clear cell renal cell carcinoma, higher PVT1 expression correlated with advanced TNM stage, histological grade, and poor survival. PVT1 knockdown promoted apoptosis, inhibited renal cancer cell proliferation, decreased Mcl-1, and increased cleaved caspase-3 and cleaved PARP. PVT1 increased Mcl-1 mRNA levels in renal cancer cells by promoting mRNA stability without influencing its transcription. in vitro, the enhanced apoptosis arising from PVT1 suppression was attenuated by overexpressing Mcl-1. In addition, in vivo experiments showed that PVT1 knockdown repressed xenograft tumor growth, while Mcl-1 overexpression partially rescued xenograft tumor growth. These results indicate the PVT1/Mcl-1 pathway inhibits renal cancer cell apoptosis in vitro and in vivo. PVT1 may thus serve as a novel biomarker, and the PVT1/Mcl-1 pathway may be a useful therapeutic target for clear cell renal cell carcinoma.


INTRODUCTION
Renal cell carcinoma (RCC) is the most common malignant neoplasm of the kidney, which accounts for 2-3% of all adult malignancies [1]. The incidence and mortality rate of RCC has increased in recent years, especially in young patients and those with high-grade disease [2,3]. RCC incidence in China has risen rapidly in populations >35 years of age, and there were 45,096 new RCC cases in China in 2011 [4]. Due to the lack of early detection and RCC prognostic markers, 25%-30% of patients have already developed metastases at the time of diagnosis. Radical nephrectomy is the principal and most effective treatment for RCC, but more than 40% of patients develop metastases after radical nephrectomy with poor prognosis [5]. Clear cell renal cell carcinoma (CCRCC) is the most common and most aggressive histologic subtype of RCC, which accounts for 75%-80% of RCC [6]. We investigated the molecular mechanisms of CCRCC formation and development to identify reliable biomarkers and novel therapeutic targets for CCRCC.

PVT1 was up-regulated in CCRCC tissues and was tightly associated with clinical significance of CCRCC
Compared with corresponding adjacent non-tumor tissues, PVT1 was up-regulated in 85.45% (47 of 55) of CCRCC tissues according to qRT-PCR ( Figure 1A). This finding was lower than the data from TCGA (97.2%) ( Figure 1B), but not significantly different. Data from TCGA demonstrated that PVT1 expression was up-regulated in CCRCC tissues compared to normal tissues (534 tumor tissues and 72 adjacent non-tumor tissues) ( Figure  1C). TCGA data showed that higher PVT1 expression was correlated with advanced TNM stage ( Figure 1D), histological grade ( Figure 1E), and poor survival ( Figure 1F). These results indicate that PVT1 might be tumorigenic and stimulate the development and progression of CCRCC.

PVT1 knockdown inhibited renal cancer cell proliferation and colony formation in vitro
To evaluate the function of PVT1 in renal cancer cell apoptosis and proliferation, two independent siRNAs against PVT1 were transfected into renal cancer cell lines 786-O and ACHN, respectively. PVT1 expression was reduced by siPVT1-1 or si-PVT1-2, which was confirmed by qRT-PCR analysis in both cell lines ( Figure 2A). CCK-8 and colony formation assays also showed that 786-O and ACHN cell proliferation and colony-forming efficiencies decreased with PVT1 knockdown ( Figure 2B and 2C).

Knockdown of PVT1 promoted renal cancer cell apoptosis by down-regulating Mcl-1
Mcl-1 is an anti-apoptotic member of the Bcl-2 family, which regulates apoptosis through both pro-apoptotic and anti-apoptotic factors [17]. Mcl-1 overexpression is one of the most common genetic abnormities in a variety of human cancers, and can be the cause of resistance to several chemotherapeutic agents [18]. In our study, flow cytometry showed that PVT1 down-regulation promoted cell apoptosis of 786-O and ACHN cells compared with the si-NC group. ( Figure 3A). Mcl-1 expression was down-regulated in the si-PVT1 group compared with si-NC group in 786-O and ACHN cells according to qRT-PCR ( Figure 3B). Western blot analysis showed that Mcl-1 was down-regulated with PVT1 knockdown, and that the expression of cleaved caspase-3 and cleaved PARP were increased in 786-O and ACHN cells ( Figure 3C). The results suggest that silencing PVT1 down-regulated Mcl-1 and promoted apoptosis.

PVT1 promoted renal cancer cell growth and inhibited apoptosis by promoting Mcl-1 in vivo
To evaluate the above phenomenon in vivo, we established xenograft tumor models in nude mice using the ACHN cell line with or without stable knockdown of PVT1 and stable Mcl-1 overexpression. We found that silencing PVT1 reduced xenograft tumor growth, which was partially rescued by Mcl-1 overexpression ( Figure  6A  revealed that Mcl-1 promotes PVT1-induced renal cancer cell growth and inhibits apoptosis in vivo ( Figure 6E).

