生物活性肽ACBP介导miRNA调节胃癌干细胞作用机制及功能研究

Gastric cancer is the second malignant tumor in the incidence and mortality of our country. Although some progress has been made in the scientific research and clinical diagnosis of gastric cancer, the incidence is still high. Nowadays, scientists are more concerned with stem cells are able to find the specific biomarkers miRNA. we take miR-1915 and miR-4308 as the target to carry out these research. To put forward the scientific hypothesis: (1) bioactive peptide ACBP may be regulated miR-1915 and miR-4308 by targeting to influence the biological behavior of gastric cancer stem cells, down regulation of Bcl-2 expression; (2) miRNA-1915 and miRNA-4308/Bcl-2 are compose to the regulatory network possibly and to restrain the proliferation of gastric cancer stem cells. The hypothesis verification work will be done from the four levels including molecular level, cell level, animal level and clinical specimens . To further elucidation that ACBP mediated microRNA could influence on the mechanism of proliferation, apoptosis and invasion of gastric cancer stem cells in different differentiation, to explore the relevant mechanism of regulation of ACBP-microRNA-stem cell target gene network and gastric cancer therapy, to confirme the anti-tumor function of miRNA1915 and miRNA4308, to provide scientific theory and experimental data for biological therapy of gastric cancer with miRNA as target.

胃癌是我国发病率及死亡率占第二位的恶性肿瘤。虽然对胃癌的科学研究和临床诊治取得了一定进展，但胃癌发病率仍然居高不下。当今，科学家们更加关注针对干细胞是否能够找到特异性生物标志物miRNA。本课题深入地以miR-1915、miR-4308为靶点开展研究。提出科学假说：（1）生物活性肽ACBP可能通过靶向调节miRNA影响胃癌干细胞生物学行为，下调Bcl-2的表达；（2）miRNA-1915、miRNA-4308/ Bcl-2可能组成调控网络，抑制胃癌干细胞增殖。项目拟从分子、细胞、动物水平以及临床肿瘤标本四个层次验证假说，深入阐明ACBP介导miRNA对分化不同的胃癌干细胞增殖、凋亡、侵袭的分子机制，探索ACBP-miRNA-干细胞-靶基因网络调控与胃癌治疗的相关机制，证实miRNA1915、miRNA4308的抗肿瘤功能，为以miRNA为靶标的胃癌生物治疗提供科学的理论与实验数据。

在分子水平调控胃癌干性细胞特性机制目前尚未阐明。微小RNAs（microRNAs）在靶信使RNA的转录后基因沉默中发挥重要作用，参与细胞增殖、分化和凋亡等所有的生物学过程。关注干细胞的研究，发现特异性生物标志物miRNA，探索新的防治途径是热点。项目在前期研究基础上以miR-1915、miR-4308为靶点开展深入研究,解决了以下问题：（1）生物活性肽ACBP通过靶向调节miRNA影响胃癌干细胞生物学行为，下调Bcl-2的表达。①流式细胞术分选CD44阳性标记的MKN-45细胞结果显示，与母本胃癌MKN-45细胞比较，阳性细胞占比例为91.10±3.76%，其中15.67±2.16%的CD44阳性标记的MKN-45细胞克隆成球，且致BALB/c-nu裸鼠成瘤，干性相关基因（Oct4、Sox2、Nanog、Bmil）表达；CD44抗体阴性标记的胃癌MKN-45细胞未见克隆形成及裸鼠致瘤。②证实了拥有自主知识产权的生物活性肽具有较强的抗肿瘤活性,抑制胃癌干性细胞增殖、促进凋亡；阻滞细胞周期于S、G2/M期。生物活性肽干预后抑制转染miR-1915-3p mimics 与miR-1915-3p inhibitor组细胞增殖，促进细胞凋亡，阻滞细胞周期于G2/M期。双荧光素酶报告基因实验证实miR-1915-3p的靶基因为Bcl-2。生物活性肽降低转染miR-1915-3p胃癌干性细胞中Bcl-2、Bcl-xl及Mcl-1的表达，促进Bax及Bak表达；减少共转染miR-1915-3p mimics与pCMV3-OFPSpark-BCL2质粒组胃癌干性细胞的凋亡；（2）miRNA-1915、miRNA-4308/ Bcl-2组成调控网络，抑制胃癌干细胞增殖；与胃癌患者TNM分期、远处淋巴结转移及预后相关；（3）生物活性肽能够显著抑制荷瘤裸鼠肿瘤生长，与上调miR-1915-3p的表达，下调miR-4308表达相关；（4）miRNA-1915以及有共同靶基因的miRNA-30c有望成为胃癌分子标记物. 本项目从分子、细胞、动物水平以及临床肿瘤标本四个层次验证假说，系统阐明ACBP介导miRNA对分化不同的胃癌干细胞增殖、凋亡、侵袭的分子机制，探索ACBP-miRNA-干细胞-靶基因网络调控与胃癌生物治疗的相关机制，为以miRNA为靶标的胃癌生物治疗提供科学的理论与实验数据