The tetraspanin protein superfamily participate in the dynamic regulation of cellular membrane compartments expressed in a variety of tumor types, which may alter the biological properties of cancer cells such as cell development, activation, growth and motility. The role of tetraspanin 7 (TSPAN7) has never been investigated in bladder cancer (BCa). In this study, we aimed to investigate the biological function of TSPAN7 and its therapeutic potential in human BCa. First, via reverse transcription and quantitative real-time PCR (qRT-PCR), we observed downregulation of TSPAN7 in BCa tissues samples and cell lines and found that this downregulation was associated with a relatively high tumor stage and tumor grade. Low expression of TSPAN7 was significantly correlated with a much poorer prognosis for BCa patients than was high expression. Immunohistochemistry (IHC) showed that low TSPAN7 expression was a high-risk predictor of BCa patient overall survival. Furthermore, the inhibitory effects of TSPAN7 on the proliferation and migration of BCa cell lines were detected by CCK-8, wound-healing, colony formation and transwell assays in vitro. Flow cytometry analysis revealed that TSPAN7 induced BCa cell lines apoptosis and cell cycle arrest. In vivo, tumor growth in nude mice bearing tumor xenografts could be obviously affected by overexpression of TSPAN7. Western blotting showed that overexpression of TSPAN7 activated Bax, cleaved caspase-3 and PTEN but inactivated Bcl-2, p-PI3K, and p-AKT to inhibit BCa cell growth via the PTEN/PI3K/AKT pathway. Taken together, our study will help identify a potential marker for BCa diagnosis and supply a target molecule for BCa treatment.

Circular RNAs (circRNAs) containing retained introns are normally sequestered in the nucleus. Dysregulation of cellular homeostasis can drive their nuclear export, which may be involved in cancer metastasis. However, the mechanism underlying circRNAs nuclear export and its role in lymph node (LN) metastasis of bladder cancer (BCa) remain unclear. Here, we identify an intron-retained circRNA, circNCOR1, that is significantly downregulated in LN metastatic BCa and is negatively associated with poor prognosis of patients. Overexpression of circNCOR1 inhibited lymphangiogenesis and LN metastasis of BCa in vitro and in vivo. Nuclear circNCOR1 epigenetically promoted SMAD7 transcription by increasing heterogeneous nuclear ribonucleoprotein L (hnRNPL)-induced H3K9 acetylation in the SMAD7 promoter, leading to inhibition of the TGFβ-SMAD signaling pathway. Nuclear retention of circNCOR1 was regulated by SUMOylation of DDX39B, an essential regulatory factor responsible for circRNA nuclear-cytoplasmic transport. Reduced SUMO2 binding to DDX39B markedly increased circNCOR1 retention in the nucleus to suppress BCa LN metastasis. By contrast, SUMOylated DDX39B activated nuclear export of circNCOR1, impairing the suppressive role of circNCOR1 on TGFβ-SMAD cascade activation and BCa LN metastasis. In patient-derived xenograft (PDX) models, overexpression of circNCOR1 and inhibition of TGFβ signaling significantly repressed tumor growth and LN metastasis. This study highlights SUMOylation-induced nuclear export of circNCOR1 as a key event regulating TGFβ-SMAD signaling and BCa lymphangiogenesis, thus supporting circNCOR1 as a novel therapeutic agent for patients with LN metastatic BCa.

Considering its speedy development and extremely low 5-year overall survival rate worldwide, bladder cancer (BCa) is one of the most common and highly malignant tumors. Increasing evidence suggests that protein-coding mRNAs and non-coding RNAs, including long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs), play an essential role in regulating the biological processes of cancer. To investigate the molecular regulation associated with poor prognosis during advanced BCa development, we constructed a competitive endogenous RNA (ceRNA) network. Using transcriptome profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases, we performed differential expression (DE) analysis, weighted gene co-expression network analysis, functional enrichment analysis, survival analysis, prediction of miRNA targeting, and Pearson correlation analysis. Through layers of selection, 8 lncRNAs-28 mRNAs and 8 miRNAs-28 mRNAs pairs shared similar expression patterns, constituting a core ceRNA regulatory network related to the invasion, progression, and metastasis of advanced clinical stage (ACS) BCa. Subsequently, we conducted real time qPCR, western blotting, and immunohistochemistry to validate expression trend bioinformatics analysis on 3, 2, and 3 differentially expressed mRNAs, lncRNAs, and miRNAs, respectively. The most significantly differentially expressed LINC01929, miR-6875-5p and ADAMTS12 were selected for in vitro experiments to assess the functional role of the LINC01929/miR-6875-5p/ADAMTS12 axis. RNA pull-down, luciferase assays, and rescue assays were performed to examine the binding of LINC01929 and miR-6875-5p. Increasing trends in COL6A1, CDH11, ADAMTS12, LINC01705, and LINC01929 expression variation were verified as consistent with previous DE analysis results in ACS-BCa, compared with low clinical stage (LCS) BCa. Expression trends in parts of these RNAs, such as hsa-miR-6875-5p, hsa-miR-6784-5p, COL6A1, and CDH11, were measured in accordance with DE analysis in LCS-BCa, compared with normal bladder urothelium. Through experimental validation, the cancer-promoting molecule ADAMST12 was found to play a key role in the development of advanced BCa. Functionally, ADAMTS12 knockdown inhibited the progression of bladder cancer. Overexpression of LINC01929 promoted bladder cancer development, while overexpression of miR-6785-5p inhibited bladder cancer development. Mechanistically, LINC01929 acted as a sponge for miR-6785-5p and partially reversed the role of miR-6785-5p. Our findings provide an elucidation of the molecular mechanism by which advanced bladder cancer highly expressed LINC01929 upregulates ADAMTS12 expression through competitive adsorption of miR-6875-5p. It provides a new target for the prognosis and diagnosis of advanced bladder cancer.