DISCUSSION
Many long non-coding RNAs have been identified in various cancer genomes, and can be used as novel biomarkers and therapeutic targets for cancer [19]. PVT1 is among those long non-coding RNAs, which is overexpressed and associated with tumorigenesis and poor prognosis in a range of cancers [11][12][13][14][15][16]20]. TCGA cohorts revealed that among all cancer types, CCRCC showed the strongest up-regulation of PVT1, and the highest level of PVT1 correlated with poor clinical outcome [21]. Our data also showed that PVT1 was increased in CCRCC tissues versus the corresponding non-tumor tissues. PVT1 might stimulate the development and deteriorate the prognosis of CCRCC. Moreover, our study revealed that PVT1 knockdown could inhibit proliferation and induce apoptosis of renal cancer cells in vivo and in vitro.
Mcl-1 is an anti-apoptotic member of the Bcl-2 family [22], and it is up-regulated in a variety of human cancers [23] and highly expressed in many human cancer cell lines [24]. The up-regulation of Mcl-1 results in resisting cell death, increasing cell proliferation, and tumor cell survival [25]. Down-regulation of Mcl-1 has shown tumor growth inhibition in colon, lung, ovarian cancer cells and lymphoma cells by inducing apoptosis. Knockdown of Mcl-1 restores sensitivity to chemotherapy in chemoresistant cells [18].
Rather than enhancing transcriptional activity of the Mcl-1 promoter, our data showed that PVT1 enhanced the stability of Mcl-1 mRNA in renal cancer cells. RNA stability is affected by various factors such as RNases, RNA binding proteins, miRNAs, and lncRNAs. Subramanian D reported in 2008 that RNA binding protein CUGBP2 promoted Mcl-1 mRNA stabilization [26]. Our next study will investigate whether PVT1-enhanced Mcl-1 mRNA stability is associated with CUGBP2.
The present study found that PVT1 expression was increased in CCRCC, correlated with advanced TNM stage, histological grade, and poor survival of CCRCC. We also showed that PVT1 inhibits renal cancer cell apoptosis by enhancing Mcl-1 mRNA stability. These findings suggest that PVT1 may be oncogenic and a CCRCC biomarker, and that the PVT1/Mcl-1 pathway may serve as a novel therapeutic target for treating CCRCC.

Cell proliferation assays
Cells were seeded at a density of 2 × 10 3 cells/ well in a 96-well plate for 24 h, then transfected with si-PVT1 or negative control. Cell proliferation was tested with CCK-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. All experiments were performed at least three times.

Colony formation assay
Cells transfected with siRNAs were plated on 6-well plates (1 × 10 3 cells per well) and maintained in proper medium containing 10% FBS for 12 days. Colonies were fixed with methanol and stained by 0.1% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China). Visible colonies were photographed and counted manually.

Western blot assay and antibodies
Total protein was isolated from cells and tumor tissues with RIPA lysis buffer (Beyotime, Jiangsu, China). Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST, incubated with primary antibodies at 4°C overnight, and further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Proteins were visualized using ECL reagents (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Protein levels were normalized against α-tubulin. Antibodies of cleaved caspase-3, cleaved PARP, and α-tubulin were obtained from Cell Signaling Technology (CST, Danvers, MA, USA) and Mcl-1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Plasmid generation
The pEX-Mcl-1 and pEX negative control vectors were purchased from Genepharma (Shanghai, China) for ectopic expression in cells. The qRT-PCR assay was conducted to evaluate Mcl-1 expression. The fragment containing human Mcl-1 promoter regions (-1693~+208) was chemically synthesized and cloned into the pGL3-Basic vector, and the resulting plasmid was named pGL3-Mcl-1. The fragment containing human PVT1 was chemically synthesized and cloned into the pmiR-RB-REPORT (Ribobio, Guangzhou, china), and the resulting plasmid was named pmiR-RB-PVT1.

Luciferase reporter assay
ACHN cells were seeded in 24-well plates and grown to 70-80% confluence, and 12hours later they were transiently transfected using Lipofectamine3000 (Invitrogen). The cells were cotransfected with pGL3-Mcl-1 and monitor plasmid pRL-TK (Promega, Madison, USA). After 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, USA). Data are represented as the fold induction after normalizing the luciferase activity of the tested sample to that of the corresponding control sample. The transfection experiments were performed three times in triplicate.

In vivo xenograft experiments
All the experimental protocols were approved by the Institutional Animal Care and Committee of Xinqiao Hospital, The Third Military Medical University, Chongqing, P. R. China. Four-week old male BALB/c nude mice were randomly divided into four groups, with six mice in each group. ACHN cells stably transfected with LV3-shNC, LV3-shPVT1, shPVT1+vector, and shPVT1+Mcl-1 (5 × 10 6 cells per mouse) were injected subcutaneously into the right flanks of the mice. Tumor growth was monitored, and tumor size were measured every three days. Tumor volume was calculated using the formula of volume = (width 2 ×length×0.5). Thirty days after injection, the mice were sacrificed and tumor weights were measured for further analysis. The primary tumors were excised, and tumor tissues were used for qRT-PCR analysis, Western blot analysis, and immunostaining analysis.

Statistical analysis
The statistical analysis was performed using SPSS 16.0 by a blinded investigator. The data were expressed as the mean ± SD. When two groups were compared, Student's t test was used. When more than two groups were compared, one-way ANOVA followed by Tukey's Test was carried out. Monte Carlo simulation was employed to compare the expression between the tumor and non-tumor tissues. The Wilcoxon's Sign Rank Test was used to compare the expression difference among histological grades and pathological stages. The Long Rank Test analyzed the overall survival difference between higher and lower expression groups. A p-value of < 0.05 was statistically significant