Abstract
Long non-coding ribonucleic acid urothelial carcinoma-associated 1 has been found to be a participant in cancer development and glucose metabolism in bladder cancer. However, the role of urothelial carcinoma-associated 1 in metabolic reprogramming in cancer remains to be clarified. In this study, we aim to elucidate the molecular mechanism underlying the regulation of glutamine metabolism by urothelial carcinoma-associated 1 in bladder cancer.
The RNA levels of urothelial carcinoma-associated 1, GLS2 and miR-16 in bladder tissues and cell lines were examined by real-time reverse transcriptase-polymerase chain reaction. The protein levels of GLS2 were detected by western blot analysis. Reactive oxygen species generation was examined by the fluorescein isothiocyanate mean value and fluorescence microscope. Glutamine consumption was analyzed using the glutamine assay kit. Additionally, we performed luciferase reporter assays to validate urothelial carcinoma-associated 1 sequence whether contains miR-16 binding site and the interaction between the 3′UTR sequence of GLS2 and mature miR-16.
Real-time reverse transcriptase-polymerase chain reaction demonstrated that the RNA level of urothelial carcinoma-associated 1 and GLS2 was positively correlated in bladder cancer tissues and cell lines. The expression of GLS2 mRNA and protein increased in cells which overexpression of urothelial carcinoma-associated 1 and decreased in cells which knocked-down of urothelial carcinoma-associated 1 cell lines. urothelial carcinoma-associated 1 reduced ROS production, and promoted mitochondrial glutaminolysis in human bladder cancer cells. Furthermore, luciferase reporter assays indicated that there was a miR-16 binding site in urothelial carcinoma-associated 1, and it showed appreciable levels of sponge effects on miR-16 as readouts in a dose-dependent manner. Moreover, the ‘seed region’ of miR-16 directly bound to the 3′UTR of GLS2 mRNA and regulated GLS2 expression level.
Together, our results revealed that urothelial carcinoma-associated 1 regulated the expression of GLS2 through interfering with miR-16, and repressed ROS formation in bladder cancer cells.
Introduction
A key goal of cancer research is to identify how changes impact tumor initiation and development. The essential hallmarks of cancer are metabolic imbalances and improved resistance to mitochondrial apoptosis. The first cue that mitochondria plays pivotal roles in cancer cell biology was discovered by Otto Warburg in the 1920s, and the phenomenon is known as the Warburg effect (1). Mitochondria, especially damaged mitochondria, are the master generators of reactive oxygen species (ROS) (2,3). ROS have long been thought as by-products of cell metabolism, further leading to genetic instability and tumor initiation and procession (4). Glutamine metabolism is very important for tumor cells to a keep redox balance and to manage the toxicity of elevated ROS levels (5). Glutamine is not an essential amino acid, but is an important nutrient source for the majority of tumor cells survival, and this phenomenon is termed as Gln addiction (6–8). Both glutamine uptake and the rate of glutaminolysis are known to accelerate in tumor cells. In glutamine metabolism, mitochondrial glutaminase (GLS) is vital for the conversion of glutamine to glutamate. In mammalian cells, glutaminase consists of two isoforms: GLS1 and GLS2, perform different functions as they are distributed in different chromosomes. GLS2 mediates the antioxidant defense function in cells by prohibiting ROS production and protecting cells from oxygen toxicity, which is known to be involved in cancer formation and development (9,10).
It is clear that up to 70% of our genome is transcribed into RNA that does not serve as protein coding genes, consist of long non-coding RNAs (lncRNAs). lncRNAs were found to be deregulated in several human cancers and show tissue-specific expression (11,12). So far, lncRNAs were also involved in a new regulatory circuitry, lncRNAs can act as competing endogenous RNAs to sequester microRNAs and regulate levels of their transcriptional targets (13,14). Bladder cancer is the fourth most common cancer (15). Urothelial carcinoma-associated 1 (UCA1) is a lncRNA, prior studies linking UCA1 to bladder cancer programs of progression (16), resistance of drug (17,18) apoptosis (17,19,20), tumor microenvironment (21) and the Warburg effect (22), which let us analyze whether UCA1 could reprogram the cellular bioenergetic state phenotype by decoying miRNAs, especially glutaminolysis in bladder cancer.
