Renal cell carcinoma (RCC) accounts for approximately 3% of all malignancies and represents the most lethal urological cancer with approximately 202,000 cases and 102,000 deaths worldwide [
]. Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC and is responsible for nearly 85% of all RCC cases [
]. The wide application of ultrasound and computed tomography has shown that about one-third of ccRCC patients with newly diagnosed disease show evidence of metastases that are associated with a poor prognosis, and the median survival time for these patients is only 13 months [
]. Despite numerous studies that have shown that many genetic and epigenetic changes are associated with the development and progression of ccRCC, the molecular mechanism of renal cancer pathogenesis is still elusive, and the prognosis remains poor. Therefore, the identification of sensitive and specific ccRCC targets and the development of novel therapeutic strategies is urgently needed.
Long noncoding RNAs (lncRNAs) are a newly discovered class of noncoding RNAs (ncRNA) that are longer than 200 nucleotides and are not translated into proteins [
]. Mounting evidence has indicated that lncRNAs play important roles in diverse biological processes, such as cell growth, cell death, stem cell pluripotency, tumorigenesis and development [
]. The rapid development of high-throughput RNA sequencing and cancer genomics also highlighted the significance of lncRNA in various human cancers [
]. However, the molecular mechanism and clinical significance of lncRNA in ccRCC remains largely unknown.
PANDAR (promoter of CDKN1A antisense DNA damage activated RNA) is a relatively new lncRNA that is localized at 6p21.2 [
]. PANDAR is induced after DNA damage in a p53-dependent pattern, and it interacts with the transcription factor NF-YA to repress the expression of pro-apoptotic genes [
]. Both DNA damage and NF-YA are closely associated with tumorigenesis [
]. Therefore, PANDAR may play an important role in the development of cancers. Recently, it has been reported that the expression of PANDAR was downregulated in non-small cell lung cancer (NSCLC) and a low level of PANDAR was associated with a poor prognosis [
]. In contrast, PANDAR was found to be upregulated in hepatocellular and in bladder carcinoma, and a high level of PANDAR was associated with a poor prognosis [
]. These studies indicate that PANDAR plays controversial roles in cancers. Moreover, the role of PANDAR in ccRCC has not been previously investigated. These findings prompted us to study the role of PANDAR in ccRCC.
In the present study, we found that PANDAR was significantly upregulated in ccRCC tissues compared to corresponding normal tissues. The upregulation of PANDAR was correlated with an advanced TNM stage and with lymph node involvement and distant metastasis. In vitro studies showed that PANDAR could regulate cell proliferation, migration and apoptosis. Furthermore, we demonstrated that PANDAR could modulate the anti-apoptotic proteins Bcl-2 and Mcl-1, as well as the PI3K/Akt/mTOR pathway.
This study was approved by the Human Ethics Committee of First Affiliated Hospital of Zhejiang University. ccRCC tissues and normal tissues were obtained from 62 patients who underwent nephrectomy or partial nephrectomy for ccRCC between 2012 and 2016. Written informed consent was obtained from all individual participants included in the study. None of the patients received local or systemic treatment before surgery. All tissues were washed with sterile PBS before being frozen in liquid nitrogen and then stored at −80 °C until analyzed. The pathological stage and grade were evaluated by an experienced pathologist.
ccRCC cell lines 7860 and Caki-1 were obtained from the Shanghai bank of cell lines (Shanghai, China). The 7860 and Caki-1 cells were cultured in RPMI 1640 and DMEM medium, respectively, at 37 degree in a humidified atmosphere of 5% CO2.
