lncRNA-RACGAP1P介导真基因调控癌细胞线粒体动态变化促进乳腺癌侵袭转移的作用及其分子机制

Based on lncRNA expression profile assay and validation results, we discovered, for the first time, that lncRNA-RACGAP1P is expressed at a high level in breast carcinoma cells and breast carcinoma tissues, and its high expression positively correlates with malignant progression in breast carcinoma. Furthermore, more importantly, the our preliminary experiments show that lncRNA-RACGAP1P mediated the parent gene RacGAP1 can promote carcinoma cell invasion/migration through phosphorylation of Drp1 and modulating mitochondrial dynamics. Based on our preliminary experiments and analysis of bioinformatics, we deduce that lncRNA-RACGAP1P activates MAPK pathway/Drp1 phosphorylation via upregulating its parent gene RacGAP1 by competitively binding miR-345-5p, conseqently promotes mitochondrial fission and tumor invasion/metastasis. However, many questions still remain to be clarified, such as regulatory relationships among lncRNA-RACGAP1P, RacGAP1 and mitochondrial dynamics, and the relations of their expressions with malignant phenotypes of breast carcinoma. By means of different genes overexpression cells and CRISPER/Cas9-mediated specific gene-depleted cells (such as DICER-depleted cells, etc.), the in vitro and in vivo studies will be employed in this study in order to demonstrate the regulation mechanisms of lncRNA-RACGAP1P in promting breast carcinoma invasion/metastasis through modulating mitochondrial dynamics, therefore providing theoretical basis and experimental support for elucidating lncRNAs function in breast carcinoma progress.

我们首次发现：lncRNA-RACGAP1P在乳腺癌细胞和组织中高表达且其与癌细胞侵袭和患者恶性进展正相关，更重要的是预实验显示lncRNA-RACGAP1P介导其真基因RacGAP1,引起Drp1磷酸化调控线粒体动态变化促进癌细胞侵袭/迁移。据预实验结果和生信分析，我们提出科学假说lncRNA-RACGAP1P通过miR-345-5p调控RacGAP1表达，激活MAPK通路/Drp1磷酸化调控癌细胞线粒体状态促进细胞侵袭转移。预实验结果和科学假说中关于lncRNA-RACGAP1P-真基因-线粒体动态变化之间的调控，及其与乳腺癌恶性表型关系等问题尚需深入研究，故我们拟用基因转染和CRISPR/Cas9技术构建的特定基因缺失细胞系等，经体内、外实验明确lncRNA-RACGAP1P介导真基因调控线粒体状态促进乳腺癌侵袭转移的作用和机制，为揭示lncRNAs在乳腺癌进展中发挥的功能提供依据。

乳腺癌是危害妇女健康的最常见恶性肿瘤，肿瘤的侵袭和转移是影响其疗效，导致肿瘤病人死亡的主要因素。近年，长链非编码RNA（lncRNAs）研究成为肿瘤研究的热点，但它们在乳腺癌发病机制中的潜在作用及机制尚未完全阐明。.本课题利用lncRNAs芯片分析发现lncRNA RACGAP1P（RACGAP1的假基因）在乳腺癌组织中表达上调，且经qRT-PCR证实之。随后，我们在102例乳腺癌患者标本的实验发现lncRNA RACGAP1P和其真基因RACGAP1两者的表达均与患者淋巴结转移、远处转移、TNM分期和预后差呈正相关。通过体外实验和动物实验研究了lncRNA RACGAP1P和RACGAP1在乳腺癌的生物学功能和分子机制，实验证实lncRNA RACGAP1P竞争性结合miR-345-5p，调控其对应亲本基因RACGAP1的表达促进线粒体分裂，进而促进乳腺癌的侵袭和转移。.另外，体外实验发现乳腺癌细胞中RACGAP1的过表达可导致线粒体分裂，促进线粒体自噬，加快线粒体更新速率，并增加有氧糖酵解ATP的生成，进而促进乳腺癌细胞侵袭和转移。为了探究RACGAP1调控线粒体质量控制的分子机制，我们设计了一些实验。实验结果发现RACGAP1在有丝分裂后期能够招募ECT2，激活ERK-DRP1通路，从而促进线粒体融合。PGC-1a是一种关键的线粒体生物合成调节器，过表达RACGAP1也增加了PGC-1a的表达，这可能是由于RACGAP1诱导线粒体自噬强度增加所致。PGC-1a增加了线粒体中DNMT1的富集，线粒体DNMT1能够增强线粒体DNA的甲基化并上调线粒体基因组的转录。我们的研究为探索乳腺癌患者中lncRNA RACGAP1P/RACGAP1过表达相关的肿瘤机制提供了一个新的角度。