Background: Circular RNAs (circRNAs), a novel type of noncoding RNA (ncRNA), are covalently linked circular configurations that form via a loop structure. Accumulating evidence indicates that circRNAs are potential biomarkers and key regulators of tumor development and progression. However, the precise roles of circRNAs in renal cell carcinoma (RCC) remain unknown. Methods: Through circRNA high-throughput sequencing of RCC cell lines, we identified the circRNA TLK1 (circTLK1) as a novel candidate circRNA derived from the TLK1 gene. qRT-PCR detected the mRNA, circRNA and miRNA expression levels in RCC tissues and cells. Loss-of function experiments were executed to detect the biological roles of circTLK1 in the RCC cell phenotypes in vitro and in vivo. RNA-FISH, RNA pull-down, dual-luciferase reporter, western blot and immunohistochemistry assays were used to investigate the molecular mechanisms underlying the functions of circTLK1. Results: circTLK1 is overexpressed in RCC, and expression is positively correlated with distant metastasis and unfavorable prognosis. Silencing circTLK1 significantly inhibited RCC cell proliferation, migration and invasion in vitro and in vivo. circTLK1 was mainly distributed in the cytoplasm and positively regulated CBX4 expression by sponging miR-136-5p. Forced CBX4 expression reversed the circTLK1 suppression-induced phenotypic inhibition of RCC cells. Moreover, CBX4 expression was positively correlated with VEGFA expression in RCC tissues. CBX4 knockdown significantly inhibited VEGFA expression in RCC cells. Conclusion: Collectively, our findings demonstrate that circTLK1 plays a critical role in RCC progression by sponging miR-136-5p to increase CBX4 expression. circTLK1 may act as a diagnostic biomarker and therapeutic target for RCC.

Accumulating evidences suggest that longnon-coding RNAs (lncRNAs) play functional roles in development of different cancers, including cancer initiation and progression. Metastasis associated lung adenocarcinoma transcript 1(MALAT1) is a well-known lncRNA which was previously shown to be a direct target of miR-125b in bladder cancer (BCa) and to promote cancer progression and invasion. However, little is known whether MALAT1 can also target miR-125b. In the present study, using CRISPR-based technologies and qRT-PCR, we show that MALAT1 is capable of suppressing mature miR-125b and increasing the expression of its target genes (Bcl-2 and MMP-13), but has no effect on pri-miR-125b and pre-miR-125b. We observe that the biotin-labeled MALAT1-RNA probe is able to pull down Ago2 and miR-125b and that the negative regulation of miR-125b by MALAT1 is dependent on Ago2. Importantly, the results of flow cytometry assay and transwell assay reveal that the MALAT1-mediated cancer progression is in part due to specific suppression of miR-125b and activation of its two target genes. All together, these data suggest that the “MALAT1-miR-125b-Bcl-2 / MMP-13” axis plays an important role in the progression of BCa, thereby may provide a potential therapeutic strategy for the treatment of human BCa.

Background: Long non-coding RNAs (lncRNAs) have been proved to act as key molecules in cancer development and progression. Dysregulation of lncRNAs is discovered in various tumor tissues and cancer cells where they can serve as oncogenes or tumor suppressors. Long non-coding RNA HOXD-AS (HOXD cluster antisense RNA 1) has recently been identified to be involved in the development of several cancers including neuroblastoma, adenocarcinomas and breast cancer. However, the role of HOXD-AS1 in bladder cancer remains unknown. Methods: The synthetic tetracycline-controllable shRNA was used to modulate the level of HOXD-AS1 by adding different concentrations of doxycycline (dox). RT-qPCR was used to detect the expression level of HOXD-AS1. Cell proliferation was determined by CCK-8 assay and EdU incorporation experiment when HOXD-AS1 was knocked down. We used wound-healing assay for detecting the effect of HOXD-AS1 on cell migration. Eventually, cell apoptosis was determined by caspase 3 ELISA assay and flow cytometry assay. Results: In this study, we found that the expression level of HOXD-AS1 was significantly increased in bladder cancer tissues and cells. Furthermore, high expression of HOXD-AS1 was significantly related to tumor size, histological grade and TNM stage. In vitro assays confirmed that knockdown of HOXD-AS1 suppressed cell proliferation/migration and increased the rate of apoptotic cell in bladder cancer cells. At last, we used the important element of synthetic biology, tetracycline(tet)-controllable switch, to construct tet-controllable shRNA vectors which can modulate the expression of HOXD-AS1 in a dosage-dependent manner. Conclusions: Our research suggested that high expression of HOXD-AS1 may be involved in the bladder cancer carcinogenesis through inhibiting the phenotypes and activating endogenous cancer-related molecular pathways. Therefore, HOXD-AS1 may act as an oncogene and provide a potential attractive therapeutic target for bladder cancer. In addition, the synthetic tetracycline-controllable shRNA may provide a novel method for cancer research in vitro assays.

Previous studies have suggested that EZH2 is up-regulated in bladder cancer tissues and identified it as a biomarker for poor prognosis. However, the biological functions of EZH2 in bladder cancer cells remain unknown. In this research, we discovered that EZH2 expression is irrelevant to the TNM stage and poor prognosis of bladder cancer patients. But suppression of EZH2 can slowdown the progression of bladder cancer cells. Moreover, we used the technology of synthetic biology to construct the tetracycline-controllable artificial microRNA-HOTAIR + EZH2, which can decrease the expression of HOTAIR and EZH2 in a doxycycline dosage-dependent manner. And we also found that HOTAIR expression was positively correlated with EZH2 expression. Tetracycline-controllable artificial microRNA-HOTAIR + EZH2 can inhibit the proliferation and migration of bladder cancer cells. Meanwhile, the apoptosis rate of bladder cancer cells was increased. Taken together, our research showed the cancer-promoting effects of EZH2 and created a novel method to rescue the development of bladder cancer cells.