METHODS: The NEAT1 expression in CRC tissues and CRC cells was analyzed by RT-qPCR. The genes co-expressed with NEAT1 in CRC were obtained from UALCAN, which were intersected with the transcription factors targeting NEAT1 from hTFtarget. Dual-luciferase assay, RT-qPCR, and ChIP were conducted to analyze the transcriptional regulatory relationship between BHLHE40 and NEAT1. LoVo and HCT-15 cells knocking down BHLHE40 and overexpressing NEAT1 were subjected to MTT, Transwell, Western blot, and flow cytometry to examine the malignant aggressiveness of CRC cells. The effects of knocking down BHLHE40 and overexpressing NEAT1 on tumor and lung metastasis were investigated in mice using HE and immunohistochemical analyses.
RESULTS: NEAT1 and BHLHE40 were significantly overexpressed in CRC tissues and cells. BHLHE40 has a binding relationship with the NEAT1 promoter. Knockdown of BHLHE40 resulted in a reverted malignant phenotype in vitro and slowed tumor growth and metastasis dissemination in vivo, which were reversed by NEAT1 overexpression. Overexpression of BHLHE40 increased Wnt/β-catenin pathway activity, but knockdown of NEAT1 decreased Wnt/β-catenin pathway activity.
CONCLUSIONS: BHLHE40 mediates the transcriptional activation of NEAT1, which activates the Wnt/β-catenin pathway and promotes the CRC progression.
方法:通过RT-qPCR分析CRC组织和CRC细胞中的NEAT1表达。在CRC中与NEAT1共表达的基因来自UALCAN,与来自hTFtarget的靶向NEAT1的转录因子相交。双荧光素酶测定,RT-qPCR,和ChIP分析BHLHE40与NEAT1的转录调控关系。对敲低BHLHE40和过表达NEAT1的LoVo和HCT-15细胞进行MTT,Transwell,蛋白质印迹,和流式细胞术检测CRC细胞的恶性侵袭性。使用HE和免疫组织化学分析在小鼠中研究了敲低BHLHE40和过表达NEAT1对肿瘤和肺转移的影响。
结果:NEAT1和BHLHE40在CRC组织和细胞中显著过表达。BHLHE40与NEAT1启动子具有结合关系。敲除BHLHE40导致体外恶性表型逆转,并减缓体内肿瘤生长和转移扩散,被NEAT1过表达逆转。过表达BHLHE40可增加Wnt/β-catenin通路活性,但是NEAT1的敲减降低了Wnt/β-catenin通路的活性。
结论:BHLHE40介导NEAT1的转录激活,激活Wnt/β-catenin通路,促进CRC进展。