Long non-coding RNA CCAT2 promotes cell proliferation and invasion through regulating Wnt/β-catenin signaling pathway in clear cell renal cell carcinoma

Abstract

Clear cell renal cell carcinoma (ccRCC) is a common urologic malignancy. Long non-coding RNA colon cancer–associated transcript 2 (CCAT2) has been suggested as serving pivotal roles in tumorigenesis. However, the clinical significance and biological role of CCAT2 in ccRCC remains elusive. The purpose of this study is to identify the function of CCAT2 in ccRCC and its possible molecular mechanism. Expression of CCAT2 was analyzed in 61 ccRCC tissues and two ccRCC cell lines (786-O and ACHN) by quantitative reverse transcription polymerase chain reaction. The functional roles of CCAT2 in ccRCC were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, Transwell assay, and flow cytometric analysis. The influence of CCAT2 on tumorigenesis was monitored by in vivo mice xenograft model. The activation of Wnt/β-catenin signaling pathway was evaluated by the TOP/FOP Wnt luciferase reporter assay and western blot assay. CCAT2 expression was markedly higher in ccRCC cell lines and tissues, being positively associated with tumor size and tumor stage in ccRCC patients. Patients with higher CCAT2 expression had a markedly poorer overall survival than did patients with low CCAT2 expression. Knocking down CCAT2 expression led to reduced cell proliferation and increased apoptosis of ccRCC cells in vitro as well as the activation of Wnt/β-catenin signaling pathway, and CCAT2 overexpression remarkably enhanced these oncogenic properties. In vivo mice xenograft model also showed that knocking CCAT2 expression inhibited the growth of ccRCC xenografts. In conclusion, these results indicated that CCAT2 may play a critical role in ccRCC progression and will be further considered as a biomarker for predicting the survival of ccRCC patients and a potential therapeutic target for ccRCC intervention.

Keywords

Long non-coding RNA CCAT2 clear cell renal cell carcinoma Wnt/β-catenin signaling pathway prognosis cell cycle

Introduction

Renal cell carcinoma (RCC) remains the most common type of kidney cancer in adults (>85%), accounting for approximately 2%–3% of adult malignancies and causes about 100,000 deaths worldwide every year,¹ and the incidence of this disease has gradually elevated in recent years. RCC is heterogeneous and can be classified into four clinical subtypes, including clear cell renal cell carcinoma (ccRCC), papillary RCC (pRCC), chromophobe RCC (chRCC), and renal oncocytoma (RO). Among them, ccRCC is the most predominant histological subtype and accounts for approximately 70%–80% of all RCC cases.² In recent years, although great efforts and progressions have been made in surveillance and clinical treatment strategies, there is a very poor prognosis for patients with metastatic ccRCC of only 1–2 years overall survival after curative resection.³ Accordingly, it is of utmost importance to further elucidate the molecular mechanisms of ccRCC progression and metastasis and develop more effective therapeutic approaches.

The human genome sequencing project showed that 70% of the genome is transcribed, but only up to 2% of the human genome serves as blueprints for proteins.⁴ As a group of non-protein-coding RNAs that regulate gene expression at the transcriptional or post-transcriptional level,⁵ long non-coding RNAs (lncRNAs) have been considered to play important roles in multiple pathological conditions, including cancer progression.⁶ In ccRCC research, lncRNAs have also received great attention. Several deregulated lncRNAs, such as CADM1-AS1,⁷ SPRY4-IT1,⁸ MALAT1,⁹ lnc-ZNF180-2,¹⁰ Linc00152,¹¹ and TCL6,¹² have been previously reported to be closely associated with the initiation and progression of ccRCC.

LncRNA colon cancer–associated transcript 2 (CCAT2), mapped to 8q24 genomic region, was originally identified as a lncRNA associated with metastasis and survival in non-small-cell lung cancer.¹³ Aberrant CCAT2 expression was also found in multiple types of human malignancies, including esophageal squamous cell carcinoma,¹⁴ breast cancer,¹⁵ non-small-cell lung cancer,¹⁶ and glioma.¹⁷ However, to the best of our knowledge, the biological functions and the roles CCAT2 plays in ccRCC has not been well studied so far.

Accordingly, based on the previous researches, the purpose of our study was to investigate the expression profiles of CCAT2 in ccRCC and its involvement in the oncogenic properties of ccRCC cells. Our results provided new insights into the biological functions of CCAT2 as well as its underlying mechanisms in ccRCC.

