| INTRODUC TI ONBladder cancer (BCa) is the most common malignant tumour of the urinary tract and the 10th most common type of carcinoma worldwide. Approximately 549 000 new cases and 200 000 deaths were estimated by GLOBOCAN in 2018. 1 In 2019, approximately 80 470 patients (including 61 700 men) were diagnosed with BCa and 17 670 patients (including 12 870 men) died from BCa in the United States; thus, BCa ranks the forth in incidence and eighth in mortality in men. 2 The increasing trend in these numbers constantly urges researchers to better understand the mechanisms underlying the pathogenesis of BCa to identify potential therapies against BCa. m 6 A, a modification first identified in mRNA-enriched RNA fractions in 1974, 3 refers to methylation of the N6 position of adenosine bases, which are widely distributed in the mammalian mRNA. 4,5 With the application of available methods for detecting m 6 A, insights into the regulatory mechanism have been revealed in recent years. m 6 A RNA modification is a dynamic and reversible posttranscriptional modification process maintained by a multicomponent Abstract N6-Methyladenosine (m 6 A) modification, the most prevalent modification of eukaryotic messenger RNA (mRNA), is involved in the progression of various tumours.However, the specific role of m 6 A in bladder cancer (BCa) is still poorly understood.In this study, we demonstrated the tumour-promoting function and specific regulatory mechanism of m 6 A axis, consisting of the core 'writer' protein METTL3 and the major reader protein YTHDF2. Depletion of METTL3 impaired cancer proliferation and cancer metastasis in vitro and in vivo. Through transcriptome sequencing, m 6 A methylated RNA immunoprecipitation (MeRIP) and RIP, we determined that the METTL3/YTHDF2 m 6 A axis directly degraded the mRNAs of the tumour suppressors SETD7 and KLF4, contributing to the progression of BCa. In addition, overexpression of SETD7 and KLF4 revealed a phenotype consistent with that induced by depletion of the m 6 A axis. Thus, our findings on the METTL3/YTHDF2/SETD7/KLF4 m 6 A axis provide the insight into the underlying mechanism of carcinogenesis and highlight potential therapeutic targets for BCa. K E Y W O R D Sbladder cancer, carcinogenesis, METTL3/YTHDF2 m 6 A axis, mRNA degradation, RNA modification | 4093 XIE Et al.

BackgroundCurcumin induces apoptosis and autophagy in different cancer cells. Moreover, chemical and biological experiments have evidenced that curcumin is a biologically active iron chelator and induces cytotoxicity through iron chelation. We thus hypothesized that curcumin may induce apoptosis and autophagy in castration-resistant prostate cancer (CRPC) cells through its iron-chelating properties.Materials and methodsCRPC cells were loaded with curcumin alone or in combination with ferric ammonium citrate (FAC). Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was assessed by flow cytometry, terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay and caspase activity. Autophagy status was analyzed by the detection of autophagosomes and light chain 3-II (LC3-II) using transmission electron microscopy and Western blot. Iron-binding activity of curcumin was assessed by spectrophotometry and MTT assay. The expression levels of transferrin receptor 1 (TfR1) and iron regulatory protein 1 (IRP1) were examined by Western blot.ResultsCurcumin induced apoptosis and autophagy in CRPC cells. Combining curcumin with autophagy inhibitors (3-methyladenine [3-MA]) synergized the apoptotic effect of curcumin. Moreover, curcumin bound to FAC at a ratio of ~1:1, as assessed by spectrophotometry and MTT assay. Apoptosis and autophagy induced by curcumin were counteracted by equal amounts of FAC. At apoptosis- and autophagy-inducing concentrations, curcumin enhanced the expression levels of TfR1 and IRP1, indicative of iron deprivation induced by curcumin.ConclusionTogether, our results indicate that curcumin induces apoptosis and protective autophagy in CRPC cells, which are at least partially dependent on its iron-chelating properties.

Bladder neck preservation (BNP) during radical prostatectomy (RP) may improve postoperative urinary continence, although its overall effectiveness remains controversial. We systematically searched PubMed, Ovid Medline, Embase, CBM and the Cochrane Library to identify studies published before February 2016 that assessed associations between BNP and post-RP urinary continence. Thirteen trials (1130 cases and 1154 controls) assessing BNP versus noBNP (or with bladder neck reconstruction, BNR) were considered suitable for meta-analysis, including two randomized controlled trials (RCT), six prospective and five retrospective studies. Meta-analysis demonstrated that BNP improved early urinary continence rates (6 mo, OR = 1.66; 95% CI, 1.21–2.27; P = 0.001) and long-term urinary continence outcomes (>12 mo, OR = 3.99; 95% CI, 1.94–8.21; P = 0.0002). Patients with BNP also had lower bladder neck stricture frequencies (OR = 0.49; 95% CI, 0.29–0.81; P = 0.006). Anastomotic leak rates, positive surgical margins and biochemical failure rates were comparable between the two groups (P>0.05). There were no differences in baseline characteristics except for a smaller average prostate volume (WMD = −2.24 ml; 95% CI, -4.27 to -0.22; P = 0.03) in BNP patients. Our analyses indicated that BNP during RP improved early recovery and overall long-term (1 year) urinary continence and decreased bladder neck stricture rates without compromising oncologic control.

Emerging discoveries of dynamic and reversible N6-methyladenosine (m6A) modification on RNA in mammals have revealed the key roles of the modification in human tumorigenesis. As known m6A readers, insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) are upregulated in most cancers and mediates the enhancement of m6A-modified mRNAs stability. However, the mechanisms of IGF2BPs in renal cell cancer (RCC) still remain unclear. Bioinformatic analysis and RT-qPCR were performed to evaluate the expression of IGF2BPs and m6A writer Wilms tumor 1-associating protein (WTAP) in RCC samples and its correlation with patient prognosis. In vitro, in vivo biological assays were performed to investigate the functions of IGF2BPs and WTAP in RCC. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) combined with bioinformatics analysis and following western blot assay, dual-luciferase reporter assays were performed to validate the regulatory relationships between transcription factor (TF) early growth response 2 (EGR2) and potential target genes IGF2BPs. RNA sequencing (RNA-seq), methylated RNA immunoprecipitation-qPCR (MERIP-qPCR), RIP-qPCR, m6A dot blot, and dual-luciferase reporter assays combined with bioinformatics analysis were employed to screen and validate the direct targets of IGF2BPs and WTAP. Here, we showed that early growth response 2 (EGR2) transcription factor could increase IGF2BPs expression in RCC. IGF2BPs in turn regulated sphingosine-1-phosphate receptor 3 (S1PR3) expression in an m6A-dependent manner by enhancing the stability of S1PR3 mRNA. They also promoted kidney tumorigenesis via PI3K/AKT pathway. Furthermore, IGF2BPs and WTAP upregulation predicted poor overall survival in RCC. Our studies showed that the EGR2/IGF2BPs regulatory axis and m6A-dependent regulation of S1PR3-driven RCC tumorigenesis, which enrich the m6A-modulated regulatory network in renal cell cancer. Together, our findings provide new evidence for the role of N6-methyladenosine modification in RCC.