br Early studies had reported that circRNAs
Early studies had reported that circRNAs could function as efficient miRNA sponges . However, circRNAs also possessed some other biofunctions except “miRNA sponges”. CircMBL and its franking introns could be strongly and specifically bound by MBL, which contributed to the biogenesis of circRNAs . CircFoxo3 promoted MDM2-induced p53 ubiquitination and subsequent degradation by binding to p53 and MDM2, which indicate that circRNAs may function as protein scaffolds . Recently, it was reported that circ-ZNF609 could be translated to a protein in a cap-independent manner in eukaryote . In the present study, circNR3C1 was found located at cytoplasm, and multiple miRNA-binding sites were predicted in its sequences by miRNA response ele-ments (MREs) analysis, indicating that circNR3C1 might function as a miRNA sponge. Actually, our data clarified that circNR3C1 harbored four miRNA Paxilline of miR-27a-3p and could sponge miR-27a-3p as competing endogenous RNA in bladder cancer cells, which further verified “miRNA sponges” theory. However, whether circNR3C1 pos-sesses other functions in biological process, such as translation or
Fig. 6. cyclin D1 is directly targeted by miR-27a-3p. (A–B) cyclin D1 mRNA expression level was measured using qRT-PCR in EJ and T24T cells which were transfected with circNR3C1 plasmid or circNR3C1 control (vector), and miR-27a-3p-inhibtors or inhibitor NC for 48 h. The negative control was used to rule out the genomic DNA amplification. The data indicated mean ± SEM, n = 3, ns > 0.05. (C) Schematic diagram showed the structure of dual luciferase reporter plasmid of cyclin D1 5′UTR and putative binding sites of cyclin D1 5′UTR bound by miR-27a-3p, as predicted by RNAhybrid2.2. (D–E) Dual luciferase reporter assay showed that luciferase activities were significantly decreased in EJ and T24T cells after transfected with circNR3C1 or miR-27a-3p-inhibitors compared with controls for 48 h. The data indicated mean ± SEM, n = 3, *p < 0.05, **p < 0.01. (F) Three mutants (Mut 1, Mu2 and Mut 1 + Mut2) of dual luciferase reporter plasmids were designed to confirm the two candidates (position 1 and position 2) of miR-27a-3p binding sites of cyclin D1 5′UTR as shown in schematic figure. The bases colored with blue were replace by the bases colored with red in synthesizing the mutants. (G–H) The luciferase activities of cyclin D1 5′UTR were measured in EJ and T24T cells which co-transfected with circNR3C1 plasmid or miR-27a-3p-inhibitors, and three mutants or 5′UTR plasmid for 48 h, respectively. The data indicated mean ± SEM, n = 3. *p < 0.05 versus vector, **p < 0.01 versus vector. (I) The luciferase activities of cyclin D1 5′UTR were measured in EJ and T24T cells which co-transfected with circNR3C1 plasmid or vectors, and miR-27a-3p mimic or miR-NC for 48 h. The data indicated mean ± SEM, n = 3. *p < 0.05, **p < 0.01.
protein sponge, still needs to be explored.
An increasing number of evidences suggest that microRNAs are abnormally expressed in diverse human cancers and have important roles in the tumorigenesis, progression and metastasis of these cancers . Several studies have demonstrated that miR-27a-3p was
upregulated and played significant roles in promoting cell proliferation, migration, invasion and metastatic in multiple types of cancers, such as human gastric cancer , breast cancer , and renal cell carcinoma . Our recent study indicated that inhibition of miR-27a-3p by MEG3, a competing endogenous RNA, could promote PHLPP2 protein
Fig. 7. Schematic diagram of the circNR3C1-regulated pathway in bladder cancer cells. The green arrows indicate promotive function and red lines indicate suppressive function. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)