Materials and methods

Patients and specimens

A total of 61 ccRCC tissues and corresponding non-tumor tissues were obtained from patients who were diagnosed and underwent radical nephrectomy surgery in the Department of Urology, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital (Sichuan, China). None of them received preoperative chemotherapy and/or radiation therapy. All fresh tissue samples were immediately flash-frozen in liquid nitrogen and stored at −80°C for further analysis. The study was approved by the Ethical Review Board of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital and written informed consent was obtained from all participants.

Cell culture and transfection

Human ccRCC–derived cell lines 786-O and ACHN, purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 medium (HyClone, Logan, UT, USA). The normal human proximal tubule epithelial cell line HK-2, obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), was cultured in keratinocyte serum-free medium (KSFM; Gibco™ Life Technologies, Grand Island, NY, USA). The culture media were all supplemented with 10% fetal bovine serum (FBS), 50 U/mL of penicillin, and 50 µg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). All cells were maintained in a humidified incubator with 5% CO₂ at 37°C.

The short hairpin RNA targeting human CCAT2 (shRNA-CCAT2) and non-targeting shRNA (shRNA-NC) were synthesized and inserted into the pHBLV-U6 vector (Hanbio, Shanghai, China). To overexpress CCAT2, the full-length coding sequence was amplified and subcloned into the pcDNA 3.1 (+) vector (Invitrogen). These constructs were transfected into ccRCC cells using Lipofectamine 2000 reagent (Invitrogen). Transfection efficacy was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 48 h of transfection.

Total RNA extraction and qRT-PCR

Total RNA was isolated from ccRCC specimens and cell lines using TRIzol reagent (Invitrogen). The cDNA was synthesized from 200 ng of extracted total RNA using PrimeScript RT reagent Kit (Takara Bio Company, Shiga, Japan). After reverse transcription of the total RNA, qRT-PCR was conducted to detect the expression of CCAT2 using SYBR Premix Ex Taq (TaKaRa, Dalian, China) on Light Cycler 480 SYBR Green I Master (Roche, Grenzach, Germany) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the control gene. The 2^−ΔΔCt method was used to determine the relative quantification of gene expression levels. The premier sequences were as follows: CCAT2 (forward): 5′-CCCTGGTCAAATTGCTTAACCT-3′, (reverse): 5′-TTATTCGTCCCTCTGTTTTATGGAT-3′; GAPDH (forward): 5′-GTCAACGGATTTGGTCTGTATT-3′, (reverse): 5′-AGTCTTCTGGGTGGCAGTGAT-3′.

Western blot analysis

Total, cytoplasmic, and nuclear protein extractions were performed using a protein extraction kit (CWbio, Beijing, China). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were probed with specific antibodies against β-catenin (Abcam, Cambridge, UK), c-myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1 (Santa Cruz Biotechnology), GAPDH (Cell Signaling Technology, Danvers, MA, USA), Lamin A (Abcam), and a-tubulin (Abcam), followed by incubation with appropriate horseradish peroxidase–conjugated secondary antibodies. The amount of detected protein was visualized by enhanced chemiluminescence (Pierce Biotechnology Inc., Rockford, IL) followed by densitometry using the ImageJ software (NIH, Bethesda, MD, USA).

Luciferase assay

ACHN cells were seeded in 24-well plates and co-transfected with the TOP/FOP Flash expression plasmids (Biovector NTCC Ltd., Beijing, China) and Renilla TK-luciferase vector (Promega Corp., Madison, WI, USA) as a control; 48 h after transfection, the cells were harvested and lysed for a luciferase assay.

Cell viability assay

Transfected ccRCC cells were seeded on a 96-well plate at a density of 2000 cells per well and incubated at 37°C. Proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Nanjing KeyGen Biotech. Co. Ltd., Nanjing, China) at 24, 48, 72, and 96 h after transfection. The optical density (OD) was measured at 560 nm using a microtiter plate reader.

Colony formation assay

After transfection, 500 ccRCC cells per well were seeded in six-well plates. After 7 days, cells were fixed with methanol and stained with 0.1% crystal violet. Visible colonies were manually counted.

Cell-cycle analysis

Transfected ccRCC cells were washed in phosphate-buffered saline (PBS) and fixed in 70% ethanol at 4°C for 2 h. DNA staining was carried out with propidium iodide (PI) using a Cellular DNA Flow Cytometric Analysis kit (Roche Diagnostics, Basel, Switzerland). Cell-cycle profiles were generated using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with ModFit 3.0 software (BD Biosciences).

Cell apoptosis assay

Transfected ccRCC cells were stained using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (BD Biosciences). Then, the cells were analyzed through FACSCalibur flow cytometer equipped with CellQuest software (BD Biosciences). Cells were discriminated into viable cells, dead cells, early apoptotic cells, and apoptotic cells.

