LYN通过AKT/mTOR信号通路对胃癌细胞增值及凋亡的影响

Effects of LYN on Proliferation and Apoptosis of Gastric Cancer Cells through AKT/mTOR Signaling Pathway

目的:研究LYN在胃癌中的表达量,以及敲低LYN表达对胃癌细胞增殖及凋亡的影响和机制。方法:使用免疫组织化学方法检测LYN在胃癌和癌旁组织的表达;利用脂质体Lipofectamine2000将shRNA-LYN转染到AGS(胃腺癌)细胞中,以未转染的胃癌细胞为对照组。CCK8和流式细胞术分别用于检测LYN敲低对AGS细胞增殖和凋亡的影响。利用蛋白印迹分析调查LYN敲低引起的AKT/m TOR信号通路相关蛋白,细胞凋亡相关蛋白Bcl2,Bax,Caspase-9和Casapse-3的表达量的改变,对其增殖及凋亡机制进行研究。使用生物信息学软件筛选出靶基因miR-496,western blot检测转染miR-496对LYN表达的影响。进行拯救实验,分析miR-496与LYN间的相互作用。结果:LYN在胃癌组织中的高表达的样本量为58例,显著高于癌旁组织的28例(P<0.05)。转染48h和72h后sh-LYN组细胞的吸光度值(0.0992±0.0346)和(1.0953±0.0379),明显低于NC组的(1.4123±0.1161)和(1.6717±0,0120)(P<0.05)。转染后sh-LYN组细胞的凋亡率(16.3567±0.5556)显著高于NC组(P<0.05)。敲低LYN后细胞Bcl2(0.4629±0.0294),p-AKT(0.5565±0.0473),p-m TOR(0.6167±0.0459),p70S6K(0.6049±0.0570)表达量较NC对照组下降,而Bax(1.407±0.0528),Caspase-9(1.5200±0.1063),Caspase-3(1.4170±0.0751)表达量上升。转染miR-496后可以抑制AGS细胞中LYN的表达。结论:敲低LYN表达可抑制AKT/m TOR信号通路,从而达到抑制胃癌细胞增殖,促进细胞凋亡的作用,同时其对胃癌细胞的作用受MiR-496靶向调控。

Objective:To investigate the expression level of LYN in gastric cancer and the effect and mechanism of knockdown LYN expression on proliferation and apoptosis of gastric cancer cells.Methods:Immunohistochemistry was used to detect LYN expression in gastric cancer and adjacent tissues.Shrna-LYN was transfected into AGS(gastric adenocarcinoma)cells using Lipofectamine 2000,and untransfected gastric cancer cells were used as control.CCK8 and flow cytometry were used to detect the effects of LYN knockdown on AGS cell proliferation and apoptosis,respectively.Western blot analysis was used to investigate the changes in the expression levels of AKT/m TOR signaling pathway related proteins and apoptosis related proteins Bcl2,Bax,Caspase9 and Casapse3 induced by LYN knockdown,and to study its proliferation and apoptosis mechanism.Bioinformatics software was used to screen out the target gene miR-496,and western blot was used to detect the effect of transfection miR-496 on LYN expression.Rescue experiments were performed to analyze the interaction between miR-496 and LYN.Results:The sample number of LYN overexpression in gastric cancer tissues was 58 cases,which was significantly higher than that in paracancer tissues(28 cases)(P<0.05).After transfection 48 and 72h,the absorbance values of sh-LYN group were(0.0992±0.0346)and(1.0953±0.0379),which were significantly lower than those of NC group(1.4123±0.1161)and(1.6717±0.0120),(P<0.05).After transfection,the apoptosis rate of sh-LYN group(16.3567±0.5556)was significantly higher than that of NC group(P<0.05).After LYN knockdown,the expression levels of Bcl2(0.4629±0.0294),P-Akt(0.5565±0.0473),P-MTOR(0.6167±0.0459)and p70S6K(0.6049±0.0570)decreased compared with NC control group.The expression of Bax(1.407±0.0528),Caspase-9(1.5200±0.1063),caspase-3(1.4170±0.0751)increased.Transfection of miR-496 could inhibit LYN expression in AGS cells.Conclusion:Knockdown LYN expression can inhibit AKT/m TOR signaling pathway,thereby inhibiting proliferation and promoting apoptosis of gastric cancer cells,and its effect on gastric cancer cells is regulated by miR-496.