目的 探究强心苷类单体化合物21-hydroxy-neriaside的抗胃癌作用及机制。方法 采用CCK-8法考察21-hydroxy-neriaside对胃癌HGC27、MGC803、GT0603和GT112细胞增殖的影响；观察21-hydroxy-neriaside对HGC27、MGC803、GT0603和GT112细胞形态的影响；考察21-hydroxy-neriaside对HGC27、GT0603和GT112细胞集落形成的影响；采用RNA测序探究21-hydroxy-neriaside抑制胃癌细胞增殖的可能机制；采用qRT-PCR验证RNA测序结果；考察shRNA-E2F转录因子5（E2F transcription factor 5，E2F5）对GT112细胞E2F5表达的影响；采用Western blotting检测21-hydroxy-neriaside对HGC27、GT0603和GT112细胞E2F5、骨髓细胞瘤病毒癌基因（cellular-myelocytomatosis viral oncogene，c-Myc）、细胞周期D1（Cyclin D1）、细胞周期E2（Cyclin E2）和剪切型聚腺苷酸二磷酸核糖转移酶[cleaved poly (ADP-ribose) polymerase，cleaved PARP]蛋白表达的影响。结果 CCK-8实验、形态学观察和细胞克隆形成实验均表明21-hydroxy-neriaside具有抑制胃癌细胞增殖的活性，与时间和药物浓度呈正相关。RNA测序、qRT-PCR和shRNA-E2F5慢病毒转染实验表明21-hydroxy-neriaside通过调控E2F5影响细胞周期通路，从而抑制肿瘤细胞生长。Western blotting实验结果表明21-hydroxy-neriaside组细胞中E2F5、c-Myc、Cyclin D1和Cyclin E2蛋白表达水平均明显降低（P＜0.05、0.01、0.001），cleaved PARP蛋白表达水平显著升高（P＜0.001）。结论 21-hydroxy-neriaside具有抗胃癌活性，能够通过下调E2F5表达来影响细胞周期通路相关蛋白，抑制胃癌细胞生长，从而促使肿瘤细胞凋亡。

Objective To explore anti-tumor effect and mechanisms of cardiac glycoside monomeric compound 21-hydroxy-neriaside against gastric cancer. Methods The effect of 21-hydroxy-neriaside on proliferation of gastric cancer HGC27, MGC803, GT0603 and GT112 cells was investigated by CCK-8 method; The effect of 21-hydroxy-neriaside on morphology of HGC27, MGC803, GT0603 and GT112 cells was observed; The effect of 21-hydroxy-neriaside on colony formation of HGC27, GT0603 and GT112 cells was observed; RNA sequencing was used to explore the possible mechanism of 21-hydroxy-neriaside inhibiting gastric cancer cell proliferation; qRT-PCR was used to verify RNA sequencing results; The effect of shRNA-E2F transcription factor 5 (E2F5) on E2F5 expression of GT112 cells was investigated; Western blotting was used to detect the effect of 21-hydroxy-neriaside on E2F5, cellular-myelocytomatosis viral oncogene (c-Myc), cell cycle D1 (Cyclin D1), cell cycle E2 (Cyclin E2) and cleaved poly(ADP-ribose) polymerase (cleaved PARP) protein expressions in HGC27, GT0603 and GT112 cells. Results CCK-8 assay, morphological observation and cell clone formation assay showed that 21-hydroxy-neriaside had the activity of inhibiting the proliferation of gastric cancer cells, which was positively correlated with time and drug concentration. RNA sequencing, qRT-PCR and shRNA-E2F5 lentiviral transfection experiments showed that 21-hydroxy-neriaside affected cell cycle pathways by regulating E2F5, thereby inhibiting tumor cell growth. The results of Western blotting showed that the protein expression levels of E2F5, c-Myc, Cyclin D1 and Cyclin E2 in 21-hydroxy-neriaside group were significantly decreased (P < 0.05, 0.01, 0.001), and cleaved PARP protein expression level was significantly increased (P < 0.001). Conclusion 21-Hydroxy-neriaside has anti-gastric cancer activity. It can affect cell cycle pathway-related proteins by down-regulating E2F5 expression, inhibit the growth of gastric cancer cells, and promote tumor cell apoptosis.

R285.5

广东省医学科学技术研究基金资助项目（B2020031）