Introduction
Renal cell carcinoma (RCC) is a highly malignant tumor of the urinary system. It is also one of the most common clinical types [
1]. It is a malignant tumor that originates in the renal parenchyma urinary tubule epithelial system. The incidence of RCC is third among urological tumors, second to only prostate and bladder cancers [
2]. It accounts for 3% of adult malignancies and 80–90% of renal tumors, of which the male-to-female patient ratio is approximately 2:1 [
3]. Regarding the pathological types of RCC, approximately 75–80% of cases are clear cell renal cell carcinoma (ccRCC) [
4,
5]. Because nearly 1/3 of patients have localized or distant metastasis at the initial diagnosis and because ccRCC is not sensitive to radiotherapy or chemotherapy but is treatable by surgery, although the recurrence rate is still up to 20–40% after radical nephrectomy, so the 5-year survival rate of ccRCC is only approximately 20% after surgical resection [
6,
7]. Therefore, finding new targets against the proliferation and metastasis of ccRCC cells is urgently required.
LncRNAs are eukaryotic cell genome-encoded transcripts larger than 200 RNA nucleotides that lack obvious open reading frames and do not encode proteins; thus, they are called long noncoding RNAs [
8]. Although lncRNAs do not participate in the encoding of protein, they can regulate gene expression at different levels, such as the epigenetic, transcriptional and posttranscriptional levels, and play key roles in genome modification, transcriptional activation, transcriptional interference, and chromosome sedimentation [
9]. Several studies have confirmed that abnormal expression of lncRNAs is closely related to the biological behaviors of malignant tumor cells, such as growth, proliferation, invasion and metastasis [
10]. For instance, the lncRNA HOTAIR is highly expressed in a variety of cancer cells and regulates the methylation level of histone H3K27 by recruiting PRC2 to specific locus, leading to the formation of repressor chromatin complexes and the epigenetic silencing of tumor suppressor genes, which thus promotes the occurrence and progression of multiple malignancies, including colorectal cancer, cervical cancer, and RCC [
11,
12]. High expression of MEG3 can regulate the level of histone H3 methylation by recruiting the histone methyltransferase EZH2 and the histone demethylation enzyme JARID2 to the regulatory regions of the epithelial marker gene CDH1 and microRNA-200 family genes, which play a key role in the EMT process mediated by TGF-beta, which ultimately results in lung cancer and regulates EMT in lung cancer at the epigenetic level. In addition, high expression of MEG3 is closely related to the malignancy of hepatocellular carcinoma, gastric cancer, colorectal cancer and bladder cancer [
13]. In addition, a number of studies have confirmed that abnormal expression or functional changes in lncRNA expression are closely related to the formation, local progression, and distant metastasis of urinary tract tumors, such as prostate cancer [
14], bladder cancer [
15] and kidney cancer [
16].
As for our research, we are the first to report the expression pattern, biological function and potential regulatory mechanism of OTUD6B-AS1 in ccRCC. Quantitative real-time PCR (qRT-PCR) assays revealed that OTUD6B-AS1 expression was significantly decreased in ccRCC tissue samples and cell lines, suggesting antioncogene functions for OTUD6B-AS1. The probable relationship between OTUD6B-AS1 and overall survival in ccRCC patients were also determined. Furthermore, our findings indicated that the lncRNA OTUD6B-AS1 inhibited ccRCC proliferation through the inactivation of the Wnt/β-catenin pathway and suppressed the expression of EMT-related proteins.
Materials and methods
A total of 75 paired ccRCC tissue and adjacent normal tissue samples were collected from ccRCC patients who underwent radical nephrectomy at the First Affiliated Hospital of Harbin Medical University. All the samples were immediately snap frozen in liquid nitrogen for long-term preservation until RNA extraction.
