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Methylation-associated Has-miR-9 deregulation in paclitaxel- resistant epithelial ovarian carcinoma.

Li X, Pan Q, Wan X, Mao Y, Lu W, Xie X, Cheng X - BMC Cancer (2015)

Bottom Line: CCNG1, validated as a direct target of miR-9, mediates paclitaxel resistance. miR-9-1 and 3 gene hypermethylation would decrease miR-9 expression, while demethylation of miR-9 gene could restore miR-9 expression and improve paclitaxel sensitivity in chemoresistance EOC cells.Furthermore, methylation-associated miR-9 deregulation in EOC cells could be induced by paclitaxel exposure.Methylation-associated miR-9 down-regulation is probably one of the key mechanisms for paclitaxel resistance in EOC cells, via targeting CCNG1.

View Article: PubMed Central - PubMed

Affiliation: Women's Reproductive Health Laboratory of Zhejiang Province, Women's Hospital, School of Medicine, Zhejiang University, No.1 Xueshi Road, 310006, Hangzhou, Zhejiang, China. lixsure@163.com.

ABSTRACT

Background: Drug resistance is still one of the key causes of death in epithelial ovarian carcinoma (EOC) patients, however there are very few strategies to reverse chemoresistance. Here we try to clarify whether and how miR-9 takes part in the regulation of paclitaxel sensitivity.

Methods: miR-9 expressions in EOC cells and tissues were detected by Realtime PCR. The target of miR-9 was validated through dual luciferase reporter assay and Western Blot. Methylation study, RNAi technique and cytotoxicity assay were used to determine the intrinsic mechanism of miR-9 in paclitaxel sensitivity regulation.

Results: miR-9 is down-regulated in paclitaxel resistant EOC. The patients with lower miR-9, Grade 3, Stage III -IV and suboptimal surgery present shorter survival time. miR-9 and suboptimal surgery are independent prognostic factors of EOC. Modulating miR-9 expression could change paclitaxel sensitivity of EOC cells. CCNG1, validated as a direct target of miR-9, mediates paclitaxel resistance. miR-9-1 and 3 gene hypermethylation would decrease miR-9 expression, while demethylation of miR-9 gene could restore miR-9 expression and improve paclitaxel sensitivity in chemoresistance EOC cells. Furthermore, methylation-associated miR-9 deregulation in EOC cells could be induced by paclitaxel exposure.

Conclusions: Methylation-associated miR-9 down-regulation is probably one of the key mechanisms for paclitaxel resistance in EOC cells, via targeting CCNG1. Our findings may also provide a new potential therapeutic target to reverse paclitaxel resistance in EOC patients.

No MeSH data available.


Related in: MedlinePlus

CCNG1 is the direct target of miR-9 and modulates the paclitaxel sensitivity of EOC cells a. Modulating miR-9 expression changed paclitaxel sensitivity of ST30 and SKOV3 cells. ST30 cells were transfected with miR-9 mimic or negative 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 3′UTR vectors or mutant 3′UTR vectors together with miR-9 mimic or its negative control. Luciferase activity was measured 48 h after 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 times
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Fig2: CCNG1 is the direct target of miR-9 and modulates the paclitaxel sensitivity of EOC cells a. Modulating miR-9 expression changed paclitaxel sensitivity of ST30 and SKOV3 cells. ST30 cells were transfected with miR-9 mimic or negative 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 3′UTR vectors or mutant 3′UTR vectors together with miR-9 mimic or its negative control. Luciferase activity was measured 48 h after 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 times

Mentions: A significant reduction of miR-9 level was observed in SKOV3 cells after miR-9 inhibitor transfection, and a significant increase of miR-9 level was observed in ST30 cells after miR-9 mimic transfection (Additional file 2: Figure S1A, B). The cytotoxic effect of paclitaxel on EOC cell lines was assessed after transfection of miR-9 mimic or inhibitor (or negative control). miR-9 mimic induced a decreased IC50 value of paclitaxel in ST30 and A2780R cells, whereas miR-9 inhibitor brought an increased IC50 value in SKOV3 and A2780 cells (Fig. 2a, d). These data suggest that elevated miR-9 expression enhances paclitaxel cytotoxicity to drug-resistant EOC cells, while reduced miR-9 expression inhibits paclitaxel cytotoxicity to drug-sensitive EOC cells.Fig. 2


Methylation-associated Has-miR-9 deregulation in paclitaxel- resistant epithelial ovarian carcinoma.

