实用医学杂志 ›› 2021, Vol. 37 ›› Issue (9): 1099-1105.doi: 10.3969/j.issn.1006⁃5725.2021.09.001

• 基础研究 •    下一篇

miR⁃330⁃3p靶向丙酮酸激酶M2型对胃癌细胞增殖、凋亡及有氧糖酵解进程调控机制研究

刘磊, 赵羲和, 田忠   

  1. 中国医科大学附属盛京医院1普通外科,2 肿瘤科(沈阳110023)

  • 出版日期:2021-05-10 发布日期:2021-05-10
  • 通讯作者: 田忠 E⁃mail:tianzhongcmu2h@126.com
  • 基金资助:
    国家自然科学基金项目(编号:81702402,81802760)

Regulation mechanism of miR⁃330⁃3p on proliferation and apoptosis of gastric cancer cells,and aerobicglycolysis process by targeting pyruvate kinase M2

LIU Lei,ZHAO Xihe,TIAN Zhong.   

  1. Department of Gen⁃ eral Surgery,Shengjing Hospital of China Medical University,Shenyang,110023,China

  • Online:2021-05-10 Published:2021-05-10
  • Contact: TIAN Zhong E⁃mail:Tianzhongcmu2h@126.com

摘要:

目的 探讨miR⁃330⁃3p靶向丙酮酸激酶M2型(PKM2)基因对胃癌细胞增殖、凋亡及有氧糖酵解进程的影响及机制。方法 采用反转录聚合酶链式反应检测胃癌细胞SGC⁃7901、胃粘膜上皮细胞GES⁃1 miR⁃miR⁃330⁃3p表达水平,将miR⁃NC、miR⁃330⁃3p⁃inhibitor、miR⁃330⁃3p⁃mimcs、miR⁃330⁃3p⁃inhibitor+PKM2⁃NC、miR⁃330⁃3p⁃inhibitor+PKM2⁃shRNA、miR⁃330⁃3p⁃mimcs+LV⁃NC、miR⁃330⁃3p⁃mimcs+LV⁃PKM2转染至胃癌细胞中,采用CCK8 法、Transwell 实验、Annexin V/PI 法分别检测细胞增殖、侵袭、凋亡情况,检测葡萄糖利用率、乳酸生成量、己糖激酶(HK)活性、乳酸脱氢酶(LDH)活性等糖酵解指标,采用Western Blot法检测PKM2、GLUT1、HK2、Bax、Caspase⁃3蛋白的表达,采用双荧光素酶报告基因检测实验检测miR⁃330⁃3p与PKM2的靶向关系。结果 (1)胃癌细胞系miR⁃330⁃3p表达量高于胃粘膜上皮细胞,PKM2表达量低于胃粘膜上皮细胞(P < 0.05)。(2)生物信息学显示,miR⁃330⁃3p与PKM2存在结合位点;荧光素酶报告基因检测结果显示,与miR⁃NC组比较,miR⁃330⁃3p PKM2野生型荧光素酶活性降低(P < 0.05)。(3)与miR⁃NC组比较,miR⁃330⁃3p⁃inhibitor组吸光度值、侵袭细胞数增多,凋亡细胞数减少,细胞葡萄糖消耗量、乳酸生成量、HK活性、LDH活性降低,PKM2、GLUT1、HK2表达升高,促凋亡蛋白Bax、Caspase⁃3表达量降低(P < 0.05),miR⁃330⁃3p⁃inhibitor+PKM2⁃shRNA组升高或降低幅度低于miR⁃330⁃3p⁃inhibitor组(P < 0.05)。(4)与miR⁃NC组比较,miR⁃330⁃3p⁃mimcs 组miR⁃330⁃3p表达量升高(P < 0.05),吸光度值、侵袭细胞数减少,凋亡细胞增多,细胞葡萄糖消耗量、乳酸生成量、HK 活性、LDH 活性升高,PKM2、GLUT1、HK2 表达降低,促凋亡蛋白Bax、Caspase⁃3 表达量升高(P < 0.05);miR⁃330⁃3p⁃mimcs+LV⁃PKM2 组升高或降低趋势低于miR⁃330⁃3p⁃mimcs 组(P < 0.05)。结论 miR⁃330⁃3p 表达与胃癌细胞增殖、凋亡及有氧糖酵解进程相关,可能通过靶向PKM2实现。

