MITF-Mediated lncRNA CCDC183-As1 Promotes the Tumorigenic Properties and Aerobic Glycolysis of Bladder Cancer via Upregulating TCF7L2

As a primary malignancy tumor of the urology system, bladder cancer (BC) is characterized by its high recurrence and metastasis characteristics. Despite the great improvement in clinical interventions over the past decades, the outcomes of BC patients are still unsatisfactory. Novel molecular mechanisms for developing effective diagnostic and therapeutic strategies are urgently needed; therefore, we screened the lncRNA expression profile in four pairs of BC tissues, showing that CCDC183-AS1 was the most upregulated lncRNA. Subsequently, results of CCK-8, EdU, Transwell, and aerobic glycolysis detection showed that CCDC183-AS1 plays an oncogene role in BC progression. Furthermore, an investigation of the downstream and upstream factors of CCDC183-AS1 identified a novel MITF/CCDC183-AS1/miR-4731-5p/TCF7L2 axis in BC progression, which might furnish novel insights for developing effective diagnostic and therapeutic strategies for BC.


Introduction
Bladder cancer (BC) ranks the second most common cancer in the urology system with more than 550,000 newly diagnosed and 20,000 BC-related deaths every year [1]. e most prominent features of BC are the high recurrence rate and local or distant metastasis, contributing to the poor prognosis and outcomes of BC [2]. Despite the great improvement in clinical treatment including radiation, surgery, and chemotherapy, the 5-year survival rate is still poor [3]. Hence, identifying novel mechanisms of BC for developing effective diagnosis and treatment strategies is demanded.
Long noncoding RNA (lncRNA) is a type of RNA more than 200 nucleotides long without protein-coding capability [4]. lncRNA exerts its role via four mechanisms including decoys, guides, molecular scaffolds, and signaling molecules to regulate the transcription or expression of genes [5]. Currently, the molecular and biological functions of lncRNAs in various diseases have been well documented, such as cardiovascular diseases, neuropathic pain, diabetes, and human cancers [6][7][8][9]. Accumulating evidence suggests that dysregulation of lncRNAs participates in the development of BC. Chen et al. revealed that lncRNA LNMAT contributes to the lymphatic metastasis of BC [10]. He et al. demonstrated that lncRNA BLACAT2 aggravates BC-associated lymphangiogenesis and lymphatic metastasis [11]. Luo et al. found that lncRNA PR11-89 modulates tumorigenesis and ferroptosis resistance of BC through sponging miR-129-5p [12]. Tan J et al. demonstrated a novel lncRNA TUG1/miR-320a/FOXQ1 pathway in BC progression [13].
is study explored a new functional lncRNA in the progression of BC. First, we collected four pairs of BC tumor samples and comparative normal samples for the lncRNA microarray experiment, showing that lncRNA coiled-coil domain-containing 183 antisense RNA 1 (CCDC183-AS1) was markedly increased in BC tumor samples compared to normal samples. Next, the biological functions of CCDC183-AS1 in BC cells were investigated, showing that CCDC183-AS1 knockdown retarded cell viability, migration, and invasion. Furthermore, CCDC183-AS1 was involved in the modulation of aerobic glycolysis. Subsequently, our study demonstrated the molecular mechanisms including the downstream and upstream regulators of CCDC183-AS1, identifying a novel MITF/CCDC183-AS1/ miR-4731-5p/TCF7L2 axis in BC progression, which may be useful for developing new diagnostic or treatment strategies for BC.  [13]. e experiment was repeated in triplicate (fold change > 1.5 and P values < 0.05).

Western
Blotting. Western blot analysis was carried out with the primary antibody TCF7L2 (ab134275, 1 : 500) and HPR-labeled secondary antibody (ab205718, 1 : 20000). In brief, total proteins were isolated using a radioimmunoprecipitation assay (Beyotime, Jiangsu, China) and quantified using a bicinchoninic acid assay. e proteins were separated using 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (Beyotime) and then transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). 5% milk was used to block membranes, then incubated with TCF7L2 antibody for 12 hours at 4°C, followed by HPR-labeled antibody for 2 hours. An enhanced chemiluminescence system (Millipore) was used to capture the signals.

Subcellular Fractionation.
e determination of subcellular distribution was conducted using a PARIS Kit (Life Technologies, Pudong, Shanghai, China) following the manufacturer's guides.

Cell Proliferation Assay.
A CCK-8 (Beyotime, Shanghai, China) was applied to measure cell proliferation ability. e cells were cultured in a 96-well plate at 37°C for 24 h before the addition of CCK-8 solution for 1 hour. Cell absorbance was analyzed using a microplate reader (Potenov, Beijing, China) at 450 nm wavelength to calculate cell growth.

Detection of Cell Migration and
Invasion. Cells (10 6 cells/ mL, 200 μL) were seeded in an 8-μm Transwell chamber (Corning, NY, USA). e chamber (lower one) was filled with cells or 10% FBS medium (600 μL). After 24 hours, the cells were added with 4% paraformaldehyde and stained for 20 min with 0.5% crystal violet staining solution (Sigma-Aldrich). Subsequently, the cells in the chamber (upper one) were removed, and then migrated cells were recorded using an inverted fluorescence microscope (TE2000, Nikon, Japan). e chamber added with Matrigel (BD Biosciences, CA, USA) was for invasion detection.