Given the importance of UCA1 during tumorigenesis, the purpose of the present study is to explore the molecular mechanism underlying the regulation of glutamine metabolism by UCA1 in bladder cancer cells. There is a positive correlationship between RNA levels of UCA1 and GLS2 in bladder cancer tissues and cell lines. Furthermore, we identified that upregulation of UCA1 can promote glutaminolysis and inhibit ROS by improving GLS2 expression in bladder cancer cells. Finally, our results showed that UCA1 contains the miR-16 binding site, and the mRNA expression levels of GLS2 are negatively correlated with miR-16 in bladder cancer cells and tissues. Knowledge of the molecular mechanism underlying UCA1 participation in glutamine metabolism in bladder cancer will shed new light on understanding of bladder cancer and provide future clinical treatment for this disease.
Patients and methods
Clinical samples
A total of 51 tissues, consisting of 6 normal bladder tissues, 10 adjacent cancer tissues and 35 bladder cancers were obtained from patients at the First Affiliated Hospital of Xi'an Jiaotong University in China between 2006 and 2011. All human samples were used in accordance with the policies of the institutional review board of the First Affiliated Hospital of Xi'an Jiaotong University. All tissues were obtained either from transurethral resection (TUR) or cystectomy. Tumors were graded by the 2004 WHO Histologic Classification of urinary tract tumors. The clinicopathologic characteristics of the informative cases are shown in Table 1.
Summary of clinical characteristics
| Characteristics | Cases |
|---|---|
| Bladder cancer tissue | |
| Total number | 35 |
| Median age years (range) | 64 (44–76) |
| Sex | |
| Male | 30 |
| Female | 5 |
| Grade | |
| G1 | 8 |
| G2 | 16 |
| G3 | 11 |
| Operation mode | |
| TUR | 2 |
| Partial cystectomy | 3 |
| Radical cystectomy | 30 |
| Normal bladder tissue | |
| Total number | 6 |
| Adjacent cancer tissue | |
| Total number | 10 |
| Characteristics | Cases |
|---|---|
| Bladder cancer tissue | |
| Total number | 35 |
| Median age years (range) | 64 (44–76) |
| Sex | |
| Male | 30 |
| Female | 5 |
| Grade | |
| G1 | 8 |
| G2 | 16 |
| G3 | 11 |
| Operation mode | |
| TUR | 2 |
| Partial cystectomy | 3 |
| Radical cystectomy | 30 |
| Normal bladder tissue | |
| Total number | 6 |
| Adjacent cancer tissue | |
| Total number | 10 |
TUR, transurethral resection.
Summary of clinical characteristics
| Characteristics | Cases |
|---|---|
| Bladder cancer tissue | |
| Total number | 35 |
| Median age years (range) | 64 (44–76) |
| Sex | |
| Male | 30 |
| Female | 5 |
| Grade | |
| G1 | 8 |
| G2 | 16 |
| G3 | 11 |
| Operation mode | |
| TUR | 2 |
| Partial cystectomy | 3 |
| Radical cystectomy | 30 |
| Normal bladder tissue | |
| Total number | 6 |
| Adjacent cancer tissue | |
| Total number | 10 |
| Characteristics | Cases |
|---|---|
| Bladder cancer tissue | |
| Total number | 35 |
| Median age years (range) | 64 (44–76) |
| Sex | |
| Male | 30 |
| Female | 5 |
| Grade | |
| G1 | 8 |
| G2 | 16 |
| G3 | 11 |
| Operation mode | |
| TUR | 2 |
| Partial cystectomy | 3 |
| Radical cystectomy | 30 |
| Normal bladder tissue | |
| Total number | 6 |
| Adjacent cancer tissue | |
| Total number | 10 |
TUR, transurethral resection.
