Huang Qianwu, Lv Jun, Dong Ting, Liu Haijun, Xu Lei, Wu Mingcai
Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
Department of Biochemistry and Molecular Biology, Wannan Medical College, Wuhu, Anhui, China.
Dose Response. 2020 Jul 9;18(3):1559325820939022. doi: 10.1177/1559325820939022. eCollection 2020 Jul-Sep.
Cryptochrome 1 (CRY1) is a key protein that regulates the feedback loop of circadian clock. The abnormal expression of CRY1 was reported in numerous cancers, and contributed to tumorigenesis and progression. But the underlying mechanism remains undefined.
overexpression was constructed by lentivirus vector. Gene and protein expression was detected by reverse transcription quantitative polymerase chain reaction and Western blot. Cell proliferation was analyzed by CCK-8 assay. Cell migration ability was analyzed by scratch assay and transwell migration assay. The cAMP concentration was measured by intracellular cAMP assay.
Overexpression of showed slightly effect on the proliferation and migration of HGC-27 cells. Upon exposure to isoproterenol (ISO), a β-adrenergic receptor agonist, cell proliferation, and migration were inhibited while the cAMP/PKA pathway was activated and ERK1/2 phosphorylation was suppressed. overexpression reduced cAMP accumulation, retained ERK1/2 phosphorylation level and alleviated the antiproliferative effect upon exposure to ISO. However, overexpression was inoperative on the antiproliferative effect of forskolin (FSK), a direct activator of adenyl cyclase (AC), or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase (PDE) inhibitor.
Our results suggest overexpression may protect cells from the antiproliferative effects via activation of the cAMP/PKA pathway through interrupting signal transduction from G protein-coupled receptors to AC.
隐花色素1（CRY1）是一种调节生物钟反馈回路的关键蛋白。CRY1在多种癌症中存在异常表达，并促进肿瘤发生和进展。但其潜在机制仍不明确。
通过慢病毒载体构建过表达。采用逆转录定量聚合酶链反应和蛋白质免疫印迹法检测基因和蛋白表达。采用CCK-8法分析细胞增殖。通过划痕试验和Transwell迁移试验分析细胞迁移能力。采用细胞内cAMP检测法测定cAMP浓度。
CRY1过表达对HGC-27细胞的增殖和迁移影响较小。暴露于β-肾上腺素能受体激动剂异丙肾上腺素（ISO）后，细胞增殖和迁移受到抑制，同时cAMP/PKA途径被激活，ERK1/2磷酸化受到抑制。CRY1过表达减少了cAMP积累，维持了ERK1/2磷酸化水平，并减轻了暴露于ISO后的抗增殖作用。然而，CRY1过表达对腺苷酸环化酶（AC）的直接激活剂福斯高林（FSK）或磷酸二酯酶（PDE）抑制剂3-异丁基-1-甲基黄嘌呤（IBMX）的抗增殖作用无效。
我们的结果表明，CRY1过表达可能通过中断从G蛋白偶联受体到AC的信号转导，激活cAMP/PKA途径，从而保护细胞免受抗增殖作用。
