【摘要】 目的:探究微小RNA-19a(miR-19a)通过调控转化生长因子-β1(TGF-β1)/Smad信号通路对人胃癌AGS细胞增殖、侵袭和糖酵解的影响。方法:体外培养人胃癌AGS细胞，采用脂质体进行转染分别将inhibitor NC、mimics NC、miR-19a inhibitor、miR-19a mimics转染至人胃癌AGS细胞记为inhibitor NC组、mimics NC组、miR-19a inhibitor组和miR-19a mimics组，不做转染处理的为对照组，通过实时荧光定量PCR(RT-qPCR)法检测miR-19a的表达量，用细胞计数试剂盒-8(CCK-8)检测细胞活力，发现敲低miR-19a可显著抑制胃癌AGS细胞活力。所以后续实验分为对照组、inhibitor NC组、miR-19a inhibitor组、抑制剂组(miR-19a inhibitor转染+10μmol/L TGF-β1/Smad通路抑制剂LY2109761)和激活剂组(miR-19a inhibitor转染+10μmol/L TGF-β1/Smad通路激活剂SRI-011381),采用RT-qPCR法、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)、Transwell小室、乳酸、葡萄糖检测试剂盒及蛋白免疫印迹(WB)法分别对细胞miR-19a的表达、增殖率、侵袭数、乳酸含量、葡萄糖消耗水平及糖酵解、TGF-β1/Smad相关蛋白表达水平进行分析。结果:敲低miR-19a可显著抑制胃癌AGS细胞活力，所以选用转染miR-19a inhibitor进行后续通路验证实验。结果发现，对照组与inhibitor NC组在miR-19a的表达水平、细胞增殖率、侵袭数、葡萄糖消耗及乳酸水平、增殖细胞核抗原(PCNA)、己糖激酶2(HK2)、乳酸脱氢酶A(LDHA)、甘油醛-3-磷酸脱氢酶(GAPDH)、TGF-β1、p-Smad3蛋白表达水平等各项指标均无统计学差异(P>0.05)。miR-19a inhibitor组细胞miR-19a的表达、增殖率、侵袭数、葡萄糖消耗及乳酸水平、PCNA、HK2、LDHA、GAPDH、TGF-β1、p-Smad3蛋白水平低于inhibitor NC组(P<0.05)。抑制剂组细胞miR-19a的表达、增殖率、侵袭数、葡萄糖消耗及乳酸水平、PCNA、HK2、LDHA、GAPDH、TGF-β1、p-Smad3蛋白表达水平低于miR-19a inhibitor组，而激活剂组这些指标高于miR-19a inhibitor组(P<0.05)。结论:下调miR-19a可抑制人胃癌AGS细胞增殖、侵袭和糖酵解，其作用机制与阻滞TGF-β1/Smad信号转导有关。更多还原

【Abstract】 Objective:To investigate the effects of microRNA-19a(miR-19a) on the proliferation, invasion and glycolysis of human gastric cancer AGS cells by regulating transforming growth factors-β1(TGF-β1)/Smad signal pathway.Methods:Human gastric cancer AGS cells were cultured in vitro and divided into control groups.Liposomes were used for transfection.Inhibitor NC,mimics NC,miR-19a inhibitor, miR-19a mimics were transfected into human gastric cancer AGS cells and recorded as inhibitor NC group, mimics NC group, miR-19a inhibitor group and miR-19a mimics group.The expression of miR-19a was detected by real-time fluorescent quantitative PCR(RT-qPCR),and the cell viability was detected by cell counting kit-8(CCK-8).It was found that knockdown of miR-19a could significantly inhibit the activity of gastric cancer AGS cells.Therefore, the follow-up experiment was divided into control group,inhibitor NC group,miR-19a inhibitor group and inhibitor group(miR-19a inhibitor transfection + 10 μmol/L TGF-β1/Smad pathway inhibitor LY2109761),activator group(miR-19a inhibitor transfection + 10 μmol/L TGF-β1/Smad pathway activator SRI-011381). The miR-19a expression level,proliferation rate,invasion number,glucose consumption,lactic acid level,glycolysis,and expression level of related proteins were analyzed by real-time fluorescent quantitative PCR(RT-qPCR),5-acetyl-2 ’deoxyuridine(EdU),Transwell chamber,lactic acid,glucose detection kit,and western blot(WB).Results:It was found that knockdown of miR-19a could significantly inhibit the viability of gastric cancer AGS cells,so miR-19a inhibitor was transfected for subsequent pathway validation experiments. The results showed that the expression level of miR-19a,cell proliferation rate,invasion rate,glucose consumption and lactic acid level,PCNA,HK2,LDHA,GAPDH,TGF-β1,p-Smad3 protein expression level was no significant difference in control group and inhibitor NC group(P> 0. 05). The expression of miR-19a,proliferation rate,invasion number,glucose consumption,lactate level,PCNA,HK2,LDHA,GAPDH,TGF-β1,p-Smad3 protein levels of miR-19a inhibitor group were lower than those of miR-19a inhibitor group(P< 0. 05). The expression,proliferation rate,invasion number,glucose consumption,lactate level,PCNA,HK2,LDHA,GAPDH,TGF-β1 and p-Smad3 protein expression levels of miR-19a inhibitor group were lower than those of miR-19a inhibitor group(P< 0. 05).Conclusion:Down regulating the expression of miR-19a can inhibit the proliferation,invasion and glycolysis of human gastric cancer AGS cells,and its mechanism of action is to block TGF-β1/Smad signal transduction.更多还原