Cell culture and treatment
The human bladder cancer cells (UMUC2 and 5637) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), human bladder transitional cell carcinoma cell lines BLS-211 and BLZ-211 were obtained from surgical specimens from our laboratory in 1994. All cell lines were cultured in PRMI 1640 medium with l-glutamine (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% bovine calf serum. Stable cell lines with ectopic expression of UCA1 in UMUC2 cells, named as pcDNA-M and pcDNA-U (constructed with pcDNA3.1(+)/Mock and pcDNA3.1(+)/UCA1, respectively), and cell lines with knockdown of UCA1 in 5637 cells, defined as pRNAT-N and pRNAT-U (pRNAT-U6.1/Neo-NC and pRNAT-U6.1/Neo-shUCA1, respectively) were kindly provided by our colleagues Chen Yang and Yu Wang. microRNA mimics and inhibitors (RiboBio Biotech Co Ltd, Guangzhou, China) were transfected using XtremeGene siRNA Transfection Reagent (Roche Diagnostics, Indianapolis, IN, USA)) according to the protocol of the manufacturer.
RNA extraction and qRT-PCR analyses
Total RNA was extracted from cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Of note, 1–3 µg RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Perfect Real-Time; Takara, Dalian, China), quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses were performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The conditions for PCR reaction were as follows: initial denaturation at 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, 57°C for 15 s and 72°C for 20 s. ΔCt values were normalized to β-actin as the internal control. Each sample was analyzed in triplicate, and all experiments were performed in triplicates. The primer sequences (β-actin, forward 5′-TCCCTGGAGAAGAGCTACGA-3′, and reverse 5′-AGCACTGTGTTGGCGTACAG-3′; Lnc-UCA1, forward 5′-CTCTCCATTGGGTTCACCATTC-3′, and reverse 5′-GCGGCAGGTCTTAAGAGATGAG-3′; GLS2, forward 5′-TGCCTATAGTGGCG ATGTCTCA -3′, and reverse 5′-GTTCCATATCCATGGCTGACAA -3′. The primers of miR-16, miR-195, miR-15 and miR-424 were purchased from RiboBio Biotech Co Ltd.
Intracellular ROS assay
Cells were digested with trypsin, and incubated with 10 nmol/l dichlorodihydrofluorescein diacetate(DCFH-DA) at 37°C for 60 min, and then centrifuged at 600g for 5 min at 4°C. Fluorescence-labeled cells were suspended in phosphate buffered saline and fluorescence was detected with the BD FACS Calibur Flow Cytometry System (excitation: 485 nm; emission: 530 nm). ROS levels were expressed as relative DCF fluorescence by the fluorescein isothiocyanate mean value and fluorescence microscope.
Glutamine activity assay
To test the levels of glutamine, the supernatants of cell culture media were collected and analyzed using the glutamine assay kit (JianCheng, NanJing, China) according to the protocol of manufacturer. The values were measured by calculating the optical density values. Glutamine consumption was estimated based on the standard curve, and normalized to the cell number.
Western blot assay and antibodies
Briefly, blocked nitrocellulose membrane reacted with anti-GLS2 antibody (Abcam, Cambridge, UK), anti-β-actin (Cell Signaling Technology, MA, USA) at a dilution of 1:1000. The signals were visualized by the chemiluminescence detection kit (Pierce, Rockford, IL, USA) and the immunoreactive bands were tested by ChemiDoc MP System (Bio-Rad Laboratories).
Bioinformatic analysis
To explore miRNA-binding sites in UCA1, we used a web-based program known as RNAhybrid. To examine the predicted target genes and their conserved sites that match the seed region of each miRNA, we employed the TargetScan program. The sequences of the predicted mature miRNAs were verified by miRBase.
