mTOR功能性单倍体通过ERS-IRE1/α-JNK通路调控乳腺癌细胞药物敏感性的机制研究

The functional promoter variations of mTOR gene are associated with the tumorigenesis, variability of chemosensitivity. However, the mechanism is unclear until now. We for the first time found that mTOR gene haplotypes (mTOR-promoter- Hts) which constituted by -141G/T and -78 C/G polymorphisms located in the promoter region correlated to the developing breast cancer, chemosensitivity of breast cancer patients. In the breast cancer tissues, we found that the expression mTOR, MDR associated proteins ABCG2, ABCB1, ABCC1 are related to the different mTOR haplotypes carriers, and further detected the higher expression of mTOR, ABCG2, ABCB1 in the MCF7/Tax cell line, which is associated with the function of ERS-IRE1/α-JNK signialing pathway. Those results indicated that mTOR-promoter-Hts maybe affect the chemotherapeutic sensitivity and developing for breast cancer through ERS-IRE1/α-JNK signialing pathway. Our project will detect the similarities and differences between regulation function for mTOR-promoter-Hts and transcription factors through ERS-IRE1/α-JNK signialing pathway, and clarify the mechanism of mTOR gene functional haplotypes regulating ERS-IRE1/α-JNK signialing pathway to affecting chemosensitivity and multidrug resistance in breast cancer. This project may find new potential regulatory factor and provide theoretical and experimental basis in individualized diagnosis and chemotherapy treatment in breast cancer patients.

mTOR基因启动子区变异参与肿瘤发生发展及化疗敏感性调控，但相关机制不清。申请者首次发现mTOR启动子区-141G/T和-78 C/G位点构成的mTOR-promoter-Hts单倍体型与乳腺癌发生易感性及化疗敏感性密切相关；在乳腺癌组织检测到mTOR-promoter-Hts单倍体型介导mTOR差异表达及ABCG2/ABCB1/ABCC1等耐药蛋白表达；并在MCF7/Tax耐药细胞检测到mTOR、ABCB1及ABCG2蛋白高表达与ERS-IRE1/α-JNK活化相关；提示mTOR单倍体型可能通过ERS-IRE1/α-JNK通路调控乳腺癌发生及药物敏感性。本课题拟通过检测mTOR-promoter-Hts单倍体型与关键TFs差异结合对ERS-IRE1/α-JNK通路的调控作用异同，明确mTOR功能性单倍体型调控乳腺癌发生及耐药性作用与机制，为发现乳腺癌个体差异单倍体型调控因子提供依据。

本研究首先应用病例-对照研究分析mTOR转录起始点上游~1,100bp序列中的SNPs位点，结果发现-78G/C (rs2295079)、-141G/T (rs2295080)构成的2种主要单倍型mTOR-Ht1：-78C/-141G，mTOR-Ht2：-78G/-141T在乳腺癌人群中频率分别为17.0%和79.8%，此2种Hts在人群中分布频数合计高达96.9%。进一步对比分析实验组和对照组人群Haplotype分布差异后发现携带mTOR-Ht2(-78G/-141T)单倍型显著增加乳腺癌易感性；并且携带mTOR-Ht2单倍型与乳腺癌患者紫杉醇NCT化疗敏感性降低[P=0.006;OR(95%CI) =2.391(1.198- 4.772)]以及预后OS缩短相关。临床乳腺癌病理组织分析发现携带mTOR-Ht2患者的mTOR表达水平明显高于mTOR-Ht1单倍型患者，且与MDR1及CCND1相关蛋白表达呈正相关。为了深入探讨mTOR启动子区单倍体型变异与乳腺癌药物敏感性的调控作用，本实验通过CCK8实验发现在MCF7敏感细胞中转染含mTOR-Ht1或Ht2单倍体型的重组质粒后mTOR-Ht2使细胞对PTX的药物敏感性明显降低（P<0.05），呈现化疗抵抗，而在紫杉醇耐药株MCF7/PTX中并见统计学差异。流式细胞术检测发现，与转染mTOR-Ht1相比，转染mTOR-Ht2后MCF7细胞的SPF值（S期细胞%）明显增高，进一步HPLC/MS检测发现转染mTOR-Ht2后MCF7细胞的药物浓度明显增高。荷瘤鼠实验也证实了转染mTOR-Ht2后荷瘤鼠移植瘤对紫杉醇敏感性不同于mTOR-Ht1组，与药物抵抗相关联。Luc报告基因分析发现在293T细胞和MCF7细胞中分别转染mTOR-Ht1或Ht2后，mTOR-Ht2组使mTOR转录活性明显增强；ZEB1通过与mTOR-Ht2位点结合，同时KLF5与mTOR-Ht1结合，差异上调mTOR转录活性。Western blot检测也发现转染mTOR-Ht2组的p-mTOR蛋白表达增加的同时，MDR1表达水平也明显增高；表明mTOR5’功能性区单倍体变异调控mTOR表达及其介导的药物敏感性。

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