内质网应激诱导的miR-301a-3p调控胃癌曲妥珠单抗耐药发生与传递的分子机制

Trastuzumab resistance had seriously influenced the clinical effects in the therapy of HER2-positive gastric cancer, and the molecular mechanisms were still elusive. Endoplasmic reticulum (ER) stress was closely related to drug resistance of tumor, but what purpose it reserved in trastuzumab resistance of gastric cancer was not yet known. In our previous studies, we had confirmed that ER stress induced therapeutic resistance to trastuzumab in HER2-positive gastric cancer cells. The miR-301a-3p with significantly higher levels was screened using micro-array analysis and the ER stress-induced trastuzumab resistance was reversed following downregulation of miR-301a-3p expression by its inhibitor. Moreover, the resistant phenotype was also transferred to recipient gastric cancer cells via the release of exosomes carrying miR-301a-3p by gastric cancer cells under ER stress. Accordingly, we hypothesized that ER stress-dependent induction of miR-301a-3p would mediate the occurrence and transmission of trastuzumab resistance in HER2-positive gastric cancer. Firstly, we would demonstrate the molecular mechanism of miR-301a-3p regulating the occurrence of trastuzumab resistance during ER stress in vitro and in vivo by using ER stress cell or xenograft mouse models through dual-luciferase assay, etc. Secondly, we would use exosomes isolation and quantitative analysis methods to clarify the molecular mechanisms of exosome-transferred miR-301a-3p conferring the resistant phenotype to recipient gastric cancer cells. At last, the molecular mechanisms of ER stress-induced trastuzumab resistance would be revealed from theory and clinic in HER2-positive gastric cancer, which might be a potential molecular marker for trastuzumab resistance and a target for reversing resistance.

曲妥珠单抗耐药严重影响胃癌治疗的临床疗效，其分子机制尚未阐明。内质网应激（ERS）与肿瘤耐药密切相关，但它在胃癌曲妥珠单抗耐药中的作用与机制不清楚。课题组前期研究发现，ERS诱导HER2阳性胃癌细胞对曲妥珠单抗产生耐药；通过miRNA芯片筛选miR-301a-3p并下调其表达，耐药性被逆转；ERS还通过诱导胃癌细胞释放携带miR-301a-3p的外泌体将耐药性传递至受体细胞。据此，我们首次提出ERS通过诱导miR-301a-3p的表达介导胃癌曲妥珠单抗耐药的发生与传递。本项目拟通过ERS状态细胞与小鼠模型，采用双荧光素酶实验等方法，首先阐明ERS诱导的miR-301a-3p调控耐药发生的分子机制；再通过外泌体分离与定量分析等方法，研究外泌体miR-301a-3p传递耐药性的分子作用机制。研究结果将从理论与临床角度，揭示ERS调控耐药发生与传递的分子机制，为逆转耐药新靶点研究奠定分子基础。

曲妥珠单抗耐药严重影响胃癌治疗的临床疗效，其分子机制尚未阐明。缺氧、氧化应激、钙离子稳态失衡等肿瘤微环境的变化，将导致未折叠或错误折叠蛋白聚集于内质网，超出内质网的处理能力，造成内质网内稳态的失衡，从而引发内质网应激（ERS）。ERS通过激活细胞内一系列代偿机制，对肿瘤细胞发挥保护作用，从而促进其增殖、侵袭、转移及耐药等。然而，ERS在胃癌曲妥珠单抗耐药中的作用与机制并不清楚。本项目首先通过建立ERS状态胃癌细胞和动物模型，采用基因转染、CCK-8、qRT-PCR、western blot以及IHC等实验方法，从体内与体外实验阐明ERS通过诱导miR-301a-3p的表达调控胃癌曲妥珠单抗耐药的发生；其次通过生物信息学分析、Dual Luciferase Assay、RTK磷酸化抗体芯片、基因转染、qRT-PCR、western blot以及LNA-ISH等实验方法，从体内与体外实验阐明ERS诱导的miR-301a-3p通过调控LRIG1表达介导耐药发生的分子机制；再通过外泌体分离与鉴定、激光共聚焦分析、电穿孔基因转染、qRT-PCR、western blot以及IHC等实验方法，从体内与体外实验阐明ERS诱导的miR-301a-3p以外泌体为载体介导耐药性在不同状态胃癌细胞之间传递的分子作用机制；最后通过临床病例分析和标本检测，阐明ERS诱导的miR-301a-3p调控耐药发生与传递的临床分子机制。研究结果揭示了ERS诱导的miR-301a-3p以外泌体为载体，通过下调LRIG1促进IGF-1R和FGFR1过表达来激活下游MAPK和AKT信号通路，进而介导胃癌曲妥珠单抗耐药的发生以及在不同状态胃癌细胞之间的传递。研究结果从理论上进一步阐明ERS调控肿瘤耐药的分子机制，从临床角度揭示曲妥珠单抗治疗胃癌耐药性发生新的分子机制，为逆转曲妥珠单抗耐药新靶点的临床研究奠定分子基础。