Luciferase reporter assay of UCA1 and GLS2
Through an analysis of the complementary sequences between the miRNAs and GLS2 mRNA, miR-16 has been found to contain 10 nucleotides that complement bases 2562–2571 of the GLS2 mRNA. miR-16 binding sites in UCA1 have been found to contain 23 nucleotides that complement bases 217–239 of the UCA1. Thus, one 3′UTR segment of 192 bp (2408–2599) of the GLS2 gene and UCA1 full-length cDNA were inserted into the pMIR-REPORT™ Luciferase (pMIR-R-L) control vector, respectively. Target segments and mutant inserts were confirmed by sequencing. Renilla luciferase vector was used for normalization. The cells were co-transfected in 24-well plates using the FUGENE HD Transfection Reagent according to the manufacturer's specifications with 0.18 µg pMIR-R-L vector and 0.02 µg control vector. Moreover, different concentrations of microRNA mimics were used for each well. pMIR-R-L and Renilla luciferase activities were measured consecutively using the dual-luciferase reporter assay system (Promega, Madison, WI, USA), 24–48 h after transfection.
Statistical analysis
All the experiments were performed as three independent experiments. Data are expressed as the means ± SEM, were analyzed using one-way ANOVA by the SPSS 18.0 and GraphPad Prism 5 statistical software, with P values <0.05 are considered to be significant.
Results
UCA1 and GLS2 relative level is positively correlated in bladder cancer tissues and cell lines
In order to compare the expression of UCA1 in different bladder cancers, we detected RNA levels of UCA1 in 6 normal bladder tissues, 10 adjacent bladder cancer tissues and 35 bladder cancer tissues. Total RNA was extracted from these samples; quantitative real-time PCR analysis was performed to detect the expression level of UCA1. We identified that UCA1 level in bladder cancer tissues is higher than in normal bladder tissues and adjacent cancer tissues (Fig. 1A). Moreover, by comparing the relationship of RNA expression levels between UCA1 and GLS2 in bladder tissues, we found that the mRNA expression of GLS2 is positively correlated with UCA1 in bladder tissues (Fig. 1B). Furthermore, we analyzed the RNA expression levels of UCA1 and GLS2 among bladder cancer cell lines (5637, UMUC2, BLS-211 and BLZ-211). As shown in Fig. 1C, the results show that the GLS2 level in UCA1 high-expression cell lines (5637 and BLZ-211) was higher than UCA1 low-expression cell lines (UMUC2 and BLS-211) (Fig. 1D). These results demonstrated that mRNA expression levels of GLS2 are positively correlated with UCA1in bladder cancer.
Urothelial carcinoma-associated 1 (UCA1) and GLS2 relative levels are positively correlated in bladder cancer tissues and cell lines. (A) mRNA level of UCA1 in bladder cancer tissues is higher than in the normal bladder tissues and adjacent cancer tissues. (B) GLS2 level positively correlates with the UCA1 level in bladder tissues. (C) UCA1 expression levels were detected in various bladder cancer cell lines. (D) GLS2 expression levels were detected in various bladder cancer cell lines. Both UCA1 and GLS2 relative levels were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). β-Actin was used as the internal control.