RNA extraction and quantitative real-time PCR
Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was transcribed from total RNA using SuperScript III kit (Invitrogen). The primer sequences were as follows: PANDAR primers, forward: 5′- CTGTTAAGGTGGTGGCATTG-3′, reverse: 5′- GGAGGCTCATACTGGCTGAT-3′; and GAPDH primers, forward: 5′-CGCTCTCTGCTCCTCCTGTTC-3′, reverse: 5′- ATCCGTTGACTCCGACCTTCAC -3′. Quantitative real-time PCR was performed using the ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). The average value of each triplicate was used to calculate the relative amount of PANDAR using 2-ΔΔCt methods. Each sample was measured in triplicate.
Small interfering RNA (siRNA) and nonspecific control siRNA or short hairpin RNA (shRNA) were synthesized (Sangon, Shanghai, China) and transfected into cells using Lipofectamine 3000 (Invitrogen, USA). The target sequence of si-PANDAR was 5′-GCAATCTACAACCTGTCTT-3′. The cells were cultured 24 h prior to transfection. Stably transfected cells were selected using G418 (Amresco, OH, USA).
The lysates were resolved by 12% SDS-PAGE and then transferred to PVDF membranes. Primary antibodies against the following were used at 4 degree overnight: MMP-2 (Abcam, CA, USA); TIMP3 (Abcam, CA, USA); Cyclin D1 (Cellular Signaling Technology, MA, USA); Cyclin E1 (Cellular Signaling Technology, MA, USA); CDK4 (Cellular Signaling Technology, MA, USA); p21(Cellular Signaling Technology, MA, USA); Caspase-8 (Cellular Signaling Technology, MA, USA); Caspase-3 (Cellular Signaling Technology, MA, USA); cleaved PARP (Cellular Signaling Technology, MA, USA); Bcl-2 (Cellular Signaling Technology, MA, USA); Mcl-1 (Cellular Signaling Technology, MA, USA); Bax (Cellular Signaling Technology, MA, USA); p-PI3K (Cellular Signaling Technology, MA, USA); PI3K (Cellular Signaling Technology, MA, USA); p-Akt (T450) (Cellular Signaling Technology, MA, USA); p-Akt (S473) (Cellular Signaling Technology, MA, USA); Akt (Cellular Signaling Technology, MA, USA); mTOR (Cellular Signaling Technology, MA, USA) and GAPDH (Sigma, MO, USA). All chemicals were obtained from Sigma-Aldrich (MO, USA).
Cell proliferation assay
Cell proliferation was assayed using a CCK-8 kit (Beyotime Biotech, China). Briefly, 2 × 103 cells/well were seeded in 96-well plates 24 h before the start of the experiment. The cells were then transfected with the corresponding si-RNA and cultured in medium. At 0, 24, 48, 72 and 96 h after transfection, 10 μl of CCK-8 (5 mg/ml) was added to each well and the cells were cultured for 1 h, and the absorbance at 450 nm was determined.
Cell cycle analysis
Transfected cells were harvested after 48 h of incubation in 6-well plates. The cells were collected and fixed in ethanol. The cells were then washed with PBS and stained with propidium iodide (BD Bioscience) for 30 min in PBS supplemented with RNase at room temperature in the dark. The analysis was performed in triplicate, and the cell cycle distribution was evaluated using a flow cytometer (BD bioscience).
Transfected cells were harvested and double stained with an Annexin V Apoptosis Detection Kit (BD Bioscience). The cells were then analyzed using a flow cytometer (BD Bioscience).
Colony formation assay
For the colony formation assay, 1000 cells were plated into 6-well plates and incubated in media. One week later, the cells were fixed and stained with 0.1% crystal violet and the visible colonies were counted.
Cell invasion assays
The cell invasion assay was performed using 24-well insert transwell chambers (Corning, NY, USA). Cells were suspended in 200 μl of serum-free medium and were added to the upper chamber, and medium with 10% FBS was placed in the bottom chamber. The cells were then incubated for 48 h at 37 degree, and the cells on the upper surface were washed away, while the cells on the bottom surface were fixed with 20% methanol and stained with 0.1% crystal violet. The number of invaded cells was counted in five randomly selected fields using a microscope.
