CAJ | 학술논문

目的探讨Lin28A基因对胃癌细胞糖酵解的作用及其分子机制.方法利用慢病毒载体建立Lin28过表达胃癌BGC-823细胞株,以生物化学法检测细胞培养液乳酸含量,酶联免疫吸附试验(ELISA)法检测细胞内磷酸果糖(PFK)含量,蛋白质印迹法检测Lin28A、缺氧诱导因子1o(HIF-1α)、葡萄糖转运蛋白1(GLUT-1)、丙酮酸激酶M2 (PKM2)蛋白水平表达,反转录(RT)-PCR法检测let-7a RNA水平,膜联蛋白(Annexin) Ⅴ-别藻蓝蛋白(APC)单染法检测细胞凋亡.结果 Lin28A蛋白在转染组的表达水平明显高于对照组和空载组(均P＜0.001).转染组let-7a RNA表达水平(0.602±0.017)明显低于空载组(1.001 ±0.063) (P=0.005 2).转染组HIF-1 α、GLUT-1、PKM2蛋白的表达水平明显低于其他两组(均P＜0.001).对照组、空载组、转染组细胞培养液乳酸含量分别为(1.71±0.13)、(1.53±0.11)、(1.24±0.04) mmol/L,转染组明显低于对照组和空载组(P =0.017 0、0.031 0);细胞内PFK含量分别为(3.71±0.13)、(3.49±0.14)、(1.79±0.05) mmol/L,转染组明显低于对照组和空载组(P=0.014 0、0.036 0);凋亡率分别为5.02％±0.14％、7.16％±0.21％、10.39％±0.37％,转染组凋亡率明显高于对照组和空载组(P =0.000 5、0.000 7).结论 Lin28A过表达可以抑制let-7a表达,并通过抑制细胞的糖酵解促进人胃癌细胞凋亡.
목적 탐토Lin28A기인대위암세포당효해적작용급기분자궤제.방법 이용만병독재체건립Lin28과표체위암BGC-823세포주,이생물화학법검측세포배양액유산함량,매련면역흡부시험(ELISA)법검측세포내린산과당(PFK)함량,단백질인적법검측Lin28A、결양유도인자1o(HIF-1α)、포도당전운단백1(GLUT-1)、병동산격매M2 (PKM2)단백수평표체,반전록(RT)-PCR법검측let-7a RNA수평,막련단백(Annexin) Ⅴ-별조람단백(APC)단염법검측세포조망.결과 Lin28A단백재전염조적표체수평명현고우대조조화공재조(균P＜0.001).전염조let-7a RNA표체수평(0.602±0.017)명현저우공재조(1.001 ±0.063) (P=0.005 2).전염조HIF-1 α、GLUT-1、PKM2단백적표체수평명현저우기타량조(균P＜0.001).대조조、공재조、전염조세포배양액유산함량분별위(1.71±0.13)、(1.53±0.11)、(1.24±0.04) mmol/L,전염조명현저우대조조화공재조(P =0.017 0、0.031 0);세포내PFK함량분별위(3.71±0.13)、(3.49±0.14)、(1.79±0.05) mmol/L,전염조명현저우대조조화공재조(P=0.014 0、0.036 0);조망솔분별위5.02％±0.14％、7.16％±0.21％、10.39％±0.37％,전염조조망솔명현고우대조조화공재조(P =0.000 5、0.000 7).결론 Lin28A과표체가이억제let-7a표체,병통과억제세포적당효해촉진인위암세포조망.
Objective To explore the effects of over-expression of Lin28A on glycolysis of gastric cancer cells and the underlying mechanism.Methods Human gastric cancer BGC-823 cells in vitro were stably transfected with lentivirus-mediated over-expression of Lin28A.Lactic acid levels of cell culture medium were detected with biochemical method,phosphofructose kinase (PFK) levels in cells with ELISA,the expression levels of Lin28A,hypoxia-inducible factor (HIF)-1α,glucose transporter (GLUT)-1,and pyruvate kinase M2 (PKM2) were detected using Western blotting,and let-7a RNA using reverse transcription-PCR.Annexin Vallophycocyanin (APC) staining method was used to assess cell apoptosis.Results Compared with the normal control (CON) group and the negative control (NC) group,Lin28A protein level in the over-expression (OE) group was significantly higher (both P ＜0.001).RNA expression level of let-7a was significantly lower in the OE group than in the NC group (0.602 ±0.017 vs.1.001 ±0.063,P =0.005 2).The levels of the HIF-1α,GLUT-1,PKM2 proteins were all significantly lower in the OE group than in the CON and the NC groups (all P ＜ 0.001).The lactic acid levels of cell culture medium in the CON,the NC,and the OE groups were (1.71 ± 0.13),(1.53 ±0.11),(1.24 ±0.04) mmol/L,which was significantly lower in the OE group than in the CON group and the NC group (P =0.017 0,0.031 0).Intracellular PFK level in the OE group was also significantly lower than in the CON group and the NC group [(1.79 ± 0.05) mmol/L vs.(3.71 ± 0.13) rmmol/L,P =0.014 0;(1.79 ± 0.05) mmol/L vs.(3.49 ± 0.14) mmol/L,P =0.036 0].Apoptosis rates of the CON,the NC,and the OE groups were 5.02％ ±0.14％,7.16％ ±0.21％,10.39％ ±0.37％,respectively,which was significantly higher in the OE group than in the CON group and the NC group (P =0.000 5,0.000 7).Conclusion Over-expression of Lin28A can inhibit the expression of let-7a,and induce apoptosis of human gastric cancer cells by suppressing glycolysis of the cells.