宫颈癌细胞中沉默hnRNPA2/B1基因导致洛铂、伊立替康药物敏感性的改变及其作用机制研究

Lobaplatin is an new antitumor drug with independent intellectual property rights.Our study showed that the hnRNPA2 / B1 in cervical cancer Caski cells expressed lower after the treatment of lobaplatin by two-dimensional electrophoresis and mass spectrometry analysis. Studies have shown that the expression levels of HnRNPA2 / B1 in intracellular have closely relationship with tumor proliferation and differentiation and regulate the apoptosis of tumor cells . In this study we first use the lentivirus packaging hnRNPA2 / B1 shRNA and transfect it to cervical cancer cells Caski and Hela , and use RT-PCR 、Western-blot technology to detect the mRNA and protein levels of Bax, BCL-2 genes and observe the cell clone formation, transfer and the changes of the invasive ability. At the same time,MTT and flow cytometry technology are used respectively to detect the cell apoptosis after the treatment of lobaplatin and Irinotecan .At the meanwhile, we will establish the nude mice tumor models and will observe the tumor volume changes between the experimental groups and the control groups after chemotherapy , our purpose is to look for the mechanism of hnRNPA2 / B1 regulating the apoptosis in the cervical cancer cell .

洛铂是具有自主知识产权的国家一类抗肿瘤新药。课题组前期用洛铂作用宫颈癌Caski细胞，经双向电泳及质谱技术分析鉴定差异表达蛋白，发现hnRNPA2/B1 表达下调。有研究表明，hnRNPA2/B1在细胞内的表达水平与细胞增殖分化及肿瘤的发生密切相关，并对肿瘤细胞的凋亡具有调控作用。本研究首次采用慢病毒包装hnRNPA2/B1 shRNA稳定转染宫颈癌细胞，应用RT-PCR、Western blot检测转染后宫颈癌细胞Bax、bcl-2等凋亡相关基因在mRNA及蛋白质水平的表达变化，观察细胞的克隆形成、转移、侵袭能力的改变。应用MTT和流式细胞仪分别检测对照组、实验组洛铂、伊立替康介导宫颈癌细胞凋亡作用的改变；并建立裸鼠成瘤模型, 达到成瘤标准后局部化疗，比较转染组及未转染组肿瘤体积的差异，对宫颈癌细胞中沉默hnRNPA2/B1基因导致洛铂、伊立替康药物敏感性的改变及其作用机制进行探讨

本研究首次采用慢病毒包装hnRNPA2/B1 shRNA稳定转染宫颈癌Caski、Hela细胞，探讨hnRNPA2/B1基因沉默对宫颈癌细胞生物学行为的影响，以及对宫颈癌细胞化疗药物敏感性的影响，从体外-体内-信号通路对hnRNPA2/B1基因在宫颈癌细胞中的作用及机制进行探讨。.课题组成功构建含有hnRNP A2/B1 shRNA的慢病毒载体，并稳定转染宫颈癌Caski及hela细胞。沉默hnRNP A2/B1基因后，能够降低宫颈癌细胞的平板克隆形成能力、迁移能力、侵袭能力；并且能够抑制宫颈癌细胞的周期，使宫颈癌细胞处于G1期，抑制宫颈癌细胞的增殖生长能力；沉默hnRNP A2/B1基因还能明显降低宫颈癌细胞对洛铂、伊立替康的IC50值，明显增强宫颈癌细胞对化疗药物的敏感性，RNA 干扰可能代表一个重要的基因治疗策略。.在此基础上, 课题组继续探讨hnRNP A2/B1基因沉默后对人宫颈癌Hela及CaSki细胞中PI3K/AKT通路中相关蛋白表达的影响,以及对裸鼠移植瘤生长的影响。细胞水平及裸鼠移植瘤中Western blot结果均显示hnRNP A2/B1沉默组中PI3K/AKT信号通路中的相关蛋白p21、p27和cleaved caspase-3的表达明显上调，且p-AKT的表达均降低 (P<0.05)。MTT和划痕实验显示各组细胞中的PI3K/AKT信号通路均可分别被LY294002和IGF-1抑制及激活。免疫组化结果显示，PCNA和Ki-67在裸鼠体内明显下调 (P<0.05)，同时，hnRNP A2/B1基因敲除对裸鼠移植瘤的生长有明显的抑制作用。.结论：沉默 hnRNP A2/B1 基因能够通过 PI3K/AKT 通路抑制宫颈癌细胞的增殖、侵袭、细胞周期以及诱导细胞凋亡,初步阐明了宫颈癌中hnRNP A2／B1基因的作用机制。