Lentivirus generation and infection
Short hairpin RNA (shRNA) directed against human lncRNA PANDAR (sh-PANDAR) or scrambled oligonucleotides (negative control, sh-NC) were cloned into the LV-3 (pGLVH1/GFP + Puro) vector (a generous gift from Dr. Xinhua Lv, Zhejiang University). The 293 cells were co-transfected with Lenti-Pac HIV Expression Packaging Mix and the lentiviral vectors (Life Technologies Ltd., Carlsbad, CA, USA). After 48 h, lentiviral particles in the supernatant were harvested and filtered by centrifugation at 500 x g for 15 min.
In vivo experiments
All of the experimental protocols were approved by the Animal Care and Use Committee of Quzhou People’s Hospital. The experiment is in compliance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Four-week-old BALB/c nude mice were randomly divided into two groups, with 4 mice in each group. The 7860 cells that were stably transfected with sh-NC or sh-PANDAR (5 × 10
6 cells per mouse) were injected subcutaneously in the right flanks of mice. 27 days later, the mice were then sacrificed by cervical dislocation, and the tumors were removed and weighed.
All statistical analyses were performed using SPSS 18.0 (IBM, Chicago, IL). A Paired Samples t-test was applied to analyze the difference in PANDAR expression between ccRCC tissues and adjacent normal tissues. The CCK-8 assay data were analyzed by ANOVA and Independent Samples t Test was used to analyze other data. Data from at least three independent experiments that were performed in triplicate are presented as the means ± standard deviations (SD). The significance of the differences between groups was estimated using Student’s t-test. OS rates were calculated using the Kaplan-Meier method with the log-rank test for comparisons. The Cox proportional hazards model was used in the multivariate and univariate analysis. Significance was defined as
P < 0.05.
ccRCC is one of the most deadly genitourinary malignancies. The prognosis for renal cell carcinoma is quite poor because most ccRCC patients are diagnosed at a later stage when treatment is not effective [
]. Therefore, the identification of novel prognostic biomarkers and therapeutic targets may have an enormous potential to improve the outcomes of ccRCC.
It is now estimated that only 2% of the human genome can be translated into proteins, whereas 60–70% of genome is transcribed into non-coding RNAs (ncRNAs) [
]. Among these ncRNAs, lncRNAs, which are longer than 200 nucleotides, are important new members of ncRNAs [
]. In recent years, lncRNAs have received great attentions because lncRNAs are involved in tumor development and therefore possess the potential to be biomarkers and prognosis factors [
]. Research regarding the functions of lncRNAs in ccRCC is diverse. For example, lncRNA ZNF180–2 and MALAT1 were found to be upregulated in ccRCC tissues and are associated with poor prognosis [
]. Conversely, the lncRNA CADM1-AS1 functions as a tumor suppressor in ccRCC [
]. Moreover, it has been reported that several novel lncRNAs were dysregulated in ccRCC, but there is no correlation between lncRNA expression and the clinicopathological features of ccRCC [
PANDAR is a newly identified lncRNA that is localized at chromosome 6 and has a length of 1506 nucleotide [
]. The role of PANDAR in tumorigenesis is still controversial. For instance, PANDAR was downregulated in non-small cell lung cancer (NSCLC), and the low level of PANDAR indicated a poor prognosis [
]. In contrast, PANDAR was significantly upregulated in bladder cancer [
]. Currently, there are no reports on the clinical relevance of PANDAR to ccRCC. In the present study, we sought to determine whether there was any difference in the expression of PANDAR between ccRCC tissues and adjacent normal tissues. We found that the expression levels of PANDAR in ccRCC tissues were significantly higher. In addition, we demonstrated that increased PANDAR expression was positively correlated with an advanced TNM stage, lymph node metastases, distant metastases and poor prognosis. Moreover, the expression of PANDAR is higher in ccRCC cell lines than in normal renal cell lines. These results suggest that PANDAR may play a role in the development of ccRCC. To understand the biological functions of PANDAR in ccRCC, we evaluated cell proliferation, the cell cycle, apoptosis and invasion after silencing of PANDAR in 7860 and Caki-1 cells. After the downregulation of PANDAR, both cell lines exhibited a marked decrease of cell proliferation and invasion. We also observed a cell cycle arrest in the G0/G1 phase, which is similar to the findings of Sang et al. who demonstrated that the silencing of PANDAR caused a G0/G1 phase arrest in breast cancer [
]. In addition, we observed that the knockdown of PANDAR led to greater apoptosis in both cell lines. To unveil the potential mechanisms by which PANDAR promotes proliferation, invasion and the inhibition of apoptosis, we measured proteins that are involved in these biological processes.