关键词: miR?330?3p, 丙酮酸激酶M2型, 胃癌, 糖酵解, 增殖, 凋亡

Abstract:

Objective To explore the effects and mechanism of miR ⁃ 330⁃3p on the proliferation and apoptosis of gastric cancer cells,and aerobic glycolysis process by targeting pyruvate kinase M2(PKM2)gene. Methods The expression levels of SGC⁃7901 in gastric cancer cells and GES⁃1 miR⁃miR⁃330⁃3p in gastric muco⁃ sal epithelial cells were detected by reverse transcription⁃polymerase chain reaction(RT⁃PCR). The miR⁃NC,miR⁃ 330⁃3p⁃inhibitor,miR⁃330⁃3p⁃mimcs,miR⁃330⁃3p⁃inhibitor+PKM2⁃NC,miR⁃330⁃3p⁃inhibitor+PKM2⁃shRNA miR⁃330⁃3p⁃mimcs+LV⁃NC and miR⁃330⁃3p⁃mimcs+LV⁃PKM2 were transfected into gastric cancer cells. The proliferation,invasion and apoptosis of cells were detected by CCK8,Transwell assay and Annexin V/PI method. The glycolysis indexes such as glucose utilization rate,production of lactic acid,activities of hexokinase(HK and lactate dehydrogenase(LDH)were detected. The expression of PKM2,GLUT1,HK2,Bax and Caspase ⁃ 3 proteins was detected by Western Blot. The targeted relationship between miR⁃330⁃3p and PKM2 was detected by double luciferase reporter gene assay. Results (1)The expression level of miR⁃330⁃3p in gastric cancer cell line was higher than that in gastric mucosal epithelial cells,while expression level of PKM2 was lower(P < 0.05).(2 reporter gene assay showed that compared with that in miR⁃NC group,activity of PKM2 wild⁃type luciferase was decreased in miR⁃330⁃3p group(P < 0.05).(3)Compared with those in miR⁃NC group,absorbance and number of invasion cells were increased in miR⁃330⁃3p⁃inhibitor group;number of apoptosis cells was decreased;cellular glucose consumption,production of lactic acid,HK and LDH activities were decreased;expression of PKM2 GLUT1 and HK2 was increased,and expression level of pro⁃apoptosis proteins(Bax and Caspase⁃3)was decreased (P < 0.05). The increase or decrease amplitude in miR⁃330⁃3p⁃inhibitor+PKM2⁃shRNA group was lower than that in miR⁃330⁃3p⁃inhibitor group(P < 0.05).(4)Compared with that in miR⁃NC group,expression level of miR⁃ 330⁃3p was increased in miR⁃330⁃3p⁃mimcs group(P < 0.05),absorbance and number of invasion cells were decreased,number of apoptosis cells was increased,cellular glucose consumption,production of lactic acid,HK and LDH activities were increased,expression of PKM2,GLUT1 and HK2 was decreased,and expression levels of pro⁃apoptosis proteins(Bax,Caspase⁃3)were increased(P < 0.05). The increase or decrease amplitude in miR⁃ 330⁃3p⁃mimcs+LV⁃PKM2 group was lower than that in miR⁃330⁃3p⁃mimcs group(P < 0.05). Conclusion The expression of miR⁃330⁃3p is related to proliferation and apoptosis of gastric cancer cells,and aerobic glycolysis pro⁃ cess,

Key words:

miR ? 330 ? 3p, pyruvate kinase M2 type, gastric cancer, glycolysis, proliferation, apoptosis