Transwell assay

Transfected ccRCC cells (1 × 10⁵) in serum-free media were added into the upper chamber of an insert (8-µm pore size; Corning Incorporated, Corning, NY, USA) pre-coated with Matrigel (without Matrigel for cell migration assay). Meanwhile, 500 µL of Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS was put into the lower chamber. After incubation for 48 h, the cells remaining on the upper chamber were scraped off. The number of cells that had migrated or invaded the membrane in each group was fixed with methanol, stained with crystal violet, imaged, and counted.

In vivo tumorigenesis assay

The animal use and care were performed following the guidelines by the U.S. National Institute of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the animal care and ethics committee of Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital Medical University. Female athymic BALB/c nude mice (aged 5 weeks), provided by SLAC Laboratory Animal Co. Ltd. (Shanghai, China), were randomized to the control or experimental group (six mice per group). ACHN cells stably expressing shRNA-CCTA2 or shRNA-NC (5 × 10⁶ in 0.1 mL of sterilized saline) were implanted subcutaneously into the left armpit of nude mice. Tumors were measured with calipers every 3 days and the volume was calculated by length × width² × 0.5. All mice were sacrificed 4 weeks after cell inoculation and the tumors were dissected and weighed.

Statistical analysis

All statistical analyses were carried out using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 6.01) software (GraphPad Software, Inc., La Jolla, CA, USA). Data are presented as the mean ± SD of at least three independent experiments. The statistical significance between groups was determined using the Student’s t-test. Association between expression level of CCAT2 and clinicopathologic parameters of ccRCC patients was evaluated using chi-square test. A log-rank test was used to analyze the statistical differences in survival based on Kaplan–Meier curves. Results were considered to be statistically significant at values of p < 0.05, and all p values were two-sided.

Results

CCAT2 expression is increased in ccRCC and correlated with poor survival

The qRT-PCR was performed to detect CCAT2 expression levels in ccRCC cell lines and clinical samples, normalized to GAPDH. The results showed that ccRCC cell lines of 786-O and ACHN expressed remarkably higher levels of CCAT2 than the normal human proximal tubule epithelial cell line HK-2 (Figure 1(a)). Furthermore, the expression of CCAT2 was evidently increased in 61 ccRCC tissues compared with paired noncancerous tissues (Figure 1(b)). We further determined the association between CCAT2 and clinicopathological characters. A median value of CCAT2 expression (6.06) in 61 ccRCC tissues was used to classify these patients into high-expression and low-expression groups. As demonstrated in Table 1, CCAT2 was closely associated with tumor size (p = 0.025) and tumor stage (p = 0.005). However, no significant correlations were found between CCAT2 expression and other clinicopathological factors, including gender, age, histological grade, and lymph nodes metastasis.

Figure 1.

CCAT2 expression is increased in ccRCC and correlated with poor survival. (a) The expression levels of CCAT2 in two ccRCC cell lines (786-O and ACHN) and immortalized normal human proximal tubule epithelial cell line HK-2 were detected by quantitative RT-PCR. (b) The expression levels of CCAT2 in 61 pairs of ccRCC tissues and matched adjacent non-tumor tissues were investigated by quantitative RT-PCR. The data are expressed as mean ± SD. The p value was assessed by Student’s t-test with p < 0.05. (c) The Kaplan–Meier curve indicated that ccRCC patients with high expression of CCAT2 had a worse overall survival compared to patient with low expression of CCAT2. The p value was assessed by log-rank test.

Table 1.

Correlation between CCAT2 expression and clinicopathological parameters of 61 ccRCC patients.

Parameter	Total (n = 61)	CCAT2 expression		p value
Parameter	Total (n = 61)	High (n = 26)	Low (n = 35)	p value
Gender				0.791
Male	34	15	19
Female	27	11	16
Age (years)				0.481
<60	29	11	18
≥60	32	15	17
Tumor size (cm)				0.025
<4	38	12	26
≥4	23	14	9
Histological grade				0.097
G1–2	40	14	26
G3–4	21	12	9
Tumor stage				0.005
T1–2	36	10	26
T3–4	25	16	9
Lymph nodes metastasis				0.120
No	48	18	30
Yes	13	8	5

ccRCC: clear cell renal cell carcinoma.

Furthermore, Kaplan–Meier survival analysis was conducted to detect the prognostic value of CCAT2 in ccRCC patients. As shown in Figure 1(c), ccRCC patients with low CCAT2 expression manifested longer overall survival compared with those with high CCAT2 expression (p = 0.002).

