Ative control(IC50 = 820.89 ?21.62 nM VS 2424.56 ?56.83nM, P = 0.001), SKOV3 cells were transfected with miR-9 inhibitor or negative control (IC50 = 122.74 ?10.12 nM VS64.63 ?2.74 nM, P = 0.000), the cytotoxicity of paclitaxel on EOC cells were assessed by MTS assay. b. Western blot analysis of CCNG1 in ST30 cells transfected with miR-9 mimic or negative control. GAPDH was used as house-keeping gene. c. Dual luciferase reporter assay. 293 T cells were transfected with CCNG1 -wild type 3UTR vectors or mutant 3UTR vectors together with miR-9 mimic or its negative control. Luciferase activity was measured 48 h after FPS-ZM1MedChemExpress FPS-ZM1 cotransfection. A decrease of the luciferase activity was observed in miR-9 overexpressing cells compared with control (* P = 0.008). d. Modulating miR-9 expression changed paclitaxel sensitivity of A2780 and A2780R cells. A2780 cells were transfected with miR-9 inhibitor or negative control (IC50 = 95.644 ?12.03 nM VS 38.16 ?6.18 nM, P = 0.000), A2780R cells were transfected with miR-9 mimic or negative control(IC50 = 194.94 ?9.36 nM VS 774.03 ?49.19 nM, P = 0.002). e. Western blot analysis of CCNG1 in SKOV3 cells transfected with miR-9 inhibitor, negative control or inhibitor combined with CCNG1 siRNA. GAPDH was used as house-keeping gene. f. Modulating CCNG1 expression changed paclitaxel sensitivity of ovarian carcinoma. Knockdown of CCNG1 alone enhanced paclitaxel cytotoxcity to ST30 cells (IC50 = 1468.50 ?32.19 nM VS 2545.84 ?168.83 nM, P = 0.000), while deleption CCNG1 reversed the role of miR-9 inhibitor on the paclitaxel sensitivity of SKOV3 cells (IC50 = 65.35 ?13.47 nM VS 177.36 ?20.88 nM, P = 0.001). The experiments were repeated three timesmiR-9 could inhibit CCNG1 expression in ST30 cells, while down-regulated miR-9 enhanced CCNG1 expression in SKOV3 cells (Fig. 2b, e). Using dual luciferase reporter assay, we found that the relative luciferase activities were significantly reduced in cells transfected with CCNG1 WT- 3UTR/miR-9 mimic vectors compared with those transfected with CCNG1 WT-3UTR/miR-9 mimic control (Fig. 2c). Furthermore, Realtime RT-PCR suggested that mRNA expression of CCNG1 in ST30 cells was significantly higher than that in SKOV3 (2.14 fold), which was contrary to the miR-9 expression trends in SKOV3 and ST30 cells (Additional file 2: Figure S1C). These data validate that miR-9 can directly bind to 3 UTR of CCNG1 and CCNG1 is regulated by miR-9.Li et al. BMC Cancer (2015) 15:Page 6 ofCCNG1 depletion enhances the paclitaxel sensitivity of EOC cellsCCNG1 was initially identified as a p53-regulated transcript induced by DNA damage [15]. Although its precise role on cellular growth control is still controversial, CCNG1 has been regarded as an oncogene [16, 17]. CCNG1 gene copy number is an independent marker of postsurgical survival in EOC patients who have received chemotherapy with taxanes and platinum compounds [18]. Thus it suggests that CCNG1, the target of miR-9, probably modulates the paclitaxel-sensitivity of EOC. To validate this hypothesis, we knocked down CCNG1 in ST30 cells through transfecting CCNG1 siRNA. The roles of CCNG1 siRNAs were confirmed using realtime RT-PCR and Western blot, and the most effective siRNA was chosen (Additional file 2: Figure S1D, E). IC50 of paclitaxel in ST30 cells was significantly decreased after CCNG1 depletion (Fig. 2f ), which confirmed that knockdown of CCNG1 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28549975 alone would enhance paclitaxel cytotoxicity to EOC cells. Furthermore, when miR-9 inhibitor was tr.