|本期目录/Table of Contents|

沉默或抑制TAK1表达能够通过NF-κB信号通路改善卵巢癌细胞的紫杉醇耐药性

《现代肿瘤医学》[ISSN:1672-4992/CN:61-1415/R]

期数:
2022年04期
页码:
576-582
栏目:
论著（基础研究）
出版日期:
2022-01-12

文章信息/Info

Title:
Silencing or inhibiting TAK1 expression can improve paclitaxel resistance in ovarian cancer cells through the NF-κB signaling pathway
作者:
王青慧1; 李 波1; 胡传翠1; 聂明朝1; 郑小妹2; 金克龙1
1.海南省妇女儿童医学中心妇产科，海南 海口 570206；2.海南医学院第一附属医院妇产科，海南 海口 570102
Author(s):
WANG Qinghui1; LI Bo1; HU Chuancui1; NIE Mingchao1; ZHENG Xiaomei2; JIN Kelong1
1.Department of Obstetrics and Gynecology,Hainan Women and Children's Medical Center,Hainan Haikou 570206，China;2.Department of Obstetrics and Gynecology,the First Affiliated Hospital of Hainan Medical College,Hainan Haikou 570102，China.
关键词:
卵巢癌; 转化生长因子β活化激酶1; 紫杉醇; NF-κB信号通路
Keywords:
ovarian cancer; transforming growth factor-β (TGF-β)-activated kinase 1(TAK1); paclitaxel; NF-κB signaling pathway
分类号:
R737.31
DOI:
10.3969/j.issn.1672-4992.2022.04.004
文献标识码:
A
摘要:
目的：探究利用小干扰RNA（small interfering RNA，siRNA）或转化生长因子β活化激酶1［transforming growth factor-β (TGF-β)-activated kinase 1，TAK1］抑制剂5Z-7-oxozeaenol进行靶向沉默或抑制TAK1表达对卵巢癌细胞紫杉醇（taxol）耐药性的影响及相关作用机制。方法：应用RT-PCR与Western blot检测TAK1在正常卵巢上皮细胞与卵巢癌细胞系中的表达，并筛选TAK1 mRNA与蛋白表达水平较高与较低的卵巢细胞OVCAR3和SKOV3进行研究；使用siRNA转染技术下调OVCAR3和SKOV3细胞中的TAK1表达，RT-PCR进行验证转染效率；将OVCAR3或SKOV3细胞分为三组：si-NC+taxol组、si-TAK1+taxol组和si-NC+taxol+5Z-7-oxozeaenol组；CCK-8法检测紫杉醇对各组卵巢细胞的半数抑制浓度（IC50值），Tunel荧光染色检测紫杉醇对各组细胞的介导凋亡发生率，Western blot检测各组细胞中NF-κB信号通路IκBα（p-IκBα/IκBα）与p65（p-p65/p65）的磷酸化水平和凋亡相关蛋白cleaved caspase-3的表达；使用SKOV3细胞建立卵巢癌裸鼠皮下移植瘤，并将小鼠分为四组：control组、si-NC+taxol组、si-TAK1+taxol组和si-NC+taxol+5Z-7-oxozeaenol组，监测肿瘤生长，免疫组化检测TAK1在肿瘤组织中的表达，并再次使用Tunel荧光染色与Western blot检测各组裸鼠肿瘤组织中的细胞凋亡率与NF-κB信号通路IκBα、p65的磷酸化水平和cleaved caspase-3蛋白的表达。结果：与正常卵巢上皮细胞相比，TAK1 mRNA与蛋白表达水平在卵巢癌细胞系中的表达均明显升高（P＜0.05），且在OVCAR3细胞中的表达水平最高，而在SKOV3细胞中表达最低；利用siRNA转染成功下调了OVCAR3与SKOV3细胞中TAK1表达；与si-NC+taxol组相比，si-TAK1+taxol组与si-NC+taxol+5Z-7-oxozeaenol组紫杉醇的IC50值明显降低（P＜0.05），细胞凋亡率显著升高（P＜0.05），细胞中IκBα与p65的磷酸化水平均明显下降（P＜0.05），而cleaved caspase-3的蛋白表达水平明显升高（P＜0.05）；裸鼠体内实验结果表明，与control组相比，si-NC+taxol组、si-TAK1+taxol组与si-NC+taxol+5Z-7-oxozeaenol组的肿瘤重量和生长速度均明显降低（P＜0.05），且肿瘤组织中TAK1的蛋白表达强度明显下降（P＜0.05），NF-κB信号通路IκBα、p65的磷酸化水平亦显著降低（P＜0.05），而细胞凋亡率与cleaved caspase-3蛋白表达水平明显增加（P＜0.05），其中与si-NC+taxol组相比，si-TAK1+taxol组与si-NC+taxol+5Z-7-oxozeaenol组中上述检测指标的变化趋势更为显著（P＜0.05）。结论：本研究通过体内外实验表明TAK1是卵巢癌发生紫杉醇耐药性的重要分子，而通过沉默或抑制TAK1的表达可能通过抑制NF-κB信号通路的活化水平促进紫杉醇诱导的细胞凋亡，从而提高肿瘤对紫杉醇的化疗敏感性。
Abstract:
Objective:To investigate the effect of targeted silencing or inhibiting TAK1 expression of small interfering RNA (siRNA) or transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol on taxol resistance of ovarian cancer cells and its mechanism.Methods:RT-PCR and Western blot were used to detect the expression of TAK1 in normal ovarian epithelial cells and ovarian cancer cell lines,and OVCAR3 and SKOV3 cells with higher and lower expression levels of TAK1 mRNA and protein were screened for further study.The expression of TAK1 in OVCAR3 and SKOV3 cells was