Cell lines and cell culture
The human ccRCC cell lines (786-O, Caki-1, 769-P, OS-RC-2, and ACHN) and human renal tubular epithelial cells (HK-2) were all purchased from the Cell Resources Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. ACHN cells were grown in RPMI-DMEM (KeyGen, Nanjing, China); HK-2, 786-O, Caki-1, 769-P, and OS-RC-2 cells were grown in RPMI 1640 medium (KeyGen, Nanjing, China) containing 10% FBS (Life Technologies, Australia). All cells were grown at 37 °C in a humidified 5% CO2 atmosphere.
Cell transfection
A plvx-OTUD6B-AS1 vector and an empty plvx-vector (vector) were commercially synthesized by GenePharma (Shanghai, China). For transient transfection, ACHN and OS-RC-2 cells were plated into six-well plates (2 × 105/well) and routinely maintained for 24 h at 37 °C. Then, the cells were transfected with plvx-OTUD6B-AS1 or the empty plvx-vector according to the manufacturer’s protocol. Subsequent experiments were performed at 48 h posttransfection.
Treatment of ACHN and OS-RC-2 cells with 5-aza-2-deoxycytidine
ACHN and OS-RC-2 cells were seeded into 6-well plates at 3 × 10
5 cells per well and cultured in RPMI-DMEM or RPMI 1640 medium containing 5 μM 5-aza-2-deoxycytidine (5-Aza-CdR, Sigma-Aldrich, USA) for 5 days according to a previously reported protocol [
17,
18]. The cells treated with 5-Aza-CdR were harvested and used for detection in plvx-OTUD6B-AS1 expression assays.
Cell proliferation assay
Cell proliferation was assayed using an MTT assay. The transfected cells were plated into 96-well plates (5000 cells/well). Cell proliferation was detected every 24 h according to the manufacturer’s protocol. Briefly, 20 μl of MTT solution was added to each well and incubated for 4 h at 37 °C. The solution was then discarded, and 150 μl of DMSO was added. After shaking for 15 min at room temperature, the absorbance of the solution was measured with a spectrophotometer at 490 nm.
Total RNA extraction and quantitative real-time PCR
Total RNA was extracted from the ccRCC tissue samples and cell lines using TRIzol reagent (Ambion, Life Technologies, USA) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit (TOYOBO Life Science, Shanghai, China) for the detection of OTUD6B-AS1 expression. The relative expression of OTUD6B-AS1 was detected using FastStart Universal SYBR Green Master Mix (ROX) and normalized to that of GAPDH. The primer sequences were as follows: 5’-GACATATCCGGGTGACGTTTTT-3′ (sense) and 5’-TTGTTCCACTGTCTTCTGGCATT-3′(antisense) for OTUD6B-AS1 and 5’-CACCCACTCCTCCACCTTTGA-3′ (sense) and 5’-ACCACCCTGTTGCTGTAGCCA-3′ (antisense) for GAPDH. The results were analyzed using the -ΔCt method. All the results are expressed as the mean ± SD of three independent experiments.
ACHN and OS-RC-2 cells (0.1 × 103 cells per well) were seeded in a six-well plate and cultured for 10 days after treatment. Colonies were then fixed with 4% paraformaldehyde for 15 min and stained for 10 min with 0.5% crystal violet. Then, the number of colonies was counted using ImageJ, and images were taken under an Olympus microscope (Tokyo, Japan).
Cell immunofluorescence staining
Treated cells were fixed in 4% paraformaldehyde for 30 min, and then 0.5% Triton X-100 was used to permeabilize the cells at room temperature for 20 min. After blocking with 5% BSA, slides were incubated overnight at 4 °C with a primary antibody against ki-67 (1:1000, Abcam, Cambridge, UK). Then, the slides were incubated with a fluorescent secondary antibody for 1 h in a wet box at room temperature in the dark. Finally, DAPI was used to counterstain the nuclei. Images were taken under an Olympus microscope (Tokyo, Japan).
Hoechst staining
After transfection, cells were fixed for 10 min with a fixation solution and washed twice with phosphate-buffered saline (PBS) for 3 min each time, and then the liquid was drained. Finally, 0.5 ml of Hoechst staining solution (Wanleibio, Shenyang, China) was added for 5 min to dye the cells. Images were taken under an Olympus microscope (Tokyo, Japan). It can be seen that the nuclei of apoptotic cells are densely stained or fragmented densely stained. Cyan represents cells that have undergone apoptosis.
