LncRNAs在结直肠癌发生及肝转移中的作用机制研究

Colorectal cancer (CRC) is one of the most lethal malignancies in our live. recently studies show that long non-coding RNAs (LncRNAs) paly a important role in various biological progressions, especially in the development of cancer. It gradually become a key point in the area of cancer. Recentlly, our reserch by lncRNAs array and real-time PCR demonstrate that LNC15726 and LNC14590 are significantly correlated with the development and hepatic metastasis in CRC clinical samples and CRC cell lines, although the biological mechanism remains elusive. Therefore, our study will exame a large of CRC patients to conform the difference of LNC15726 and LNC14590 in CRC. RNAi mediated by lentivirus vector is applied for LNC15726 or LNC14590 downregulation or overexpressing in sevaral CRC cell lines, Immunoblotting , transmission electron microscopy, flow cytometry and cell invasion and migration assays are applied to to detect the proliferation and invasion influnce of LNC15726 or LNC14590 in CRC cells, and in vivo animal imaging is applied to exame the proliferation and invasion influnce of LNC15726 or LNC14590 in an orthotopic model of colon cancer for in vivo monitoring of local tumor growth and dissemination in real time, biological analysis, western bloting, RNA pull-down and chip-on-chip is applied to detect the key protein or RNA regulated by LNC15726 or LNC14590, and the mechanism are also elusived in these experiments. In conclusion, our study will provide new insight into diagnosis and therapy for CRC.

结直肠癌是严重威胁人类生命和生活质量的恶性肿瘤。近期研究表明，长链非编码RNA在各种生物学过程尤其是肿瘤的发生、发展中发挥着重要的作用，逐渐成为国内、外肿瘤学领域关注的焦点。最近我们通过lncRNAs芯片，结合临床结直肠癌标本（203例）及结肠癌细胞系（10种）实时定量PCR检测，初步证明LNC15726、LNC14590与结直肠癌发生及肝转移的关系，但其确切的分子生物学作用机制仍需进一步研究。因此，本项目在前期研究的基础上，进一步扩大临床标本检测（300例），并通过慢病毒介导的RNA干扰技术沉默/过表达差异LncRNAs，细胞化学、电镜、流式、侵袭迁移等实验及裸鼠结肠原位癌模型活体成像观察检测差异LncRNAs对结肠癌细胞增殖、侵袭能力的影响，生物信息学分析、蛋白印迹、免疫共沉淀等技术探索差异LncRNAs作用的靶蛋白或RNA及其分子机制，进而为结直肠癌的早期诊断和靶向治疗提供新的途径。

结直肠癌是严重威胁人类生命和生活质量的恶性肿瘤。长链非编码RNA在各种生物学过程尤其是肿瘤的发生、发展中发挥着重要的作用，成为国内、外肿瘤学领域关注的焦点。我们通过lncRNAs芯片，结合临床结直肠癌标本（203例）及结肠癌细胞系（10种）实时定量PCR检测，初步证明LNC15726、LNC14590与结直肠癌发生及肝转移的关系，但其确切的分子生物学作用机制仍需进一步研究。因此，本项目在前期研究的基础上，进一步扩大临床标本检测（300例），结果显示LNC14590与结直肠癌发生显著正相关，LNC15726与结直肠癌肝转移显著负相关。通过慢病毒介导的RNA干扰技术沉默/过表达差异LncRNAs（ LNC14590、LNC15726），采用细胞化学、电镜、流式、侵袭迁移等实验及裸鼠结肠原位癌模型活体成像观察检测差异LncRNAs（ LNC14590、LNC15726）对结肠癌细胞增殖、侵袭能力的影响，结果显示差异LNC14590表达水平变化与结肠癌细胞增殖能力成显著正相关，差异LNC15726表达水平变化与结肠癌细胞侵袭、迁移能力成显著负相关。通过生物信息学分析、蛋白印迹、免疫共沉淀等技术探索差异LncRNAs作用的靶蛋白或RNA及其分子机制，结果显示LNC15726下调可引起侵袭相关蛋白MMP2/9表达上调，且自噬（LC3）及凋亡（PARP）相关蛋白表达降低，LncRNA15726下调促进结直肠癌生长及肝转移的分子生物学机制与其抑制结肠癌细胞凋亡及自噬发生并促进MMP2/9表达上调相关，相关靶蛋白为SYNM；LncRNA14590下调可引起侵袭相关蛋白MMP2/9表达下调，且自噬（LC3）及凋亡（PARP）相关蛋白表达显著升高，LncRNA14590下调抑制结直肠癌生长及肝转移的分子生物学机制与其诱导结肠癌细胞凋亡及自噬发生并抑制MMP2/9表达相关，相关靶蛋白为CEACAM1。