长链非编码RNA-肺腺癌转移相关转录本1通过磷脂酰肌醇3-激酶/蛋白激酶B/雷帕霉素靶蛋白通路调控有氧糖酵解代谢促进胃癌细胞增殖

Long non-coding RNA-metastasis associated lung adenocarcinoma transcript 1 regulates aerobic glycolysis through the phosphatidylinositol 3 kinase/inhibited protein kinase B/mammalian target of rapamycin pathway to promote the proliferation of gastric cancer cells

目的探索长链非编码RNA(lncRNA)-肺腺癌转移相关转录本1(MALAT1)对胃癌细胞增殖和有氧糖酵解的作用及其机制。方法使用实时定量反转录聚合酶链反应(RT-qPCR)检测人胃黏膜上皮细胞GES-1和胃癌细胞系MGC-803、SGC-7901、MKN45、AGS内lncRNA-MALAT1的表达水平;采用小干扰RNA(siRNA)敲低SGC-7901细胞lncRNA-MALAT1的表达水平后,细胞计数试剂盒(CCK-8)法检测胃癌细胞的增殖能力,酶标比色法检测细胞葡萄糖摄取、乳酸水平和三磷酸腺苷(ATP)含量,RT-qPCR检测有氧糖酵解相关基因的mRNA表达,蛋白质印迹法(Western blot)检测磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号通路的活化。两样本间的比较采用t检验。结果lncRNA-MALAT1在胃癌细胞中的表达量升高,其中SGC-7901组表达倍数明显高于GES-1组[(14.970±1.429)倍比1.000倍,t=16.880,P<0.01];转染靶向lncRNA-MALAT1的siRNA进入SGC-7901细胞后,沉默组lncRNA-MALAT1的表达倍数明显小于对照组[(0.560±0.110)倍比1.000倍,t=5.126,P<0.01];成功敲低lncRNA-MALAT1后,CCK-8实验显示沉默组吸光度值低于对照组(0.863±0.061比1.393±0.085,t=8.766,P<0.01),葡萄糖摄取率实验显示沉默组吸光度值低于对照组(0.563±0.097比1.010±0.105,t=5.399,P<0.01),ATP检测实验显示沉默组吸光度值低于对照组(0.583±0.155比1.037±0.120,t=4.003,P<0.05),乳酸产生率实验显示沉默组吸光度值低于对照组(0.573±0.127比0.973±0.083,t=4.572,P<0.05),沉默组己糖激酶2的mRNA表达明显低于对照组[(0.510±0.125)倍比1.000倍,t=5.980,P<0.01],沉默组M2型丙酮酸激酶的mRNA表达明显低于对照组[(0.573±0.160)倍比1.000倍,t=4.313,P<0.05],沉默组乳酸脱氢酶的mRNA表达明显低于对照组[(0.693±0.060)倍比1.000倍,t=4.400,P<0.05]和沉默组丙酮酸脱氢酶激酶1的mRNA表达明显低于对照组[(0.537±0.100)倍比1.000倍,t=5.408,P<0.01],沉默组p-PI3K/PI3K的蛋白相对表达量低于对照组[(0.519±0.070)倍比(0.805±0.071)倍,t=4.977,P<0.01],沉默组p-Akt/Akt的蛋白相对表达量低于对照组[(0.543±0.080)倍比(0.862±0.058)倍,t=5.590,P<0.01],沉默组p-mTOR/mTOR的蛋白相对表达量低于对照组[(0.341±0.075)倍比(0.853±0.040)倍,t=10.380,P<0.01]。结论lncRNA-MALAT1通过PI3K/Akt/mTOR信号通路来促进胃癌细胞有氧糖酵解和体外增殖。

Objective To explore the effect of long non-coding RNA(lncRNA)-metastasis associated lung adenocarcinoma transcript 1(MALAT1)on the proliferation and aerobic glycolysis of gastric cancer cells and underlying mechanism.Methods The expression level of lncRNA-MALAT1 in human gastric epithelial cells GES-1 and gastric cancer cell lines MGC-803,SGC-7901,MKN45 and AGS was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).After using small interfering RNA(siRNA)to knock down the expression level of lncRNA-MALAT1 in SGC-7901 cells,the proliferation ability of gastric cancer cells was detected by cell counting kit-8(CCK-8)assay.The glucose uptake,lactate level and adenosine triphosphate(ATP)content were determined by enzyme-labeled colorimetry.The mRNA expression of aerobic glycolysis related genes was detected by RT-qPCR,and the activation of phosphatidylinositol 3 kinase(PI3K)/inhibited protein kinase B(Akt)/mammalian target of rapamycin(mTOR)signal pathway was detected by Western blotting.The significance of differences between groups was analyzed by student’s t test.Results The expression of lncRNA-MALAT1 in gastric cancer cells was increased,and the expression multiple of SGC-7901 group was significantly higher than that of GES-1 group[(14.970±1.429)times vs.1.000 time,t=16.880,P<0.01].After the transfection of lncRNA-MALAT1 siRNA into SGC-7901 cells,the expression of lncRNA-MALAT1 in the silence group was significantly lower than that in the control group(0.560±0.110 vs.1.000 time,t=5.126,P<0.01).After knocking down lncRNA-MALAT1,CCK-8 assay showed that the absorbance value of knockdown group on the 4th day was lower than that of control group(0.863±0.061 vs.1.393±0.085,t=8.766,P<0.01).The glucose uptake rate experiment showed that the absorbance value of knockdown group was lower than that of control group(0.563±0.097 vs.1.010±0.105,t=5.399,P<0.01).The ATP generation experiment showed that the absorbance value of knockdown group was lower than that of control group(0.583±0.155 vs.1.037±0.120,t=4.003,P<0.05).The lactate production rate experiment showed that the absorbance value of the knockdown group was lower than that of the control group(0.573±0.127 vs.0.973±0.083,t=4.572,P<0.05).The mRNA expression of hexokinase 2 in the knockdown group was significantly lower than that of the control group[(0.510±0.125)times vs.1.000 time,t=5.980,P<0.01].The mRNA expression of M2 pyruvate kinase in knockdown group was significantly lower than that in control group[(0.573±0.160)times vs.1.000 time,t=4.313,P<0.05].The mRNA expression of lactate dehydrogenase in the knockdown group was significantly lower than that in the control group[(0.693±0.060)times vs.1.000 time,t=4.400,P<0.05].The mRNA expression of pyruvate dehydrogenase kinase 1 in knockdown group was significantly lower than that in control group[(0.537±0.100)times vs.1.000 time,t=5.408,P<0.01].The relative expression of p-PI3K/PI3K protein in the silence group was lower than that in the control group[(0.519±0.070)time vs.(0.805±0.071)times,t=4.977,P<0.01].The relative expression of p-Akt/Akt protein in the silence group was lower than that in the control group[(0.543±0.080)times vs.(0.862±0.058)times,t=5.590,P<0.01].The relative expression of p-mTOR/mTOR protein in the silence group was lower than that in the control group[(0.341±0.075)times vs.(0.853±0.040)times,t=10.380,P<0.01].Conclusion LncRNA-MALAT1 promotes aerobic glycolysis and proliferation of gastric cancer cells in vitro through the PI3K/Akt/mTOR signal pathway.