RESUMO
Trimethylamine N-oxide (TMAO) promotes atherosclerosis in association with the functions of endothelial cells. Clock and Bmal1, as two main components of molecular circadian clock, play important regulatory roles during progression of atherogenesis. However, whether Clock and Bmal1 are involved in the regulation of endothelial proliferation disturbed by TMAO are unclear. We observed that cell proliferation of human umbilical vein endothelial cells (HUVECs) was inhibited after exposed to TMAO for 24â¯h. Besides, TMAO caused increased expression of lncRNA-NEAT1, Clock and Bmal1, and inhibited MAPK pathways. While MAPK pathways were blocked, the expression of Clock and Bmal1 was elevated. NEAT1 showed a circadian rhythmic expression in HUVECs, and its overexpression reduced cell proliferation. Knockdown or overexpression of NEAT1 might decrease or increase the expression of Clock and Bmal1 respectively, while raised or suppressed the expression of MAPK pathways correspondingly. Asparagus extract (AE) was found to improve the TMAO-reduced HUVECs proliferation. Moreover, it ameliorated the disorders of NEAT1, Clock, Bmal1, and MAPK signaling pathways induced by TMAO. Therefore, our findings indicated that NEAT1 regulating Clock-Bmal1 via MAPK pathways was involved in TMAO-repressed HUVECs proliferation, and AE improved endothelial proliferation by TMAO, proposing a novel mechanism for cardiovascular disease prevention.
Assuntos
Asparagaceae/química , Ritmo Circadiano/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Metilaminas/toxicidade , Extratos Vegetais/farmacologia , RNA Longo não Codificante/fisiologia , Fatores de Transcrição ARNTL/antagonistas & inibidores , Fatores de Transcrição ARNTL/biossíntese , Fatores de Transcrição ARNTL/genética , Aterosclerose/genética , Aterosclerose/fisiopatologia , Proteínas CLOCK/biossíntese , Proteínas CLOCK/genética , Divisão Celular/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Metilaminas/farmacologia , Caules de Planta/química , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologiaRESUMO
Circadian rhythm plays an important role in diverse physiological processes. Abnormal expression of circadian rhythm genes is associated with increased risk of disease, including different types of cancer. The cancer stem cell (CSC) hypothesis suggests that there is a small subset of stemlike cells within tumors that are responsible for tumor initiation. However, the biological effect of circadian rhythm on CSCs remains largely unknown. Studies have highlighted that the circadian rhythm protein CLOCK controls key aspects of various diseases. In the present study, lung cancer stemlike cells were successfully enriched using a sphere formation assay. Next, it was observed that CLOCK mRNA and protein expression levels in the A549 and H1299 sphere cells were notably increased compared with those in the corresponding parental cells. In addition, flow cytometry was performed to isolate CD133+ cells and, consistently, CLOCK expression was also found to be markedly upregulated in CD133+ lung cancer cells. Subsequently, to determine the effect of CLOCK on lung cancer stem cells in detail, CLOCK was knocked down using targeted short inhibiting RNA and the results demonstrated that the sphereforming ability of the A549 and H1299 cell lines was reduced. In addition, CSClike properties, including the expression of CD133, CD44, sex determining region Ybox 2, Nanog and octamerbinding transcription factor 4, were markedly decreased in the A549 and H1299 sphere cells following knockdown of CLOCK. Epigallocatechin3gallate (EGCG), a green tea polyphenol, has been reported to be a potential anticancer phytochemical. EGCG was found to repress CLOCK expression in A549 and H1299 sphere cells. In addition, EGCG also decreased the ratio of CD133+ cells. The Wnt/ßcatenin pathway was notably inactivated by the knockdown of CLOCK in A549 and H1299 sphere cells. Subsequently, using a xenograft model, it was demonstrated that EGCG suppressed the CSClike characteristics of lung cancer cells by targeting CLOCK. In conclusion, the present study demonstrated that EGCG inhibited the selfrenewal ability of lung cancer stemlike cells by targeting CLOCK.