具核梭杆菌通过靶向lncRNA NEAT1促进结肠癌细胞增殖的机制研究

Fusobacterium nucleatum promotes the development of colorectal carcinoma by targeting lncRNA NEAT1

本研究旨在探讨具核梭杆菌对人结肠癌细胞增殖和凋亡的影响及其机制。取对数生长期的人结肠癌HCT116和SW480细胞,设置对照组、菌液组(加入过滤后的菌液)、具核梭杆菌组(采用具核梭杆菌感染)、小干扰核富集转录体1(small interfering nuclear-enriched abundant transcript 1,si-NEAT1)组(转染si-NEAT1)和具核梭杆菌+si-NEAT1组(转染si-NEAT1后采用具核梭杆菌感染)。采用定量鲎试验检测菌液中的内毒素含量,荧光实时定量聚合酶链反应检测细胞中的RNA表达量,CCK-8法检测细胞增殖能力,流式细胞术检测细胞凋亡率,蛋白免疫印迹法检测凋亡相关蛋白caspase-3、Bcl-2和Bax的表达量。结果显示,具核梭杆菌感染结肠癌细胞后,结肠癌细胞的增殖能力提高,凋亡率降低,caspase-3和Bax表达量降低,Bcl-2表达量升高,NEAT1表达量升高。与对照组相比,转染si-NEAT1后结肠癌细胞的增殖能力降低,凋亡率升高,caspase-3和Bax表达量升高,Bcl-2表达量降低。结果表明,敲减NEAT1可逆转具核梭杆菌对结肠癌细胞增殖能力和凋亡率的影响,提示具核梭杆菌通过靶向上调NEAT1的表达促进结肠癌细胞的增殖并抑制其凋亡。

The aim of this study was to investigate the effects of Fusobacterium nucleatum(F. nucleatum) on the proliferation and apoptosis of human colorectal cancer(CRC) cells as well as its mechanism. Human CRC cell lines HCT116 and SW480 at logarithmic growth stage were divided into the control group, bacteria solution group(added filtered bacterial solution), F. nucleatum group(infected with F. nucleatum), small interfering nuclear-enriched abundant transcript 1(si-NEAT1) group(transfected with si-NEAT1) and F. nucleatum+si-NEAT1 group(infected with F. nucleatum after transfection with si-NEAT1). Quantitative limulus test was used to detect the content of endotoxin in bacterial solution. Quantitative real-time polymerase chain reaction(qRT-PCR) was used to measure the level of RNA in cells. CCK-8 assay was performed to detect the proliferation of cells, while flow cytometry was used to calculate the apoptosis rate of cells. The expression levels of apoptosis-related proteins caspase-3, Bcl-2 and Bax were determined by Western blotting. The results showed that the proliferation of CRC cells was enhanced after the infection by F. nucleatum, and the apoptosis rate of cells was decreased. The expressions of caspase-3 and Bax were decreased, while the level of Bcl-2 was increased. In addition, the expression of NEAT1 in CRC cells was increased. After transfection with si-NEAT1, the proliferation of CRC cells was decreased, the apoptosis rate was increased, the expressions of caspase-3 and Bax were increased, while the expression of Bcl-2 was reduced. The knockdown of NEAT1 expression could reverse the effects of F. nucleatum on the proliferation and apoptosis of CRC cells. These results suggest that F. nucleatum can promote the proliferation and inhibit the apoptosis of CRC cells by up-regulating of NEAT1.