The G1-phase-related Cyclin-CDK complex is inhibited to promote cell cycle arrest [
]. Here, our finding of a decrease in cyclin D1, cyclin E1 and CDK4 in both cells after silencing of PANDAR suggests the disruption of the uncontrolled cell cycle progression of 7860 and Caki-1 cells. We also observed that MMP-2 and TIMP-3 were downregulated after the knockdown of PANDAR, and MMP-2 and TIMP-3 have been implicated in the regulation of the metabolism of the extracellular matrix, tumor progression and metastasis [
]. Thus, our data suggests that PANDAR might promote cell invasion by regulating the expression of MMP-2 gene.
On the other hand, flow cytometry analysis demonstrated that the downregulation of PANDAR resulted in the induction of apoptosis. Apoptosis is one form of programmed cell death and is considered to be a protective mechanism that eliminates mutated neoplastic cells [
]. Apoptosis is tightly regulated by pro-apoptotic and anti-apoptotic proteins such as caspases and Bcl-2 family proteins. We found that caspase-3 but not caspase-8 protein was cleaved after the silencing of PANDAR. Because Bcl-2 family proteins (Bcl-2, Mcl-1 and Bad) could affect the activation of caspase-3, we asked whether the activation of caspase-3 by the downregulation of PANDAR was due to the alteration of Bcl-2 proteins. We found that silencing of PANDAR could inhibit Bcl-2 and Mcl-1 while enhancing the expression of Bax. Therefore, PANDAR may at least affect ccRCC cell apoptosis through modulation of Bcl-2 family proteins. The present study also examined the signaling pathway that is possibly involved in apoptosis, and it was found that the silencing of PANDAR inhibited the expression of mTOR and the phosphorylation of PI3K and Akt. These results were also supported by the in vivo experiments. Taken together, these results indicate that PANDAR may promote cell proliferation via cell cycle arrest and apoptosis partly through the PI3K/Akt/mTOR pathway.
However, it is worth noting some of the limitations in the present study. First, the function of PANDAR was investigated using RNA interference, and there is a lack of gain-of-function approach, such as overexpression of PANDAR. Second, although we found that PANDAR is upregulated in ccRCC, the mechanisms underlying this dysregulation remains elusive.
We sincerely thank Penglei Mao, Xinkuan Wu, Renbing Pan, Bo Peng, Yukun Liu and Junjie Ying for their assistance in this study.
No funding was received.
Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request, but no information infringing on the privacy of the participants will be given.
LX conceived, designed the project and wrote this manuscript. YX, YT, JZ and ZL performed most of the experiments. LW, XZ and FY collected the clinical and pathological data and analyzed it. All of the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All procedures performed in these studies involving human participants were in accordance with the ethical standards of the Quzhou People’s Hospital research committee. The study was approved by the ethics committee of Quzhou People’s Hospital. Written informed consent was obtained from all individual participants that were included in the study. All of the animal experiments were performed according to the NIH animal use guidelines on the use of experimental animal. All of the animal experimental protocols were approved by the Animal Care and Use Committee of Quzhou People’s Hospital.
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