HER-2表达阳性胃癌差异miRNA鉴定与筛选及其与HER-2相互作用的机制的研究

Abnormal amplification and expression of her-2 gene is closely related to a variety of epithelial-derived malignant tumors. Basic research and clinical practice revealed that there are different molecular mechanisms of pathogenesis between gastric carcinoma of positive and negative her-2 gene amplification. In our study, the correlations would be analyzed between the expression status of her-2 gene and the clinical-pathogical features and prognosis of gastric cancer in gastric cancer resection specimens and the adjacent normal tissues. The expression level of her-2 gene will be down-regulated by siRNA interference with psiRNA-her-2 or up-regulated by stable transfection with pCDNA-her-2 in cell lines of gastric cancer.The relationship between her-2 expression level and the ability of cell proliferation and invasion would be revealed by RT-PCR, Western blot, MTT, Transwell, FCM and clonization assays. Differentially expressed miRNA molecules will be screened by comparing gastric cancer with or without her-2 gene amplification groups, gastric cancer cell line with her-2 gene amplification pre- vs. post-transfection with psiRNA-her-2, and gastric cancer cell line without her-2 gene amplification pre- vs. post-transfection with pCDNA-her-2. And then, the expected target gene of resulting miRNA will be identified by bioinformatics prediction. This study would further elucidate the pathogenesis mechanisms of gastric cancer with positive and negative her-2 gene amplification, which would provide experimental evidences for molecular pathologicalclassification and novel therapeutic targets of gastric cancer.

Her-2 基因扩增和表达异常与多种上皮源性恶性肿瘤的发病密切相关。研究表明，her-2 基因扩增阳性胃癌与阴性胃癌存在不同的分子发病机制。本研究在临床切除的胃癌和相应癌旁组织标本中分析her-2 基因扩增状态和胃癌临床病理特征及预后的关系；采用RNA 干扰技术和稳定转染pCDNA-her-2表达载体下调或上调胃癌细胞株中HER-2的表达，RT-PCR、WB、MMT、Transwell、流式及细胞克隆形成实验分析HER-2 表达水平和细胞增殖、侵袭能力的变化；采用miRNA 芯片技术比较阳性组和阴性组胃癌、psiRNA-her-2转染前和转染后的扩增阳性胃癌细胞株及pCDNA-her-2 转染前和转染后的扩增阴性胃癌细胞株的miRNA谱表达差异，筛选差异表达的miRNA 分子并预测其所调控的靶基因，通过进一步研究靶基因的功能为明确胃癌的发病机制、胃癌分子病理分型和治疗新的靶点提供实验依据。

胃癌是最常见的消化道恶性肿瘤之一，伴有her-2基因扩增阳性的胃癌约占胃癌10%，其预后相对较差，容易发生转移。其发病机制与无her-2基因扩增的胃癌有一定的差别。利用赫赛汀靶向治疗her-2扩增阳性的胃癌可以收到较好的疗效。本研究通过分析her-2扩增阴性胃癌与her-2扩增阳性胃癌的转录组差异，进而探讨两者的分子发病机制。.本研究利用二代测序进行全转录组测序比对her-2阳性胃癌及her-2阴性胃癌的转录组子的差异，通过数据分析发现，两组差异表达基因5000多个，新的lncRNA转录本245个，我们挑选了部分在胃癌中研究较少的基因如lncRNA-meg3、miR-133a、Lplunc1等进行后续体外研究，利用RT-PCR，WB检测了胃癌手术标本中的lncRNA-meg3、miR-133a及Lplunc1的表达，与测序结果相符，进而构建了lncRNA-meg3及Lplunc1稳转过表达和干扰下调载体，转染入胃癌细胞株进行了功能学研究。.1.利用荧光定量PCR检测高转移胃癌细胞株BGC-823，MKN28（低转移）及BGC-823高转移胃癌细胞株中，通过Transwell和划痕实验等检测侵袭和迁移能力的变化，结果显示：miR-133a能显著抑制胃癌细胞的侵袭和迁移。显示miR-133a 具有抑制肿瘤转移的作用。.2.利用免疫组化检测lplunc1基因在胃癌及其正常胃组织中的表达，结果显示Lplunc1表达于胃粘膜上皮和胃底腺，在胃癌中表达明显下调或缺失，这提示Lplunc1的表达下调或缺失与胃癌的发生存在一定的关系。.3.构建了lncRNA-meg3过表达和干扰下调的shRNA载体，分别转染入SGC7901及SNU5胃癌细胞株中，通过划痕和transwell实验发现，lncRNA-meg3能显著抑制胃癌细胞的侵袭和迁移，同时也发现HER-2蛋白的表达也下调，这提示lncRNA-meg3与Her-2存在某种相关性，值得进一步研究。