Research Paper Volume 11, Issue 16 pp 5975—5991

LINC00511 promotes the malignant phenotype of clear cell renal cell carcinoma by sponging microRNA-625 and thereby increasing cyclin D1 expression

Figure 5. CCND1 is a direct target gene of miR-625 in ccRCC cells. (A) A putative binding site for miR-625 in the 3′-UTR of CCND1 was predicted by starBase 3.0, TargetScan, microRNA.org, and miRDB. The mutant binding sequences for miR-625 in the 3′-UTR of CCND1 are also shown. (B) Luciferase activity was measured in A498 and 786-O cells cotransfected with a reporter plasmid carrying either the wild-type or mutant CCND1 3′-UTR and either the miR-625 mimics or miR-NC. *P < 0.05 vs. the miR-NC group. (C) RT-qPCR was performed to analyze CCND1 mRNA expression in ccRCC samples and in matched adjacent normal renal tissues. *P < 0.05 vs. normal renal tissue samples. (D) The protein levels of CCND1 were measured in the ccRCC samples and in matched adjacent normal renal tissue samples by western blotting. *P < 0.05 vs. normal renal tissues. (E) The association between miR-625 and CCND1 mRNA levels in ccRCC tissue samples was evaluated by Spearman’s correlation analysis. R2 = 0.3054, P < 0.0001. (F, G) CCND1 mRNA and protein levels in A498 and 786-O cells transfected with either the miR-625 mimics or miR-NC were investigated by RT-qPCR and western blotting, respectively. *P < 0.05 vs. the miR-NC group.