Cellular Glycolysis and Oxidative Phosphorylation
Detection.
e rate of extracellular acidification (ECAR) was used to detect glycolysis level, and the OCR was used to detect oxidative phosphorylation level using the XF96  wild-type (WT) CCDC183-AS1 and TCF7L2. e mutanttype (MUT) vectors were obtained using mutant miR-4731-5p binding sites. After transfection, the luciferase activity was measured using a microplate reader.
2.14. Statistical Analysis. GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA) and SPSS 19 were used to conduct statistical analysis. For data analysis, Student's ttests (two-tailed) and one-way ANOVA were conducted.

Journal of Oncology
Experimental data are shown as the mean ± SD. P < 0.05 was treated as statistically significant.

CCDC183-As1
Is Highly Expressed in BC. CCDC183-AS1 was markedly increased in BC tumor samples compared to the adjacent normal samples (Figure 1(a)), and this was confirmed by the data from thirty pairs of BC tissues examined by qRT-PCR (Figure 1(b)). In the BC cell lines (SW780, UMUC3, T24, HT1367, and 5637), CCDC183-AS1 expression was higher compared to the bladder cells (SV-HUC-1) (Figure 1(c)). Intracellular distribution results have shown that CCDC183-AS1 was mainly located in the cell cytosol (Figures 1(d) and 1(e)). CCDC183-AS1 might play a critical role in BC pathology.

MITF Transcriptionally Regulates CCDC183-As1
Expression in BC Cell. Accumulating evidence suggests that some translational factors may contribute to the dysregulation of lncRNAs [19,20]. Hereby, by utilizing the JASPAR database, it was found that Melanocyte Inducing Transcription Factor (MITF) might transcriptionally regulate  CCDC183-AS1 expression. Subsequently, it was revealed that the CCDC183-AS1 level was positively modulated by MITF in a dose-dependent manner (Figure 9(a)). Furthermore, CCDC183-AS1 expression decreased in MITF downregulated UMUC3 and SW780 cells (Figure 9(b)). ese data suggested that MITF regulates CCDC183-AS1. To validate that MITF is a transcription factor of CCDC183-AS1, the predicted binding sites between MITF (Figure 9(c)) and CCDC183-AS1 (Figure 9(d)) were obtained from JASPAR. Results of the dual-luciferase reporter gene assay suggested that MITF directly targets the promoter of CCDC183-AS1 in UMUC3 and SW780 cells (Figures 9(e) and 9(f )). Our results revealed that CCDC183-AS1 expression could be transcriptionally activated by MITF.

Discussion
BC is the primary malignant tumor of the genitourinary tract and a huge healthcare burden [21]. BC patients with an advanced stage or chemo-resistance phenomena have a poor prognosis and outcomes [22,23]. Due to the complex process and epigenetic abnormalities of BC, the molecular mechanisms behind BC development have been studied in-depth in the past decades, such as modifications of DNA and histone, chromatin remodeling, RNA methylation, noncoding RNAs, and ubiquitination [24][25][26][27][28][29][30]. Emerging evidence suggests that lncRNA is a hot topic in recent years [28,31,32]. Our study identified a novel functional lncRNA CCDC183-AS1, which plays an oncogene role in BC progression.
It has been well documented that lncRNA acts as a molecular sponge for microRNAs and transcriptionally regulates gene expression. Indeed, a lncRNA/miRNA/mRNA network has been studied in various diseases, especially in cancer [6,[33][34][35][36][37]. Herein, our study investigated the downstream mechanisms of CCDC183-AS1 in BC and identified the potential miRNA/mRNA axis, the novel CCDC183-AS1/miR-4731-5p/TCF7L2 axis. Furthermore, studies have confirmed that transcription factors can regulate lncRNA expression in multiple cell types [38][39][40][41]. By conducting bioinformatics analysis and luciferase reporter assays, our results suggest that MITF transcriptionally regulates CCDC183-AS1 expression in BC cells. MITF belongs to the helix-loop-helix leucine zipper (b-HLH-zip) family, the functions of which have been investigated in-depth in the development and maintenance of melanoma. e dysregulation of MITF is involved in cellular behaviors including proliferation, migration, and invasion [42][43][44][45][46]. Notably, none of them has been well studied in BC, and our results demonstrated the biological functions and tumor expression of each gene in BC progression, which enriched the research profiles for the MITF/CCDC183-AS1/miR-4731-5p/TCF7L2 axis.
TCF7L2 is an essential gene in the modulation of aerobic glycolysis (the Warburg effect) [17,18,47]. Aerobic glycolysis has a crucial role in tumor progression, maintenance, and cell transformation [48][49][50], so our study investigated whether CCDC183-AS1 exerts its function on aerobic glycolysis in BC cells. Our results suggest that CCDC183-AS1 positively regulated aerobic glycolysis in BC cells by regulating TCF7L2, which proved a new insight into the study of aerobic glycolysis in BC.
Although our results have partially demonstrated the molecular relationships among the MITF/CCDC183-AS1/ miR-4731-5p/TCF7L2 axis and revealed the biological roles of the axis in BC development, the clinical significance of each gene in BC needs large-scale human samples and data for further investigation. Also, the in vitro experiment results need to be further confirmed in vivo.
In conclusion, our results demonstrated that CCDC183-AS1 functions as an oncogene in BC progression. CCDC183-AS1 knockdown suppressed cell proliferation, migration, invasion, and aerobic glycolysis levels.
e novel MITF/ CCDC183-AS1/miR-4731-5p/TCF7L2 axis identified in BC may be a promising diagnostic or treatment target for BC in the future.
Data Availability e datasets supporting the conclusions of this article can be obtained from the corresponding author under reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
WC and MW performed primers experiments; WC and ZS worked on the analysis and interpretation of data; ZS contributed to writing, review, and/or revision of the manuscript. e final manuscript was approved by all authors.