UCA1 regulates glutaminolysis and the cell redox state in bladder cancer cells
Taking into account UCA1 plays an important role in tumor invasion, progression and glycolysis, we proposed a hypothesis that UCA1 may be involved in reprogramming of energy metabolism in cancer cells. Glutaminase 2 is a GLS enzyme catalyzing the conversion of glutamine to glutamate and it has been identified as a mediator of antioxidant defense mechanism in energy metabolism. To explore the feasible mechanisms by which UCA1 modulates glutaminolysis in bladder cancer cells, we detected the effects of UCA1 on the expression of GLS2. First, we constructed overexpression of UCA1 in UMUC2 cells, which possess low endogenous UCA1 expression levels. Real-time quantitative PCR results demonstrated that UCA1 increased GLS2 mRNA levels (Fig. 2A). In line with these results, western blot analyses indicate that GLS2 protein expression was upregulated by UCA1 (Fig. 2B). To determine whether UCA1 regulates glutamine metabolism in human bladder tumor cells, we measured the effects of UCA1 on glutaminolysis. The glutamine uptake assay displayed that UCA1 could stimulate glutamine absorption in UMUC2 cells (Fig. 2C). In line with these results, cell viability revealed that glutamine enhanced the vitality of the cells in overexpression of UCA1 cells when compared with normal UMUC2 cells (Fig. 2D). Glutamine has long been known to play a pivotal role in the metabolism and regulation of the effects of ROS in proliferating cells, compared with other amino acids, so we tested the function of UCA1 on ROS generation, the results showed that UCA1 obviously decreased the level of ROS in UMUC2 cells (Fig. 2E). We also generated UCA1 knocked-down 5637 cell lines that have high endogenous UCA1 expression levels. Knockdown of UCA1 expression repressed both GLS2 mRNA and protein expression in 5637 cells (Fig. 2F and G). Additionally, we unveiled that glutamine uptake (Fig. 2H) and cell viability were declined in 5637 cells with knockdown of UCA1 (Fig. 2I), while cell viability was slightly lower in UCA1 knocked-down 5637 cells, and there is no significant difference. We also detected that production of ROS was significantly enhanced in UCA1 knocked-down 5637 cells (Fig. 2J). Taken together, we identified that UCA1 regulates glutamine metabolism and the cell redox state in bladder cancer cells.
Urothelial carcinoma-associated 1 (UCA1) regulates glutaminolysis and the cell redox state in bladder cancer cells. (A and F) Quantitative PCR analysis of GLS2 mRNA levels in UMUC2 cells transfected with pcDNA-M or pcDNA-U plasmids and 5637 cells transfected with pRNAT-N and pRNAT-U plasmids. (A) Overexpression of UCA1. (F) Knockdown of UCA1. (B and G) Western blot assay of the GLS2 protein level in stable cell lines with overexpression of UCA1. (B) Overexpression of UCA1. (G) Knockdown of UCA1. (C and H) Glutamine uptake assay in UMUC2 cells transfected with pcDNA-M or pcDNA-U plasmids and 5637 cells transfected with pRNAT-N and pRNAT-U plasmids. (C) Overexpression of UCA1. (H) Knockdown of UCA1. (D and I) Cell viability in stable cell lines with overexpression of UCA1. (D) Overexpression of UCA1. (I) Knockdown of UCA1. (E and J) ROS generation was detected in stable cell lines. (E) Overexpression of UCA1. (J) Knockdown of UCA1. The average values SD of three separate experiments were plotted. *P < 0.05.
UCA1 contains miR-16 binding site
We next wanted to identify the mechanism of regulating the expression of GLS2 by UCA1. miRNAs can regulate gene activity by binding to the 3′UTR of target mRNA, resulting in translational suppression and gene silencing. The binding sites of GLS2 and miRNAs were analyzed using bioinformatic software of TargetScan program. Four miRNAs were selected from predicted results, including in miR-15a, miR-16, miR-195 and miR-424. lncRNAs can act as sponges to bind specific miRNAs and modulate their function (14). We observed that UCA1 obviously decreased the level of miR-16 and miR-195, ∼35.3 and 26.6%, respectively, while the level of miR-15a and miR-424 were not changed, as measured by qRT-PCR (Fig. 3A). Moreover, the level of miR-16 and miR-195 were enhanced in UCA1 knocked-down cell lines (Fig. 3B). Furthermore, we analyzed the binding energy and sites of UCA1 and miR-16 using RNAHybrid software, and the results implied that the miR-16 mature chain is partly complementary and thus potentially binds to UCA1 (Fig. 3C). This observation allows us to speculate that UCA1 may directly bind to miR-16, and we constructed luciferase reporter plasmids containing either wild-type UCA1 or a mutant UCA1 (Fig. 3C). Then, UMUC2 cells were transfected with the miR-16 or control mimics plus wild-type UCA1 or a mutant UCA1. The results showed a dose-dependent repression of miR-16 by UCA1 (Fig. 3D). While mutating the miR-16 site in UCA1 it no longer elicited such a significant effect (Fig. 3E). Taken together, these results indicated that UCA1 contains the miR-16 binding site.
