Research Paper Volume 12, Issue 17 pp 17209—17223

Figure 5. CDK6 was directly targeted by miR-147a and was indirectly regulated by circ_0072995. (A) The target genes of miR-147a were predicted in databases (TargetScan, miRWalk, miRDB, StarBase) and there were 141 genes which overlapped in the Venn diagram. (B) PPI network analyses was performed by use of the String database, and results with a combined score of > 0.4 were selected. Among these, CDK6, AKT3, AKT2, PDPK1 were found to have had a high degree of connectivity and were selected as hub genes for miR-147a. (C) CDK6, AKT3, AKT2 expression was significantly reduced after overexpression of miR-147a, especially for CDK6. (D) There were multiple binding sites between miR-147a and CDK6. (E) The binding relationship between miR-147a and CDK6 was confirmed by dual-luciferase reporter assays. (F) miR-147a mimics markedly reduced expression of CDK6, whereas miR-147a inhibitors significantly enhanced CDK6 levels in HO8910 and A2780 cells. (G) Expression of CDK6 mRNA decreased after knockdown of circ_0072995, but was rescued by miR-147a inhibitors as determined by qRT-PCR. (H) The expression of CDK6 proteins decreased after knockdown of circ_0072995, but was rescued by miR-147a inhibitors as determined by WB.