具核梭杆菌通过激活NF-κB/NCOA7-AS1/IKK正反馈环路促进结直肠癌恶性进展

Numerous studies have confirmed that Fusobacterium nucleatum could accelerate the progression of colorectal cancer. The specific mechanism, however, is not yet clear. Our study found that long non-coding RNA NCOA7-AS1 was upregulated in colorectal cancer infected with Fusobacterium nucleatum, which promoted the malignant progression of colorectal cancer via triggering positive feedback loop of NF-κB/ NCOA7-AS1/IKK. Therefore, we hypothesized that upregulated Fusobacterium nucleatum in the intestine tissues of patients with colorectal cancer could initially stimulate the TLR4/NF-κB cascade. Transcription factor NF-κB thereafter up-regulates cytoplasmic NCOA7-AS1 expression, which in turn promotes IκB-α phosphorylation by recruiting IKK kinases. As a consequence, NF-κB pathway is further activated to promote the progression of colorectal cancer. This study aims to explore the biological fucntion of Fusobacterium nucleatum in cells, experimental animals and clinical tissue specimens through a series of methods, including bioinformatics analysis, ChIP, RIP, RNA-pulldown together with mass spectrometry detection and dual luciferase reporter gene assay. Our results suggested that Fusobacterium nucleatum promotes malignant progression of colorectal cancer via activating positive feedback loop of NF-κB/ NCOA7-AS1/IKK, which provides a new basis for the treatment of colorectal cancer.

大量研究证实具核梭杆菌可加速结直肠癌进展，但具体作用机制尚不明确。本实验组研究发现长链非编码RNA NCOA7-AS1在具核梭杆菌感染的结直肠癌中异常高表达且促进结直肠癌细胞恶性进程，并初步证实结直肠癌细胞内存在NF-κB/ NCOA7-AS1/IKK正反馈调控环路。由此提出假说：结直肠癌患者肠道中高丰度的具核梭杆菌启动TLR4/NF-κB级联反应，而后转录因子NF-κB上调NCOA7-AS1表达，同时富集于胞浆里面的NCOA7-AS1通过招募IKK激酶，促进IκB-α磷酸化，进一步激活NF-κB通路，从而促进结直肠癌进展。本项目拟从细胞、模式动物、临床组织标本三个层面，结合生物信息学、ChIP、RIP实验、RNA-pulldown联合质谱检测、双荧光素酶报告基因等技术，揭示具核梭杆菌通过激活NF-κB/NCOA7-AS1/IKK环路进而促进结直肠癌进展新机制，为治疗提供新靶点。

Mindin作为一种细胞外基质分子，广泛研究表明其在免疫系统的发育过程中起到重要的作用，但是在ConA（刀豆蛋白）导致的免疫性肝损伤中的作用尚未明确。.本研究首先通过ConA构建小鼠免疫性肝损伤，通过检测血清ALT和AST的水平和TUNEL以及HE染色明确肝脏坏死范围。通过分离肝脏的淋巴细胞通过流式仪分析小鼠肝脏NK，NKT，CD4，CD8和单核巨噬中性的比例，以及NK，NKT，CD4，CD8的IFN- γ的比例，和各种免疫细胞的总数。而后我们通过骨髓移植构建了嵌合鼠模型。最后我们通过构建双敲小鼠，确定Mindin的作用机制。.通过小鼠尾静脉注射ConA构建小鼠免疫性肝损伤模型，我们发现小鼠肝脏Mindin的RNA水平以及血清中Mindin的水平明显上调，而后我们在Mindin-/-小鼠构建肝脏损伤，肝脏酶谱，HE染色以及TUNEL染色结果表明，Mindin-/-小鼠的肝脏损伤水平明显降低。分离小鼠肝脏淋巴细胞而后通过流式分析结果表明，Mindin-/-小鼠肝脏的NK，NKT，CD4，CD8和单核巨噬中性的总数明显减少，同时NK，CD4以及单核巨噬中性粒比例明显小于WT组小鼠，CD8的比例增高，而NKT细胞的比例无明显变化。此外，通过骨髓移植构建嵌合鼠模型，发现Mindin主要在肝脏实质细胞发挥作用。通过生物信息学分析，我们发现NK细胞来源的IFN- γ是小鼠体内IFN- γ差异的主要原因，此外通过测序分析发现，Mindin-/- 组NK中的STING的表达值明显上调。最后我们通过构建Mindin/STING-DKO双敲老鼠，同时我们在WT，Mindin，Mindin/STING三组小鼠构建模型，结果显示STING可以扭转Mindin敲除引起的肝脏损伤水平的降低。.以上结果表明，肝脏实质细胞分泌的Mindin通过作用于NK细胞，下调STING的水平从而促进NK细胞的增殖，最终增加肝脏的坏死水平。