Flow cytometry analysis of apoptosis and cell cycling
After transfection, the apoptosis rates of ACHN and OS-RC-2 cells were analyzed using flow cytometry. The cells were collected and washed twice with cold PBS. Subsequently, the cells were stained with an anti-Annexin V-PE antibody and 7-AAD dye for 15 min in the dark according to the manufacturer’s instructions. The stained cells were analyzed with a flow cytometer (BD Biosciences) and were counted using CellQuest software (BD Biosciences). After staining with propidium iodide (PI) using a CycleTESTTM PLUS DNA Reagent Kit (BD Biosciences), a flow cytometer (FACScan®; BD Biosciences) was used to analyze the cells. The cell cycle results elucidate the exact distribution of the cells in the G0-G1, S, and G2-M phases. This experiment was repeated three times.
Western blot analysis
Total protein was prepared from ACHN and OS-RC-2 cells using RIPA buffer with a proteinase inhibitor. The lysates were incubated on ice for 30 min and then centrifuged at 12,000 rpm for 15 min at 4 °C, and the protein concentration was measured by a BCA kit (Beyotime Biotechnology, Beijing, China). Equal quantities of protein were electrophoresed through a 12.5% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked and then incubated with the appropriate primary antibody and β-actin overnight at 4 °C. Subsequently, the membranes were incubated with an anti-mouse or anti-rabbit secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. The protein bands were visualized using a chemiluminescence reagent (ECL) kit (Beyotime Biotechnology).
Immunohistochemistry (IHC)
Tumor tissues from mice were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and cut into 4-μm-thick sections. The sections were deparaffinized, blocked against endogenous peroxidase activity and underwent antigen retrieval. After blocking with 5% bovine serum albumin, the sections were incubated overnight at 4 °C with a primary antibody against ki-67 (1:500, Abcam, Cambridge, UK). Then, the sections were incubated with a peroxidase-conjugated polymer for 30 min, and a DAB (Beyotime Institute of Biotechnology, Jiangsu, China) system was used for detection.
Female nude mice that were 4 weeks old were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). ACHN cells stably transfected with the empty vector or plvx-OTUD6B-AS1 were collected, and the concentration of resuspended ACHN cells was 2 × 107 cells/mL. Then, the suspended cells were injected into either side of the posterior flank of each mouse. Tumor volumes were tested every 3 days. The mice survived for up to 34 days after the injection before they needed to be sacrificed. Then, the tumors formed from the empty-vector-transfected or plvx-OTUD6B-AS1-transfected ACHN cells were removed from the mice and kept for weight measurement, immunohistochemistry (IHC) and hematoxylin-eosin staining (H&E). The protocol conformed with the regulations of Harbin Medical University’s Animal Ethics Committee.
Statistical analysis
Statistical data were analyzed using Statistical Program for Social Sciences (SPSS) 19.0 software (SPSS, Chicago, IL, USA). GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA) was used to plot all graphs. The differences between the tumor tissue and normal colonic mucosa were analyzed by a paired t-test. A receiver operating characteristic (ROC) curve was constructed to evaluate the diagnostic values. P < 0.05 was considered statistically significant.
Discussion
In recent research, abnormal expression of lncRNAs has been discovered in various human cancers, and increasing evidence indicates that lncRNAs participate in all steps of carcinogenesis and tumor progression [
19‐
22]. A great number of studies have indicated that lncRNAs play an important role in the diagnosis and treatment of tumors, and lncRNAs can be used as new prognostic markers for tumors [
23‐
26]. The lncRNA OTUD6B-AS1 is oriented in an antisense direction to the protein-coding gene OTUD6B on the opposite DNA strand. Although previous studies have shown that OTUD6B-AS1 expression is deregulated in the skin tissue of patients with systemic sclerosis [
27], the mechanism of OTUD6B-AS1 in tumors and other diseases, especially RCC, is unknown. In this study, we identified that the expression of the lncRNA OTUD6B-AS1 was downregulated in ccRCC tissue compared with matched paracancerous tissue, and the downregulation of OTUD6B-AS1 expression was associated with worse clinical characteristics and poor overall survival. We also confirmed the effects of OTUD6B-AS1 on ccRCC cells. The overexpression of OTUD6B-AS1 inhibited the proliferation, migration and invasion of ccRCC cells in vitro and in vivo and promoted apoptosis in ccRCC cells. Collectively, our results demonstrated that OTUD6B-AS1, acting as an antioncogene, may be a potential diagnostic and prognostic biomarker as well as a therapeutic target in ccRCC.
