YWHAE过表达活化Rac1信号诱导结肠癌细胞EMT促侵袭转移的机制研究

Our previous study confirmed that expression of YWHAE and Rac1 in colon cancer cells were downregulated after treatment of anti-cancer drugs diallyl disulfide.Further research revealed that the upregulated YWHAE of Rac1 gene transcription and protein expression level were correlated with the downregulated expression of EMT( epithelial-mesenchymal transitions, EMT) markers ecadherin, and snail upregulated expression as well as tumor metastasis status though the mechanism remains unknown. Accordingly, this project intends to detect the mRNA transcription and protein expression levels of YWHAE,Rac1 and EMT markers in metastatic and non- metastatic tumor tissues, respectively; also to detect the changes in EMT and other metastatic markers with Western blot, fluorescent quantitative RT-PCR detection technology in cultured colon cancer cells after silencing the expression of YWHAE and Rac1 gene by RNA interference technique. The cancer cell invasion and metastasis capacity were also determined with Transwell technology, involved signal transduction pathway were detected with signal chip technology and further determined with Western blot technology. Then YWHAE gene expression, activated Rac1 signal transduction pathway, and induction of EMT to promote colon cancer cell invasion and metastasis were tested in experimental mice models.

我们前期研究证实抗癌药二烯丙基二硫处理结肠癌细胞后，YWHAE及Rac1表达下调。进一步的实验发现在结肠癌组织中，YWHAE、Rac1基因转录水平及蛋白表达上调，与上皮间质转化（EMT）标记物Ecadherin表达下调、Snail表达上调相关；且与肿瘤的转移明显相关，但具体机制尚不清楚。据此，本项目拟首先利用免疫组化等技术检测YWHAE、RAC1及EMT标记物在转移、非转移组结肠癌组织中mRNA及蛋白表达情况；继而培养结肠癌细胞，采用RNA干扰技术沉默YWHAE、Rac1表达后，Western blot、荧光定量RT-PCR技术检测EMT及转移标记物的变化，应用Transwell与细胞移动实验检测癌细胞侵袭转移能力的改变、信号芯片技术探查可能涉及的信号传导通路，western blot 技术进一步证实；最后利用裸鼠成瘤实验验证YWHAE基因、Rac1信号通路与结肠癌EMT及侵袭转移的关系。

项目背景：YWHAE、RAC1在结直肠癌中存在高表达，与癌的EMT及转移相关，YWHAE的促癌作用可能与其活化RAC1信号有关；这两者的关系尚不明确，YWHAE是否通过调节RAC1信号诱导结肠癌细胞EMT，从而促进肿瘤转系尚有待研究。主要内容：本项目通过免疫组化、RNA干扰等技术探讨YWHAE、RAC1基因表达与结肠癌进展的关系，研究沉默YWHAE、RAC1表达后结肠癌EMT及细胞功能学变化，探讨YWHAE、RAC1相互间的关系及调节结肠癌转移可能的分子机制。重要结果及关键数据：1、YWHAE在结肠癌组织中高表达, 在癌旁黏膜正常腺体中低表达或表达缺失(P <0.001), 发生转移的结肠癌YWHAE表达要明显高于未转移的结肠癌组织(P <0.05);沉默YWHAE 基因表达后, 结肠癌细胞长速度明显减慢(P <0.01), 细胞凋亡增加, 细胞周期阻滞于G1期, 细胞克隆形成率显著降低(P <0.01)；裸鼠成瘤及转移率明显减少(P <0.01)。超过900个基因可能卷入YWHAE沉默抑制结肠癌细胞增殖的过程，CDK6表达下调，FOS表达上调，涉及P53\AKT信号通路。YWHAE沉默抑制结肠癌细胞转移可能与EMT无关，2、沉默RAC1基因表达后，结肠癌细胞生长速度明显减慢、细胞克隆形成率显著下降、细胞凋亡增加、细胞周期阻滞在G1期（p<0.05），裸鼠成瘤率及转移率降低。沉默前后，超过1200个基因出现表达差异，上皮间质转化受到抑制，vimentin、cyclind1、c-myc表达下调，E-CADHERN、BAD表达上调，这一过长可能涉及P53\MAPK\NFKB信号通路变化。结论：1、已证实YWHAE表达与结肠癌转移相关；沉默YWHAE表达下调CDK6及上调FOS基因的表达，诱导结肠癌细胞周期捕获及细胞凋亡，涉及P53\AKT信号通路活化或抑制，与RAC1表达及结肠癌细胞EMT无关。2、已证实RAC1表达通过诱导结肠癌细胞EMT促进肿瘤转移，通过调节cyclind1、BAD基因表达诱导结肠癌细胞周期捕获及细胞凋亡，涉及P53\MAPK信号通路活化或抑制。这些实验结果为进一步阐明结肠癌发生发展的分子机制奠定了基础，为结肠癌的靶向治疗提供了新思路。