CCAT2 promotes the malignant phenotypes of ccRCC cells

Then, to elucidate the role of CCAT2 in ccRCC progression, we decreased CCAT2 expression by transfecting shRNA-CCAT2 in ACHN cells (Figure 2(a)). In vitro experiments in ACHN cells showed that inhibition of CCAT2 inhibited cell proliferation in MTT assay (Figure 2(b)) and reduced colony formation ability (Figure 2(c)). In flow cytometry analysis, the shRNA-CCAT2 ACHN cells exhibited increased apoptosis rate and cell-cycle arrest in G0/G1 phase when compared with shRNA-NC cells (Figure 2(d) and (e)). Finally, in the experiment of Transwell migration and invasion assays, inhibition of CCAT2 could significantly suppress the migration and invasion abilities of ACHN cells (Figure 2(f)).

Figure 2.

Inhibition of CCAT2 suppresses the malignant phenotypes of ccRCC cells. (a) The expression level of CCAT2 was detected in ACHN cells after transfected with shRNA-CCAT2 or shRNA-NC by qRT-PCR. (b)–(f) Cell proliferation, colony formation, cell-cycle distribution, cell apoptosis, cell migration, and invasion were determined in ACHN cells after transfected with shRNA-CCAT2 or shRNA-NC. The data are expressed as mean ± SD. The p value was assessed by Student’s t-test with p < 0.05.

To verify the above results, we further overexpressed CCAT2 in 786-O cells using transfecting the pcDNA3.1 plasmid vector (Figure 3(a)). Our results showed that when CCAT2 was overexpressed, the proliferation and colony formation capacities of 786-O cells were significantly enhanced (Figure 3(b) and (c)). Flow cytometry analysis also showed decreased cell apoptosis rate and cell-cycle arrest in G0/G1 phase when compared with control cells (Figure 3(d) and (e)). In Transwell migration and invasion assays, overexpression of CCAT2 promoted the migratory and invasive abilities of 786-O cells (Figure 3(f)). The above in vitro experiments performed in the two ccRCC cell lines suggested that inhibition of CCAT2 may suppress the malignant phenotypes of ccRCC cell lines, and overexpression of CCAT2 will bring about more aggressive phenotypes of ccRCC cells.

Figure 3.

Overexpression of CCAT2 enhances the malignant phenotypes of ccRCC cells. (a) The expression level of CCAT2 was detected in 786-O cells after transfected with pcDNA-CCAT2 or pcDNA-NC by qRT-PCR. (b)–(f) Cell proliferation, colony formation, cell-cycle distribution, cell apoptosis, cell migration, and invasion were determined in 786-O cells after transfected with pcDNA-CCAT2 or pcDNA-NC. The data are expressed as mean ± SD. The p value was assessed by Student’s t-test with p < 0.05.

CCAT2 activates Wnt/β-catenin signaling in ccRCC cells

To determine the association between CCAT2 expression and the activation level of Wnt/β-catenin signaling pathway, TOP/FOP flash reporters were used to assess the impacts of CCAT2 on Wnt/β-catenin signaling pathway in ACHN cells. The change of cell luciferase activity was shown in Figure 4(a). While CCAT2 expression was silenced, the activation of the Wnt/β-catenin signaling pathway was suppressed. We then performed western blot analysis to further verify whether suppressing or overxpressing CCAT2 would affect Wnt/β-catenin signaling pathway. Western blot showed that knockdown CCAT2 expression noticeably inhibited the expression levels of β-catenin, c-myc, and cyclin D1 in ACHN cells (Figure 4(b)). In addition, we found that overexpression of CCAT2 evidently increased expression of β-catenin, c-myc, and cyclin D1 in 786-O cells (Figure 4(b)), indicating that CCAT2 could affect the activity of Wnt/β-catenin pathway in ccRCC cells.

Figure 4.

CCAT2 activates Wnt/β-catenin signaling in ccRCC cells. (a) Luciferase reporter assay using TOP flash vectors was performed to detect β-catenin transcription factor/lymphoid enhancer binding factor (TCF/LEF) promoter activity in ACHN cells. (b) The expression levels of β-catenin, c-myc, and cyclin D1 in two ccRCC cell lines (786-O and ACHN) were detected by western blot analysis. (c) The expression levels of β-catenin in the cytoplasmic and nuclear fractions in two ccRCC cell lines (786-O and ACHN) were detected by western blot analysis. The data are expressed as mean ± SD. The p value was assessed by Student’s t-test with p < 0.05.

As we all know, Wnt/β-catenin pathway was inactive due to inhibition of β-catenin expression and translocation to nucleus. Fractionation experiments demonstrated remarkable changes in β-catenin protein levels in the nucleus when CCAT2 expression was increased or decreased (Figure 4(c)), indicating that CCAT2 may inhibit the nuclear translocation of β-catenin in ccRCC cells.