Li X, Pan Q, Wan X, Mao Y, Lu W, Xie X, Cheng X - BMC Cancer (2015)

CCNG1 is the direct target of miR-9 and modulates the paclitaxel sensitivity of EOC cells a. Modulating miR-9 expression changed paclitaxel sensitivity of ST30 and SKOV3 cells. ST30 cells were transfected with miR-9 mimic or negative 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 3′UTR vectors or mutant 3′UTR vectors together with miR-9 mimic or its negative control. Luciferase activity was measured 48 h after 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 times
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Fig2: CCNG1 is the direct target of miR-9 and modulates the paclitaxel sensitivity of EOC cells a. Modulating miR-9 expression changed paclitaxel sensitivity of ST30 and SKOV3 cells. ST30 cells were transfected with miR-9 mimic or negative 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 3′UTR vectors or mutant 3′UTR vectors together with miR-9 mimic or its negative control. Luciferase activity was measured 48 h after 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 times
Mentions: A significant reduction of miR-9 level was observed in SKOV3 cells after miR-9 inhibitor transfection, and a significant increase of miR-9 level was observed in ST30 cells after miR-9 mimic transfection (Additional file 2: Figure S1A, B). The cytotoxic effect of paclitaxel on EOC cell lines was assessed after transfection of miR-9 mimic or inhibitor (or negative control). miR-9 mimic induced a decreased IC50 value of paclitaxel in ST30 and A2780R cells, whereas miR-9 inhibitor brought an increased IC50 value in SKOV3 and A2780 cells (Fig. 2a, d). These data suggest that elevated miR-9 expression enhances paclitaxel cytotoxicity to drug-resistant EOC cells, while reduced miR-9 expression inhibits paclitaxel cytotoxicity to drug-sensitive EOC cells.Fig. 2

Bottom Line: CCNG1, validated as a direct target of miR-9, mediates paclitaxel resistance. miR-9-1 and 3 gene hypermethylation would decrease miR-9 expression, while demethylation of miR-9 gene could restore miR-9 expression and improve paclitaxel sensitivity in chemoresistance EOC cells.Furthermore, methylation-associated miR-9 deregulation in EOC cells could be induced by paclitaxel exposure.Methylation-associated miR-9 down-regulation is probably one of the key mechanisms for paclitaxel resistance in EOC cells, via targeting CCNG1.

View Article: PubMed Central - PubMed

Affiliation: Women's Reproductive Health Laboratory of Zhejiang Province, Women's Hospital, School of Medicine, Zhejiang University, No.1 Xueshi Road, 310006, Hangzhou, Zhejiang, China. lixsure@163.com.

ABSTRACT

Background: Drug resistance is still one of the key causes of death in epithelial ovarian carcinoma (EOC) patients, however there are very few strategies to reverse chemoresistance. Here we try to clarify whether and how miR-9 takes part in the regulation of paclitaxel sensitivity.

Methods: miR-9 expressions in EOC cells and tissues were detected by Realtime PCR. The target of miR-9 was validated through dual luciferase reporter assay and Western Blot. Methylation study, RNAi technique and cytotoxicity assay were used to determine the intrinsic mechanism of miR-9 in paclitaxel sensitivity regulation.

Results: miR-9 is down-regulated in paclitaxel resistant EOC. The patients with lower miR-9, Grade 3, Stage III -IV and suboptimal surgery present shorter survival time. miR-9 and suboptimal surgery are independent prognostic factors of EOC. Modulating miR-9 expression could change paclitaxel sensitivity of EOC cells. CCNG1, validated as a direct target of miR-9, mediates paclitaxel resistance. miR-9-1 and 3 gene hypermethylation would decrease miR-9 expression, while demethylation of miR-9 gene could restore miR-9 expression and improve paclitaxel sensitivity in chemoresistance EOC cells. Furthermore, methylation-associated miR-9 deregulation in EOC cells could be induced by paclitaxel exposure.

Conclusions: Methylation-associated miR-9 down-regulation is probably one of the key mechanisms for paclitaxel resistance in EOC cells, via targeting CCNG1. Our findings may also provide a new potential therapeutic target to reverse paclitaxel resistance in EOC patients.

No MeSH data available.


Related in: MedlinePlus