In addition to the abnormal proliferation and differentiation, the invasion and migration of tumor cells also play crucial roles in the process of tumor progression, and EMT is one of the main pathways that regulates invasion and migration. The Wnt signaling pathway, which participates in cell proliferation and regeneration and plays an important role in the development of embryos and the process of EMT, is a highly conserved signaling pathway that is composed of a series of oncogene and antioncogene proteins [
28]. The most classical pathway is the Wnt/β-catenin signaling pathway. β-Catenin can combine with E-cadherin to form an E-cadherin/β-catenin complex to maintain the stability of intercellular adhesion structures and cell polarity. The Wnt signaling pathway can inhibit the phosphorylation of GSK3β and the degradation of β-catenin in the cytoplasm and can upregulate the expression of transcription factors, such as Snail and twist, thus inhibiting E-cadherin transcription and increasing the intracellular free β-catenin level, which can activate and stabilize EMT transcription factors in the nucleus and induce EMT. In normal mature cells, the Wnt signaling pathway is inactive, but there is abnormal activation of the Wnt signaling pathway in a variety of tumors, and the inhibition of the Wnt/β-catenin signaling pathway can reduce invasion, metastasis and the occurrence of drug resistance [
29‐
32]. For example, the abnormal activation of the Wnt/β-catenin signaling pathway in liver cancer can promote the malignant transformation of normal hepatocytes [
33]; there is also abnormal activation of the Wnt signaling pathway and accumulation of β-catenin in colorectal cancer, and the proliferation of colon cancer cells can be inhibited by inhibiting the activity of the Wnt signaling pathway [
34‐
36]. In different tumors, the role of the Wnt/β-catenin signaling pathway varies. For example, in tongue squamous cell carcinoma, Sox8 can combine with the FZD7 promoter region of the Wnt receptor and activate the Wnt/β-catenin signaling pathway regulated by FZD7, thus regulating tumor cell chemosensitivity, stem cell stemness and EMT [
37]. As mentioned above, we explored the mechanism by which OTUD6B-AS1 overexpression affects ccRCC by performing experiments to demonstrate that OTUD6B-AS1 decreased the activity of the Wnt/β-catenin pathway and suppressed the expression of EMT-related proteins in ccRCC cells.
In the current work, we evaluated the function of OTUD6B-AS1 in ccRCC progression and found a negative correlation between ccRCC malignancy and OTUD6B-AS1 expression. However, one of the yet undetermined mechanisms not addressed in the current study is whether OTUD6B-AS1 can function as a competitive endogenous RNA (ceRNA) to regulate microRNAs (miRNAs) during ccRCC development. For example, the lncRNA MIAT can competitively bind with miR-29c and modulate the expression of LOXL2 to regulate the progression of ccRCC [
38]. LINC01133 can be combined with miR-106a-3p and then affect the expression of APC, thereby regulating gastric cancer progression [
39]. Since ceRNAs represent a novel aspect of regulation, especially during tumorigenesis [
40], ongoing work is strongly needed to determine whether OTUD6B-AS1 could be a candidate ceRNA during ccRCC progression. Moreover, with the progress of molecular biology technology, gene therapy for tumors is attracting increasing attention, and how to apply our genes to clinical treatment still needs further study. In addition our data were only obtained with cell lines, and that cell line models have several limitations,so there’s still a lot of work to be done to study OTUD6B-AS1 in depth. This is also the focus of our future research.