Suppression of CCAT2 attenuates the growth of ccRCC xenografts in vivo

To confirm the above established effects of CCAT2 on ccRCC cells in vivo, we analyzed the effect of knockdown of CCAT2 on the tumorigenicity of ACHN cells using the xenograft model in nude mice. As exhibited in Figure 5(a), the growth of tumors generated from ACHN cells transfected with shRNA-CCAT2 was greatly suppressed at each time point than those cells transfected with shRNA-NC. At day 28, the tumors were dissected. The size and weight of tumors were dramatically decreased by the knockdown of CCAT2 in ACHN cells (Figure 5(b)). These results revealed that knockdown of CCAT2 suppressed the growth of ccRCC xenografts in vivo.

Figure 5.

Inhibition of CCAT2 attenuates the growth of ccRCC xenografts in vivo. (a) The tumor growth was monitored every 3 days. (b) Quantitative determination of the weight of the excised tumors in different groups of nude mice. The data are expressed as mean ± SD. The p value was assessed by Student’s t-test with p < 0.05.

Discussion

LncRNAs are messenger RNA (mRNA)-like transcripts with >200 nucleotides in length that lack coding protein function.¹⁸ In the past decades, overwhelming evidence have reported that lncRNAs were implicated in a wide range of biological functions, such as cell proliferation, cell apoptosis, and cell metastasis. The relationship between lncRNAs deregulation and cancer development has become one of the focuses of cancer studies. This work demonstrated the pattern of CCAT2 expression in ccRCC carcinogenesis and its potential prognostic and diagnostic significances in ccRCC patients and further investigated its effect and mechanism in ccRCC cells.

In this study, CCAT2 is identified as a promising oncogene for ccRCC. Expression of CCAT2 in the ccRCC tissues was significantly elevated than that in the corresponding normal tissues. CCAT2 expression level was positively correlated with aggressive clinicopathological characteristics and unfavorable prognosis of ccRCC patients, and the upregulation of CCAT2 was found both in ccRCC tissues and ccRCC cell lines. Aberrant cell proliferation and deregulated cell-cycle progression are the two main features of most human cancer cells.¹⁹ In vitro mechanistic studies have provided the evidences that CCAT2 overexpression enhanced cell proliferation, invasion, and migration. Furthermore, CCAT2 overexpression also induced S phase progression in the cell cycle and inhibited cell apoptosis. Consistently, downregulation of CCAT2 inhibited ccRCC tumor growth in a nude mouse xenograft model. To the best of our knowledge, this might be the first study to report the biological function of CCAT2 in ccRCC, which contribute to improve our understanding of the mechanisms underlying ccRCC progression and development.

Aberrant activation of the canonical Wnt/β-catenin signaling pathway is often observed during the initiation and progression of ccRCC.^20,21 Wnt/β-catenin signaling is one of the important molecular cascades to regulate a broad range of cellular events, such as cell proliferation, invasion, and differentiation through regulating the ability of the multifunctional β-catenin protein, which is a crucial signaling molecule in the Wnt/β-catenin pathway.²² β-catenin accumulates in the cytoplasm and then translocates to the nucleus and subsequently activates downstream target genes, including c-myc and cyclin D1. Dysregulation of β-Catenin is also an independent predictor of oncologic outcomes in ccRCC patients.²³ Previous study showed that abnormal expression of CCAT2 could influence the Wnt signaling pathway in breast cancer.¹⁵ In addition, Guo et al.²⁴ demonstrated that CCAT2 overexpression promotes glioma cell proliferation and tumorigenesis potential through activating Wnt/β-catenin signal pathway. In our study, we also observed that downregulation of CCAT2 in ccRCC cells inhibited the nuclear translocation of β-catenin, which led to suppression of Wnt/β-catenin signaling pathway.

We are aware of two potential limitations in our work. First, the clinical part was a retrospective validation, and the cohort of ccRCC patients was relatively small. Second, although we revealed the association between CCAT2 and Wnt/β-catenin signaling in ccRCC, the underlying mechanisms by which CCAT2 promotes ccRCC development and progression remain to be thoroughly clarified.

In conclusion, this study, for the first time, proves that CCAT2 expression is increased in ccRCC and its association with aggressive clinicopathological features and poor patient’s prognosis. CCAT2 expression influenced biological behaviors of ccRCC cells through inactivation of Wnt/β-catenin signaling. Our findings suggest that CCAT2 may become a novel molecular target for the prognosis and therapy for ccRCC. Nevertheless, further investigation with a larger sample size is required to support our results.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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