Abstract
Bladder cancer (BCa) is an aggressive malignancy because of its distant metastasis and high recurrence rate. Circular RNAs (circRNAs) exert critical regulatory functions in cancer progression. However, the expression patterns and roles of circRNAs in BCa have not been well investigated. In this study, we first screened circRNA expression profiles using a circRNA microarray of paired BCa and normal tissues, and the expression of circST6GALNAC6 was confirmed by qRT-PCR and fluorescence in situ hybridization (FISH). MTT, colony formation and Transwell assays were performed to measure cell proliferation, migration and invasion. We investigated the regulatory effect of circST6GALNAC6 on miRNA and its target genes to explore the potential regulatory mechanisms of circST6GALNAC6 by chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), MS2-tagged RNA affinity purification (MS2-TRAP), immunofluorescence (IF) and dual luciferase activity assays. A nude mouse xenograft model was used to examine the functions of circST6GALNAC6/STMN1 in tumour metastasis in vivo. We found that 881 circRNAs were significantly dysregulated in BCa tissues compared to normal tissues. circST6GALNAC6(hsa_circ_0088708) was downregulated in BCa tissues and cells. Overexpression of circST6GALNAC6 effectively inhibited the cell proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) in vitro and suppressed BCa metastasis in vivo. Mechanistically, we showed that the SP1 transcription factor, which binds to the circST6GALNAC6 mRNA transcript, activates circST6GALNAC6 transcription. Next, we verified that circST6GALNAC6 serves as a sponge that directly binds miR-200a-3p to regulate stathmin (STMN1) expression. Furthermore, we found that STMN1 is involved in circST6GALNAC6/miR-200a-3p axis-regulated BCa EMT and metastasis. Thus, our findings indicate an important underlying mechanism in BCa metastasis by which SP1-induced circST6GALNAC6 sponges miR-200a-3p to promote STMN1/EMT signalling. This mechanism could provide pivotal potential prognostic biomarkers and therapeutic targets for BCa.
Highlights
Bladder cancer (BCa) is one of the most prevalent malignancies throughout the world, and millions of people die from the disease every year[1,2]
The results showed that the expression of these 3 circRNAs was consistent with the microarray results obtained by qRT-PCR analysis in BCa tissues and cells (Supplement Fig. 1)
We detected the expression of this transcript as circST6GALNAC6 by amplifying it from the cDNA of J82 cells using the divergent primer strategy and the splice junction of circST6GALNAC6 was verified by Sanger sequencing (Fig. 1D), Because circRNAs are resistant to RNase R treatment, we performed RNase R digestion assay
Summary
Bladder cancer (BCa) is one of the most prevalent malignancies throughout the world, and millions of people die from the disease every year[1,2]. Epithelial–mesenchymal transition (EMT) is a process in which epithelial cells acquire mesenchymal features by decreasing the expression of epithelial markers such as Ecadherin and increasing the expression of mesenchymal markers such as N-cadherin and vimentin[7]. It is a critical process for the invasion and metastasis of tumour cells, and many molecules are involved in this process[7]. Previous studies have shown that miR-200a promotes BCa invasion by targeting Dicer, while miR-200b suppresses EMT and prostate cancer growth and metastasis. The upstream regulators and whether there are additional downstream targets of the miR-200 family in BCa are poorly understood
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