UCA1 contains miR-16 binding site. (A, B) Quantitative reverse transcriptase-polymerase chain reaction analysis of microRNA levels in UMUC2 cells transfected with pcDNA-M or pcDNA-U plasmids and 5637 cells transfected with pRNAT-N and pRNAT-U plasmids. (A) UMUC2 cells; (B) 5637 cell lines. (C) Putative miR-16-binding sequence of UCA1. The wild-type or mutant miR-16-binding UCA1 sequence was cloned into pMIR luciferase reporter. (D, E) The luciferase activity normalized relative to Renilla luciferase activity was determined after transfection 24–48 h. (D) UCA1 transfected into UMUC2 cells, together with miR-16 mimics at a final concentration of 20, 50 or 100 nM; (E) UCA1 or UCA1 mutation sites in miR-16 binding sites. The average values SD of three separate experiments were plotted. *P < 0.05. UCA1m, UCA1 mutation; m16, miR-16.
GLS2 is negatively correlated with miR-16 in bladder cancer cells and tissues
To assess the effects of miR-16 on GLS2 activity, we transfected miR-16 mimics and inhibitors into UMUC2 cells, to specifically overexpress and knockdown the endogenous expression of miR-16. As shown in Fig. 4A–D, GLS2 expression activity was greatly repressed by miR-16 mimics and was sharply enhanced by miR-16 inhibitors at both the mRNA and protein levels. Furthermore, to validate the interaction between the 3′UTR sequence of GLS2 and mature miR-16, we constructed luciferase reporter plasmids containing either wild-type GLS2 3′UTR or a mutant GLS2 3′UTR (Fig. 4E). We performed luciferase reporter assay and the observation showed that miR-16 directly binds to the GLS2 3′UTR. Only transfection of the wide-type GLS2 3′UTR decreased luciferase activity, the inhibitory effect of miR-16 on GLS2 was retarded by mutating the miR-16 site in the GLS2 3′UTR (Fig. 4F). Moreover, as we predicted, there is a negative correlation between the levels of GLS2 and miR-16 in bladder tissues (Fig. 4G). Collectively, these results demonstrated that GLS2 is negatively regulated by miR-16 in bladder cancer cells and tissues.
GLS2 is negatively correlates with miR-16 in bladder cancer cells and tissues. (A and C) Quantitative real-time PCR assay of GLS2 mRNA levels in UMUC2 cells transfected with miR-16 mimics or inhibitors. (A) UMUC2 cells transfected with miR-16 mimics; (C) UMUC2 cells transfected with miR-16 inhibitors. (B and D) Western blot analysis of GLS2 protein expression levels n UMUC2 cells transfected with miR-16 mimics or inhibitors. (B) UMUC2 cells transfected with miR-16 mimics; (D) UMUC2 cells transfected with miR-16 inhibitors. (E) Putative miR-16-binding sequence of GLS2 3′UTR. Mutation was generated on the GLS2 mRNA 3′UTR sequence in the complementary site for the seed region of miR-16. The wild-type or mutant miR-16 binding GLS2 mRNA 3′UTR sequence was cloned into pMIR luciferase reporter. (F) The luciferase activity normalized relative to Renilla luciferase activity was determined after transfection 24–48 h, GLS2 3′UTR or GLS2 mutation 3′UTR were cotransfected with miR-16 mimics or control into UMUC2 cells. (G) The RNA levels of GLS2 are negatively correlated with miR-16 in bladder tissues. GLS2m, GLS2 mutation.
