| Objectives Long non-coding RNA(lnc RNA)HOTAIR is highly expressed in ovarian cancer and closely related to poorer overall survival of patients.In the present study,we intended to explore the function of HOTAIR in paclitaxel resistance in ovarian cancer as well as the mechanism of paclitaxel resistance induced by HOTAIR via regulating cell cycle checkpoint kinase CHEK1.Methods 1.Ovarian cancer cell lines SKOV3,ES2,A2780 and CAOV3 were treated with paclitaxel at different concentrations for 24 h,48h,and72 h.The expression level of HOTAIR was detected by q RT-PCR.2.Lentivirus harboring sh RNA targeting HOTAIR and plasmid expressing HOTAIR were used to stably down-regulate and up-regulate HOTAIR in SKOV3 and ES2 cells,respectively.HOTAIR expression was verified by q RT-PCR.Colony formation assay,MTT assay,Ed U and flow cytometry were used to detect the effects of HOTAIR on drug resistance,proliferation and cell cycle.The expression of CHEK1 and P-glycoprotein(P-gp)were detected by q RT-PCR and Western blot.3.An overexpression plasmid was used to transiently up-regulate CHEK1 in SKOV3 sh HOTAIR cells.Western blot was used to assess the CHEK1 protein level.The alterations in paclitaxel resistance in SKOV3 sh HOTAIR cells induced by up-regulation of CHEK1 was evaluated by MTT assays.4.SKOV3 sh HOTAIR/SKOV3 sh NC cells were inoculated into nude mice subcutaneously to establish a subcutaneous ovarian cancer model.After tumorigenesis,the nude mice treated with SKOV3 sh HOTAIR/SKOV3 sh NC cells were randomly divided into two groups,six mice for each group,for treatment with solvent control or paclitaxel respectively every three days for 5 times in total,with weight and tumor volume measured.After the mice were executed,the protein level of CHEK1,p-CHEK1,Ki-67 in each group was detected by Immunohistochemistry?Results 1.The expression of HOTAIR was significantly increased in ovarian cell lines after treatment of paclitaxel.Comparing to control(DMSO)treatment,HOTAIR expression of four ovarian cell lines are increased from 29.31±1.06 times to 94.34±6.16 times after treated with paclitaxel.However,no dose dependence or time dependence related to the the expression level of HOTAIR were observed in ovarian cancer cells.Cell lines got the most significant increase in HOTAIR after following treatment: 0.1 ?M paclitaxel for 48 hours for SKOV3 cells;0.4 ?M paclitaxel for 24 hours for ES2 cells;0.4 ?M paclitaxel for 48 hours for A2780 cells;0.1 ?M paclitaxel for 48 hours for CAOV3 cells.As dose regimens for following cell experiments.2.HOTAIR promotes paclitaxel resistance in ovarian cancer.1)The up –and down-regulated efficiency of HOTAIR were verified by q RT-PCR after a stable up-regulated ovarian cancer cell line ES2lv-HOTAIR and a stable down-regulated ovarian cancer cell line SKOV3 sh HOTAIR were established.Compared to ES2lv-NC,the expression of HOTAIR was increased by 14.30±1.96 times in ES2lv-HOTAIR;Compared to SKOV3 sh NC,the expression of HOTAIR in SKOV3 sh HOTAIR was decreased by(74.77±4.16)%;2)HOTAIR affects the cytotoxicity of paclitaxel to ovarian cancer: the result of MTT assay showed that the IC50 of ES2lv-HOTAIR was higher than that of ES2lv-NC(IC50: 15.95±1.19 vs.7.66±2.48 n M);and the IC50 of SKOV3 sh HOTAIR is lower than that of SKOV3 sh NC(IC50: 9.81±3.747 n M vs.18.20±6.24 n M);3)HOTAIR promotes paclitaxel resistance in ovarian cancer: Colony formation assays showed that after treated with paclitaxel in different concentration,the clone-forming ability of ES2lv-HOTAIR is stronger than ES2lv-NC,and the clone-forming ability of SKOV3 sh HOTAIR is weaker than SKOV3 sh HOTAIR.4)Down-regulation of HOTAIR enhanced paclitaxel's inhibitory effect of proliferation in ovarian cancer cells: Ed U assays demonstrated that after treatment with paclitaxel,the proliferation rate of ES2lv-HOTAIR cells was about 1.75 times higher than ES2 lvNC,and the proliferation rate of SKOV3 sh HOTAIR cells was only about 37% as that of SKOV3 sh NC cells.5)Down-regulation of HOTAIR enhanced the cell cycle arrest effect of paclitaxel in ovarian cancer cells.Flow cytometry results showed that compared with SKOV3 sh NC cells,the proportion of G2/M phase cells in SKOV3 sh HOTAIR cells was significantly higher than SKOV3 sh NC(G2/M proportion:(52.23±3.45)% vs(73.37±1.10)%,p**<0.01).3.HOTAIR promotes paclitaxel resistance in ovarian cancer by regulating the protein level of CHEK1.1)Western blot showed that the expression of CHEK1 was increased in ES2lv-HOTAIR cells,and decreased in SKOV3 sh HOTAIR cells,but no significant alterations in m RNA level of CHEK1 as well as the protein level of P-g P were observed in these two cell lines.2)The resistance of ovarian cancer cells to paclitaxel was partly restored after CHEK1 was up-regulated in SKOV3 sh HOTAIR cells.After transfected with control plasmid,the IC50 of SKOV3 sh HOTAIR was lower than SKOV3 sh NC(4.18±0.90 n M vs.25.56±1.60);After CHEK1 was up-regulated,the IC50 of SKOV3 sh HOTAIR was restored,with the IC50 increased to 13.64 ± 0.96 n M.4.In vivo validation of the function of HOTAIR in promoting paclitaxel resistance in ovarian cancer 1)Compared to SKOV3 sh NC group,the inhibition effect of paclitaxel was enhanced in SKOV3 sh HOTAIR group;2)The growth rate of tumors in SKOV3 sh HOTAIR group was significantly slower than that in SKOV3 sh NC group,no matter treated with paclitaxel or not.3)Immunohistochemistry of each group of subcutaneous tumor showed that the expression level of CHEK1,p-CHEK1,Ki-67 protein in SKOV3 sh HOTAIR group(for control or paclitaxel treated)was significantly lower than that in SKOV3 sh NC group.Conclusions HOTAIR confers paclitaxel resistance in epithelial ovarian cancer by increasing CHEK1 expression and promoting cell cycle progression. |
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