ERp57-small interfering RNA silencing can enhance the sensitivity of drug-resistant human ovarian cancer cells to paclitaxel
ERp57-small interfering RNA silencing can enhance the sensitivity of drug-resistant human ovarian cancer cells to paclitaxel
复制标题
ERp57小干扰RNA沉默可增强耐药人卵巢癌细胞对紫杉醇的敏感性
DOI：
10.3892/ijo.2018.4628
复制
发表时间：
2019-01-01
影响因子：
5.2
通讯作者：
Guan, Yifu
中科院分区：
文献类型：
--
作者：
Li, Shou;Zhao, Xiaoyun;Guan, Yifu
内质网蛋白57（ERp57）已被确定与人类卵巢癌的化疗耐药性相关。然而，其在化疗耐药表型中的生物学作用仍不清楚。在本研究中，我们研究了ERp57与紫杉醇耐药细胞行为的关联，并使用ERp57 - 小干扰RNA（siRNA）沉默技术检测了化疗耐药的人类卵巢癌细胞对紫杉醇敏感性的增强情况。采用MTT法、克隆形成试验、流式细胞术分析和Transwell试验分别检测细胞活力、细胞增殖、细胞凋亡和细胞迁移。此外，利用实时荧光定量PCR和蛋白质印迹分析比较了紫杉醇敏感的人类SKOV3卵巢癌细胞中ERp57的mRNA表达水平以及ERp57、信号转导及转录激活因子3（STAT3）、磷酸化STAT3、增殖细胞核抗原（PCNA）、核仁素、微管蛋白β3（TUBB3）、P - 糖蛋白（P - gp）、波形蛋白、Bcl - 2、Bax、Bcl - xl、p53、基质金属蛋白酶1（MMP1）、基质金属蛋白酶2（MMP2）和基质金属蛋白酶9（MMP9）的蛋白表达水平与紫杉醇耐药的对应细胞SKOV3/紫杉醇（SKOV3/tax）的差异。ERp57在紫杉醇耐药的SKOV3/tax细胞中高表达，实验结果表明，紫杉醇耐药表型主要是由于STAT3信号通路的激活。慢病毒颗粒感染导致的ERp57过表达降低了SKOV3细胞对紫杉醇的敏感性。此外，ERp57 - siRNA沉默恢复了SKOV3/tax细胞对紫杉醇的敏感性。值得注意的是，与SKOV3/tax细胞相比，ERp57 - siRNA沉默的SKOV3/tax细胞的半数抑制浓度（IC50）值降低至原始水平，集落存活率显著降低。此外，ERp57 - siRNA沉默与紫杉醇共同处理可抑制STAT3信号通路，并下调下游蛋白的表达水平。值得注意的是，ERp57 - siRNA和100 nM紫杉醇共同处理下调了Bcl - 2、Bcl - xl、MMP2、MMP9、TUBB3和P - gp的表达水平，并上调了Bax蛋白的表达。此外，共同处理促进了p53异构体向p53/p47的转变。生物信息学分析支持了ERp57与卵巢癌耐药相关的实验观察结果。本研究表明，ERp57是治疗紫杉醇耐药的人类卵巢癌的一个潜在治疗靶点。
ERp57 has been identified to be associated with the chemoresistance of human ovarian cancer. However, its biological roles in the chemoresistance phenotype remain unclear. In the present study, the association of ERp57 with paclitaxel-resistant cellular behavior was investigated and the sensitivity enhancement of chemoresistant human ovarian cancer cells to paclitaxel was examined using ERp57-small interfering (si)RNA silencing. Cell viability, cell proliferation, cell apoptosis and cell migration were detected using an MTT assay, clonogenic assay, flow cytometry analysis and transwell assay. Furthermore, mRNA expression levels of ERp57 and protein expression levels of ERp57, STAT3, phosphorylated STAT3, PCNA, nucelolin, TUBB3, P-gp, vimentin, Bcl-2, Bax, Bcl-xl, p53, MMP1, MMP2 and MMP9 of paclitaxel-sensitive human SKOV3 ovarian cancer cells were compared with paclitaxel-resistant counterpart SKOV3/tax using the real-time PCR and western blot analysis. ERp57 was highly expressed in the paclitaxel-resistant SKOV3/tax cells, and experimental results concluded that the paclitaxel-resistance phenotype was due primarily to the activation of the STAT3 signaling pathway. ERp57 overexpression by lentiviral particle infection decreased the sensitivity of SKOV3 cells to paclitaxel. Furthermore, ERp57-siRNA silencing restored paclitaxel sensitivity of SKOV3/tax cells. Notably, the IC50 value of ERp57-siRNA silenced SKOV3/tax cells was reduced to the original level and colony survival was significantly decreased in comparison with that of SKOV3/tax cells. Additionally, co-treatment of ERp57-siRNA silencing and paclitaxel could inhibit the STAT3 signaling pathway and downregulate the expression levels of downstream proteins. Notably, ERp57-siRNA and 100 nM paclitaxel co-treatment downregulated Bcl-2, Bcl-xl, MMP2, MMP9, TUBB3 and P-gp expression levels and upregulated the expression of Bax protein. Furthermore, co-treatment promoted change of the isoform of p53 to p53/p47. Bioinformatics analyses supported the experimental observations that ERp57 was associated with drug resistance in ovarian cancer. The present study implies that ERp57 is a potential therapeutic target for the treatment of paclitaxel-resistant human ovarian cancer.