叶酸受体α介导ERK信号通路对宫颈癌变的作用及其机制研究

Although a number of studies have suggested that folate receptor alpha (FRα) levels are high in cervical carcinoma cells, and are positively associated with the development and progression of cervical cancer, yet the underling mechanisms are not clear precisely. ERK, known as extracellular signal-regulated kinases, are key signaling factor involved in the regulation of normal cell proliferation, survival and differentiation. Overexpression and activation of cellular oncogenes by the ERK/MAPK cascade have been reported in many human carcinomas, but the relationship between ERK signaling pathway and cervical cancer is inconclusive. The reports by various experiments suggested that FRα might affect cell proliferation via ERK signalling pathway and was associated with the expression change of key transcription factors in the pathway. It is on these basis that we hypothesize that FRα might confer a growth advantage to the development and progression of cervical carcinoma by generating regulatory in ERK signaling pathway. In order to confirm this hypothesis, on the basis of detecting the function and expression of FRα, we will explore the mechanism of FRα via ERK signaling pathway with the technology of molecular biology in the patients during the different stage pathogenesis of cervical canceration, including normal cervices, cervical intra-epithelial neoplasias (CINs) and cervical cancer. Our study will focus on key factors Src and ERK1/2 in ERK signaling pathway and nuclear transcription factors c-fos, c-jun and hnRNP E1，and measure their transfer levels and the change of downstream effects, then analyse the correlation of the above biological events. Meanwhile, we will combine vitro experiments by siRNA and inhibitor intervention further to confirm this hypothesis. According to these results, this study aims to evaluate the effects and possible molecular biology mechanisms of FRα-mediated ERK signaling pathway in progressing cervical carcinomas, as well as, provide evidence for etiology and pathogenesis of cervical cancer and afford novel insights into the cancer management.

叶酸受体α（FRα）在宫颈癌细胞中高表达，与宫颈癌的发生演变密切相关，但确切机制不明；ERK信号通路在多种肿瘤的发生发展中起重要调节作用，但与宫颈癌的关系仍未确定。鉴于FRα与ERK通路中许多关键因子的异常表达有关，且在肿瘤发生中均有不可忽视的作用，提示，FRα势必通过介导ERK信号通路在宫颈癌变中发挥作用。为了验证这一假设，本研究选择从宫颈正常、癌前病变到癌症不同阶段人群为研究对象，在界定FRα功能与表达水平的前提下，以ERK信号通路关键因子Src、ERK1/2及核转录因子c-fos、c-jun 和 hnRNP E1为靶点，检测其转录水平和下游效应的表达变化，分析以上生物事件发生的相互关联。同时结合体外实验，采用siRNA干扰和抑制剂干预等方法对这种关联加以验证。以此综合评价FRα介导ERK通路在宫颈癌变中的作用及其机制，为宫颈癌的病因和发病机制研究提供理论依据，为宫颈癌防治开拓新思路。

叶酸受体α（FRα）在多种上皮性肿瘤组织和细胞中高表达，与宫颈癌的发生演变密切相关，但确切机制不明；ERK信号通路在多种肿瘤的发生发展中起重要调节作用，但与宫颈癌的关系仍未确定。本研究采用体外实验方法，以FRα阳性宫颈癌细胞Hela为研究对象，采用FRα siRNA干扰、Src和ERK抑制剂干预的方法形成干扰前后和抑制前后的对比，分别采用CCK8、流式细胞术、Real-time PCR、western blot等方法检测细胞增殖、凋亡、周期、FRα和ERK信号通路关键因子Src、ERK1/2及核转录因子c-fos、c-jun的mRNA和蛋白表达水平。结果显示，下调FRα、抑制Src和抑制ERK均能抑制宫颈癌细胞的增殖、促进凋亡、阻滞细胞周期在G0/G1期（P<0.05）；ERK信号通路中的Src、ERK1/2、c-Fos、c-Jun因子的mRNA表达水平均增高（P<0.05）；下调FRα后p-ERK、p-c-Fos蛋白表达水平降低（P<0.05），p-Src蛋白表达水平增高（P<0.05），而p-c-Jun蛋白表达水平无统计学意义（P>0.05）；抑制Src后p-Src、p-ERK蛋白表达水平降低（P<0.05），p-c-Fos蛋白表达水平降低，但无统计学意义（P>0.05），p-c-Jun蛋白表达水平增高（P<0.05）；抑制ERK后p-ERK蛋白表达水平降低（P<0.05），p-c-Fos蛋白表达水平降低，但无统计学意义（P>0.05），p-c-Jun蛋白表达水平增高（P<0.05）。通过以上结果，作出如下结论：①FRα在宫颈癌Hela细胞中高表达。②FRα可通过介导ERK信号通路对宫颈癌变发挥作用，FRα通过对ERK信号通路中关键因子ERK1/2、c-Jun、c-Fos的蛋白表达起上调作用，促进了其磷酸化，继而引发了宫颈癌细胞的生长与增殖，在宫颈癌细胞的生长发育和细胞周期调节等方面发挥着重要作用。③Src和ERK可作为宫颈癌生物治疗的靶点。④FRα对ERK上游的Src表现为上调作用，提示在FRα对ERK信号通路的激活中可能有其他因子存在，而Src的上调及抑制实验后信号因子mRNA水平的升高，可能与受到ERK通路中被激活因子的负反馈调节有关。⑤本研究为以FRα为载体进行宫颈癌靶向治疗提供了重要的理论依据。