腺病毒携带的黑色素瘤分化相关基因-7调节生长抑素受体-2基因对胆囊癌细胞侵袭转移机制的研究
The mechanism of Ad.mda-7 regulated somatostatin receptor 2 gene in the invasion and metastasis of gallbladder cancer cells
翟东升 1李俊 1张晓梅 2申铭 3郑建伟3
作者信息
- 1. 湖北民族大学附属民大医院肝胆外科,恩施 445000
- 2. 武汉市第五医院消化内科,武汉 430051
- 3. 华中科技大学同济医学院附属同济医院胆胰外科,武汉 430030
- 折叠
摘要
目的 探讨腺病毒携带的黑色素瘤分化相关基因-7(Ad.mda-7)调节胆囊癌细胞生长抑素受体-2(SSTR2)基因表达对胆囊癌细胞SGC-996生长及细胞周期的影响.方法 构建携带MDA-7/白细胞介素(IL)-24基因的腺病毒载体,转染胆囊癌细胞SGC-996,使用细胞计数试剂盒(CCK-8)法检测胆囊上皮细胞、胆囊癌细胞实验组细胞增殖率,观察各实验组细胞增殖抑制的变化、FCM流失细胞术分析各实验组细胞周期特异性.实时定量聚合酶链反应(Real-time PCR)检测Ad.mda-7转染后,各实验组细胞MDA-7、SSTR2基因的表达,蛋白质印迹法(Western blot)检测p21、Cyclin A、CDK2、Cyclin B蛋白等细胞周期蛋白(Cyclin)及蛋白激酶B(Akt)细胞通路蛋白的表达.结果 Ad.mda-7可以有效转染胆囊癌细胞SGC-996及胆囊NF细胞,稳定表达MDA-7的mRNA及蛋白.转染MDA-7基因后,胆囊癌细胞SGC-996组细胞存活率[(43.48±9.75)％]低于空白对照组[(89.16±7.85)％]和阴性 Ad.vec 对照组[(87.97±8.63)％],Ad.mda-7 明显受到抑制SGC-996组细胞存活率(P＜0.05).流式细胞仪检测Ad.mda-7作用胆囊癌各实验组细胞24 h后细胞周期变化,Ad.mda-7 SGC-996细胞组G2/M期[(49.62±3.12)％]高于于空白组[(13.54± 1.13)％]、阴性Ad.vec对照组[(15.39±6.04)％],Ad.mda-7 SGC-996实验组细胞周期被阻滞于G2/M期(P＜0.05).Western blot进行蛋白定量分析,Ad.mda-7转染后胆囊癌SGC-996细胞后,实验组SSTR2蛋白表达上调,p21、Cyclin A、CDK2、Cyclin B蛋白表达逐渐增加,而Cyclin D1、CDK4、Cyclin D3、CDK6蛋白的表达逐渐减少,诱导肿瘤细胞细胞周期被阻滞于G2/M期.同时,Akt、ERK蛋白的表达呈明显增加,实验组p-Akt、p-ERK、p-JNK蛋白表达量明显下降.结论 Ad.mda-7转染胆囊癌细胞SGC-996后,明显抑制肿瘤细胞的生长,Ad.mda-7上调SSTR2蛋白的表达,并且通过上调细胞内Akt信号通路蛋白的表达,抑制肿瘤细胞的生长.
Abstract
Objective To investigate the effect of Ad.mda-7 regulated somatostatin receptor 2(SSTR2)gene expression on growth and cell cycle of gallbladder cancer cells(SGC-996 cell line).Methods The adenovirus vector carrying the MDA-7/interleukin(IL)-24 gene was constructed,and SGC-996 cells were transfected.Cell proliferation rate in experimental groups of gallbladder epithelial cells and gallbladder cancer cells was detected by cell counting kit-8(CCK-8)method and the changes of cell proliferation inhibition in each experimental group.Analysised the cell cycle in each experimental group used FCM cell-loss cytometric.Expression of MDA-7 and SSTR2 genes in each experimental group after Ad.mda-7 transfection was detected by Real-time polymerase chain reaction(PCR).Expression of Cyclin protein and protein kinase B(Akt)cell pathway proteins such as p21,Cyclin A,CDK2,and Cyclin B pro-teins was detected by Western blotting.Results Ad.mda-7 efficiently transfected gallbladder cancer cells SGC-996 and gallbladder normal fibroblast(NF)cells,stably expressed MDA-7 mRNA and protein.Gall-bladder carcinoma cells transfected with MDA-7 gene,group of Ad.mda-7 SGC-996 cell[(43.48± 9.75)％]was lower than the group of Ad.mda-7 SGC-996 cell blank control[(89.16±7.85)％]and negative control groups[(87.97±8.63)％].the group of Ad.mda-7 SGC-996 cell proliferation was signifi-cantly inhibited(P＜0.05).Flow cytometry detected cell cycle changes after 24 h in each experimental group.G2/M phase in the Ad.mda-7 SGC-996 cell group[(49.62±3.12)％]was higher than the blank group[(13.54±1.13)％],negative Ad.vec control group[(15.39±6.04)％],transfected Ad.mda-7 SGC-996 cells was blocked at G2/M phase of cell cycle relative to the blank group(P＜0.05).After Ad.mda-7 transfection of gallbladder cancer SGC-996 cells.Western blotting was performed protein quantitative analysis.SSTR2 protein expression was upregulated in the experimental group.progressive increases in protein expression of p21,Cyclin A,CDK2,and Cylin B,whereas expression of Cyclin D1,CDK4,Cyc-lin D3,and CDK6 proteins was decreased.Tumor cell cycle blocked at G2/M phase.Conclusion Ad.mda-7 transfected gallbladder cancer cells SGC-996 was significantly inhibited tumor cell growth.Ad.mda-7 up-regulates the expression of SSTR2 protein and suppresses tumor cell growth by up-regulating the ex-pression of intracellular Akt signaling pathway protein.
关键词
胆囊恶性肿瘤/MDA-7/生长抑素受体-2/细胞周期蛋白Key words
Gallbladder cancer/MDA-7/Somatostatin receptor 2/Cyclin引用本文复制引用
基金项目
国家自然科学基金(30872510)
湖北省自然科学基金(2017CFB632)
湖北省自然科学基金(2018CFB660)
出版年
2024
NETL
NSTL
万方数据