选择性剪接因子TRA2α诱导乳腺癌细胞紫杉醇耐药及血管拟态形成的机制研究

Alternative splicing (AS) dysregulation is one of the molecular hallmarks of cancer. Proto-oncogenes and tumor-suppressor genes could be regulated by AS. Our previous study found that the expression level of AS factor TRA2α in viable breast cancer cells was elevated after paclitaxel treatment. In addition, the exogenous TRA2α expression could promote the increase of cancer stem cells (CSCs) subpopulation in MCF-7 cells. It is well known that CSCs play key roles in chemotherapy resistance of breast cancer. Our previous study demonstrates breast CSCs can transdifferentiate into different phenotype, they express angiogenic and vasculogenic markers and they are able to organize vasculogenic mimicry (VM) network. However, there are no studies to demonstrate the AS regulated by TRA2α in the VM formation of breast CSCs and tumor development so far and the underlying molecular mechanisms remain poorly defined. This study will construct TRA2α overexpression and TRA2α knockdown plasmid to transfect breast cancer cells and observe the role of TRA2α in CSCs regulation, paclitaxel resistance and VM formation through in vivo and in vitro experiments. The nucleotide sequence which recognized and bound by TRA2α will be found. The prognostic value of TRA2α will be demonstrated in breast cancer specimen by analyzing the relationship between TRA2α expression, AS regulated by TRA2α, CSCs phenotype, VM, paclitaxel resistance and survival time. This study will provide the new sight and target for breast cancer therapy in advanced stage.

选择性剪接与癌症关系密切，许多癌症相关基因受选择性剪接因子调控。本课题组前期研究发现给予乳腺癌细胞紫杉醇处理后，存活癌细胞的选择性剪接因子TRA2α表达显著增加，并且TRA2α表达能够促进乳腺癌干细胞（CSCs）数量的增加。研究表明CSCs是导致乳腺癌化疗耐药的关键细胞，此外，我们前期研究发现乳腺CSCs能够形成血管拟态（VM）。然而TRA2α表达及其调控的选择性剪接在乳腺CSCs形成VM及肿瘤进展中的作用目前国内外尚无研究报道。本研究将通过体内及体外实验来分析TRA2α及其调控的肿瘤相关基因的选择性剪接模式在乳腺癌细胞干性特征发展、紫杉醇耐药和VM形成中的作用，找出TRA2α结合的肿瘤相关基因的核苷酸序列；将通过人乳腺癌组织中的研究明确TRA2α表达与乳腺CSCs表型之间的关系，阐明TRA2α高表达相关的紫杉醇化疗耐药为影响乳腺癌病人生存的预后因素，并为进展期乳腺癌的治疗提供新靶点。

项目的背景：.乳腺癌是女性最常见的恶性肿瘤，对其化疗耐药机制的研究一直方兴未艾。选择性剪接与肿瘤有着密切的关系，许多肿瘤相关基因受选择性剪接调控。选择性剪接因子TRA2α在乳腺癌化疗耐药中的作用迄今尚无研究报道。.主要研究内容：.1. 细胞功能实验、基因芯片、qPCR、minigene等体外实验评价TRA2α表达在乳腺癌细胞化疗耐药中的作用，找出TRA2α调控的肿瘤相关基因的选择性剪接模式及其分子机制。.2. 免疫组织化学染色、RT-PCR 及q-PCR分析乳腺癌组织的TRA2α与肿瘤相关基因不同剪接变异体表达的相关性，以及这些分子表达情况与肿瘤临床病理资料、紫杉醇敏感或耐药和生存时间之间的关系。.3. 动物实验观察TRA2A与肿瘤相关基因不同剪接变异体表达的相关性，分析TRA2A表达在乳腺癌细胞紫杉醇耐药中的作用。.重要结果、关键数据及其科学意义：.1. 紫杉醇处理后的存活乳腺癌细胞TRA2α表达显著增加，而且TRA2α表达上调促进了乳腺癌细胞的增殖、迁移及侵袭。.2. TRA2α调控肿瘤相关基因MELK, CALU, RSRC2, CEACAM1, LMCD1和PALM的选择性剪切模式，导致其不同剪接变异体的表达。.3. TRA2α蛋白能够识别和结合肿瘤抑制基因RSRC2外显子4上游内含子的AGAA或RAAG序列，从而调控RSRC2选择性剪接，促进了包含外显子4的 RSRC2变异体的表达，此变异体为无义介导的mRNA降解（nonsense-mediated mRNA decay）变异体，能够导致RSRC2蛋白表达的下降。RSRC2蛋白表达丢失诱导了乳腺癌细胞化疗耐药，增强了癌细胞增殖、迁移及侵袭能力。.4. 体内实验和人乳腺癌组织中的分析进一步表明TRA2α与RSRC2的表达之间存在负相关性，TRA2α高表达/RSRC2低表达相关的紫杉醇化疗耐药为影响乳腺癌病人生存期的独立预后因素。.5. 本课题的完成阐明了TRA2α及其调控的选择性剪接促进乳腺癌细胞拮抗紫杉醇治疗的分子机制，能够为乳腺癌的个体化治疗提供一种新思路。