down regulated by siRNA transfection,and the transfection efficiency was verified by RT-PCR.OVCAR3 or SKOV3 cells were divided into three groups:si-NC+taxol group,si-TAK1+taxol group and si-NC+taxol+5Z-7-oxozeaenol group.CCK-8 method was used to detect the IC50 value of taxol on ovarian cells in each group.Tunel fluorescence staining was used to detect the incidence of apoptosis mediated by taxol on ovarian cells in each group.Phosphorylation levels of NF-κB signaling pathway IκBα (p-IκBα/IκBα) and p65 (p-p65/p65) and expression of apoptotic related protein cleaved caspase-3 were detected by Western blot.SKOV3 cells were used to establish subcutaneous xenograft tumor in nude mice with ovarian cancer,and the mice were divided into four groups:Control group,si-NC+taxol group,si-TAK1+taxol group and si-NC+taxol+5Z-7-oxozeaenol group.Tumor growth were monitored,and the expression of TAK1 in tumor tissues was detected by immunohistochemistry.Cell apoptosis rate,phosphorylation of NF-κB signaling pathway IκBα and p65,and cleaved caspase-3 protein expression were detected by Tunel fluorescence staining and Western blot.Results:Compared with normal ovarian epithelial cells,the expression levels of TAK1 mRNA and protein in ovarian cancer cell lines were significantly increased (P＜0.05),and the highest expression level was in OVCAR3 cells,while the lowest expression level was in SKOV3 cells.The expression of TAK1 in OVCAR3 and SKOV3 cells was down regulated by siRNA transfection.Compared with si-NC+taxol group,the IC50 value of paclitaxel in si-TAK1+taxol group and si-NC+taxol+5Z-7-oxozeaenol group was significantly decreased (P＜0.05),and the apoptosis rate was significantly increased (P＜0.05).The phosphorylation levels of IκBα and p65 were significantly decreased (P＜0.05),while the protein expression level of cleaved caspase-3 was significantly increased (P＜0.05).Compared with control group,the tumor weight and growth rate of si-NC+taxol group,si-TAK1+taxol group and si-NC+taxol+5Z-7-oxozeaenol group were significantly decreased (P＜0.05),and the protein expression of TAK1 in tumor tissue was significantly decreased (P＜0.05).The phosphorylation level of IκBα and p65 was also significantly decreased (P＜0.05),while the apoptosis rate and cleaved caspase-3 protein expression level were significantly increased (P＜0.05).Compared with the si-NC+taxol group,the change trend of the above detection indicators was more significant in the si-TAK1+taxol group and si-NC+taxol+5Z-7-oxozeaenol group (P＜0.05).Conclusion:In vivo and in vitro experiments showed that TAK1 is an important molecule for the development of taxol resistance in ovarian cancer,and silenced or inhibited TAK1 expression may promote taxol-induced apoptosis by inhibiting the activation level of NF-κB signaling pathway,so as to improve the sensitivity of tumors to taxol chemotherapy.

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备注/Memo

备注/Memo:
海南省基础与应用基础研究计划（自然科学领域）高层次人才项目（编号：2019RC391）
更新日期/Last Update: 1900-01-01