槲皮素通过调控GAS5/miR-18b-5p轴抑制神经内分泌肿瘤细胞活性的研究

Quercetin inhibits the activity of neuroendocrine tumor cells by regulating the GAS5/miR-18b-5p axis

目的探讨槲皮素抑制胃肠胰神经内分泌肿瘤(GEP-NEN)的作用机制。方法将人胰腺神经内分泌瘤细胞BON-1细胞随机分为对照组、槲皮素组(80μmol·L^(-1)槲皮素)、槲皮素+si-NC组(转染si-NC+80μmol·L^(-1)槲皮素)、槲皮素+si-生长抑制特异性基因5(GAS5)组(转染si-GAS5+80μmol·L^(-1)槲皮素)。用实时荧光定量聚合酶链反应(qRT-PCR)法检测B淋巴细胞瘤-2基因(Bcl-2)和Bcl-2相关X蛋白(Bax)mRNA相对表达水平,用荧光原位杂交(FISH)实验检测细胞中GAS5和miR-18b-5p的阳性表达情况。结果双荧光素酶报告基因结果发现,GAS5与miR-18b-5p靶向结合。对照组、槲皮素组、槲皮素+si-NC组、槲皮素+si-GAS5组细胞的GAS5表达水平分别为1.00±0.13、1.72±0.19、1.78±0.14和1.16±0.11,miR-18b-5p表达水平分别为1.00±0.15、0.67±0.08、0.72±0.06和0.95±0.11,Bax mRNA相对表达水平分别为1.00±0.12、2.17±0.25、2.32±0.28和1.37±0.15,Bcl-2 mRNA相对表达水平分别为1.00±0.15、0.41±0.05、0.37±0.06和1.21±0.13。槲皮素组的上述指标与对照组比较,在统计学上差异均有统计学意义(均P<0.05);槲皮素+si-GAS5组的上述指标与槲皮素+si-NC组比较,在统计学上差异均有统计学意义(均P<0.05)。结论槲皮素可能通过靶向调控GAS5/miR-18b-5p分子轴抑制细胞生长减缓GEP-NEN发展。

Objective To investigate the inhibitory effect of quercetin on Gastro entero pancreatic NEN(GEP-NEN).Methods Human pancreatic neuroendocrine tumor BON-1 cells were randomly divided into control group,quercetin group(80μmol·L^(-1)quercetin),quercetin+si-NC group(transfected with si-NC+80μmol·L^(-1)quercetin),quercetin+si-growth arrest-specific+ranscript 5(GAS5)group(transfected with si-GAS5+80μmol·L^(-1)quercetin).Dual luciferase reporter gene assay was used to verify the targeted binding of GAS5 to miR-18b-5p;real-time quantitative fluorescent PCR(qRT-PCR)was used to detect the mRNA expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(Bax);positive expression of GAS5 and miR-18b-5p in cells was detected by fluorescence in situ hybridization(FISH)assay.Results Dual luciferase reporter gene results showed that GAS5 was targeted to miR-18b-5p.The GAS5 expression levels of control group,quercetin group,quercetin+si-NC group and quercetin+si-GAS5 group were 1.00±0.13,1.72±0.19,1.78±0.14 and 1.16±0.11,respectively;the expression levels of miR-18b-5p were 1.00±0.15,0.67±0.08,0.72±0.06 and 0.95±0.11 respectively;Bax mRNA expression levels were 1.00±0.12,2.17±0.25,2.32±0.28 and 1.37±0.15,respectively;Bcl-2 mRNA expression levels were 1.00±0.15,0.41±0.05,0.37±0.06 and 1.21±0.13,respectively.The above indexes were significantly different between quercetin group and control group(all P<0.05);the above indexes were significantly different between quercetin+si-NC group and quercetin+si-GAS5 group(all P<0.05).Conclusion Quercetin may slow down the development of GEP-NEN by targeting GAS5/miR-18b-5p molecular axis to inhibit cell growth.