Discussion
Accumulating evidences suggest that lncRNAs play key roles in the regulation of cell function in the mammalian genome. lncRNA biology is arousing wide attention in cancer research because their dysregulation occurs in a variety of cancers. The recent work of competitive endogenous RNAs (ceRNA) provides a new avenue to identify the specific mechanism of lncRNA (23,24). We have previously reported that UCA1 participated in cancer progression, apoptosis, the Warburg effect and microenvironment (16–22). However, whether UCA1 is involved in glutamine metabolism and the clear molecular mechanism is not fully known. In the current study, we found that UCA1 acts as a nature decoy to sequester miRNAs and exerts their effects on bladder cancer cells.
Mitochondria, considered as the main source of ROS, are vital for the cancer cells. ROS, by-products of mitochondrial metabolism, play multifaceted roles in cancer progression including cancer initiation, development, apoptosis and autophagy. In this study, we manifested that UCA1 can retard the rate of ROS formation. We then assessed mitochondrial biogenesis by counting the mtDNA copy number, and found that the mRNA level of UCA1 was positively correlated with the quantity of mtDNA. In addition, mitochondrial membrane potential (MMP) is evaluated using the JC-1 assay (data not shown), the results show that MMP did not change either in overexpression or down-regulation of UCA1 cell lines. These data suggested that UCA1 can protect mitochondrial function by reducing ROS generation, but is not involved in the regulation of early apoptosis.
Glutaminolysis is pivotal for cancer cells to maintain the redox balance and to reduce the excessive ROS levels. Glutamine metabolism may generate antioxidants such as NADPH and glutathione to contribute to the redox balance in cancer cells (25). We proposed a hypothesis that UCA1 performs its function in ROS production by altering glutamine metabolism. To that end, we tested that UCA1 exerts its role in glutaminolysis by upregulation of glutaminase. Glutaminase has two isoforms: GLS1 and GLS2, and we identified that UCA1 increases mRNA levels of both GLS1 and GLS2. GLS2 can act as an antioxidant defensor, by protecting cells from oxidative stress (10), consequently, we selected GLS2 as our molecular target.
Mounting evidence demonstrated that miRNAs play key roles in carcinogenesis by regulating the expression of oncogenes or tumor suppressors at the post-transcriptional level. Given that GLS2 is a known potential target of miR-16 (predicted by TargetScan software) and that lncRNAs can act as sponges that bind specific miRNAs and regulate their function (26,27). Additionally, it has been reported that miR-16 can act as a tumor suppressor to regulate cell proliferation and apoptosis in a variety of cancer cells including bladder cancer (28). We postulated that UCA1 may bind miR-16 and interfere with their function in bladder cancer cells. Our results indicated that UCA1 show appreciable levels of sponge effects on miR-16 as readouts in a dose-dependent manner. Meanwhile, we confirmed that the ‘seed region’ of miR-16 directly binds to the 3′UTR of GLS2 mRNA by utilizing the Dual Luciferase Reporter Assay System. Together, the data above suggested that UCA1 may serve as a natural molecular sponge to block the tumor suppressor role of miR-16 and to promote tumorigenesis in bladder cancer cells.
In this study, we showed that expression of both UCA1 and GLS2 was significantly higher in human bladder cancer samples than in normal bladder tissues, while miR-16 was down-regulated in bladder carcinomas. The expression level of miR-16 in bladder cancer is consistent with what have been reported in the literature (29). Collectively, our findings indicated that UCA1 regulates the expression of GLS2 by interfering with miR-16, and represses ROS formation in bladder cancer cells.
In conclusion, our study demonstrated that UCA1 is highly expressed in the bladder cancer tissue, binds to miR-16 and inhibits its function, acting as a molecular sponge through UCA1-miR-16-GLS2 axis, contributing to glutamine metabolism and redox state regulation.
Funding
This work was supported by National Natural Science Foundation of China (81372151).
Conflict of interest statement
None declared.
