TRP14/Beclin1/自噬通路在调控卵巢癌对紫杉醇敏感性中的作用

Recent studies have shown that autophagy involved in cancer chemoresistance,but its regulatory mechanism has not yet been fully elucidated. We found the expression of TRP14 was significantly higher in paclitaxel-resistant ovarian cancer cell lines by LC-MS/MS-based label free quantitative proteomics approach and qRT-PCR, Western Blot validation. TRP14 expression was induced accompanied by increase of Beclin1 expression and activation of autophagy when A2780 cells were treated with paclitaxel. Knockdown of TRP14 with specific shRNA decreased Beclin1 expression and inhibited cell autophagy, and improved paclitaxel sensitivity. Our previous studies suggest that TRP14 could adjust paclitaxel sensitivity of ovarian cancer through targeting Beclin1-dependent autophagy. In this study, we will apply GFP-LC3-II plasmid transfection, Western Blot and electron microscope to determine TRP14 expression and autophagy activation in ovarian cancer cells treated with paclitaxel. Autophagy and paclitaxel sensitivity are going to evaluated when TRP14 and Beclin1 expression are changed by using plasmid transfection and RNA interference. Whether TRP14 regulate autophagy through targeting Beclin1 will also been determined. And the function of TRP14/Beclin1/autophagy pathway in regulation of paclitaxel sensitivity in ovarian cancer will verified by cancer tissues and animal models ,for the purpose of revealing the key mechanism of paclitaxel resistance of ovarian cancer and providing a scientific basis to explore the key target for reversing paclitaxel resistance.

自噬异常是肿瘤耐药的机制之一，但其调控机制尚未完全阐明。我们通过定量蛋白质组学发现，耐紫杉醇卵巢癌细胞株的TRP14表达显著升高。进一步研究显示，紫杉醇可上调卵巢癌细胞TRP14表达，并诱导其调控蛋白Beclin1表达和自噬活性升高；下调TRP14表达降低Beclin1表达和自噬活性，并导致紫杉醇敏感性增加。我们的前期研究提示TRP14可能通过Beclin1调控自噬，进而影响卵巢癌细胞对紫杉醇的敏感性。本项目先以质粒转染和电镜等技术观察紫杉醇对卵巢癌细胞TRP14表达和自噬活性的影响；再以质粒转染和RNAi分别调控TRP14和Beclin1表达，观察细胞自噬活性和对紫杉醇敏感性的改变，并明确Beclin1是TRP14的靶基因；最后组织标本和动物模型验证TRP14/Beclin1/自噬通路在调控紫杉醇敏感性中的作用。旨在揭示卵巢癌对紫杉醇耐药的关键机制，为探寻逆转耐药的关键靶点提供科学依据。

紫杉醇作为卵巢癌治疗的一线化学药物，其化疗耐药是卵巢癌患者治疗失败和死亡的最主要原因之一。克服紫杉醇耐药，已成为提高卵巢癌远期生存率的关键。但是紫杉醇耐药在卵巢癌中的关键分子或机制仍不清楚。本课题研究通过无标记定量蛋白质组学方法筛选出356个紫杉醇耐药卵巢癌细胞与亲本细胞的差异表达蛋白，发现TRP14在紫杉醇耐药的卵巢癌细胞和组织中高表达，TRP14的高表达与较差的预后因素相关，且在157名卵巢癌患者中表现出较短的生存期。此外，紫杉醇暴露能诱导TRP14和BECN1的表达，增加自噬体形成，降低卵巢癌细胞紫杉醇敏感性。通过siRNA抑制或者质粒过表达TRP14相应地减少或增加了自噬反应和影响卵巢癌细胞紫杉醇敏感性。另外，通过siRNA下调BECN1能减弱TRP14过表达引起的自噬激活和卵巢癌细胞紫杉醇敏感性降低。因此，我们的发现表明TRP14通过BECN1诱导自噬，从而导致卵巢癌细胞对紫杉醇耐药，为进一步探索卵巢癌治疗中潜在的预测因子或靶点提供实验依据。