Ghrelin在小细胞肺癌组织及H446细胞中的表达及其沉默对癌细胞凋亡的影响  

Ghrelin expression in SCLC tissues and H446 cells and the influence of Ghrelin gene silence on SCLC cell apoptosis

作　　者：赵鹏[1] 张卉 苏海川[1] ZHAO Peng ZHANG Hui SU Hai-chuan(Department of Oncology, Tangdu Hospital ,Affiliated to the Fourth Military Medical University,Xi＇an 710038, China Internal Medicine, Jingyang County Hospital, Xianyang 713700, China)

机构地区：[1]第四军医大学唐都医院肿瘤科,陕西西安710038 [2]泾阳县医院内科,陕西咸阳713700

出　　处：《现代医学》2017年第2期204-210,共7页Modern Medical Journal

摘　　要：目的:探讨小细胞肺癌(SCLC)组织和H446细胞中生长激素释放肽(Ghrelin)的表达及Ghrelin沉默对细胞凋亡的影响及作用机制。方法:Ghrelin水平在SCLC和癌旁正常组织中以免疫组织化学法检测,在H446细胞中以RT-PCR和Western blot法检测,在细胞培养上清液中以ELISA法检测。Ghrelin以siRNA干扰法沉默,沉默效果以RT-PCR法验证;流式细胞术检测细胞凋亡;Western blot检测active-caspase-3及Bcl-2水平;RT-PCR检测miR-21水平;MTT法测定细胞活力;脂质体转染法实现miR-21过表达。结果:Ghrelin在SCLC组织中表达明显高于癌旁正常组织,在H446细胞及其培养上清中表达显著高于BEAS-2B细胞。Ghrelin沉默后,H446细胞凋亡率显著高于其他组,伴随active-caspase-3水平增高及Bcl-2水平下降,同时细胞活力下降,且miR-21水平显著低于其它组;H446细胞经GHS-R拮抗剂或抗体处理后,miR-21水平在Ghrelin基因/GHS-R共抑制细胞中达到最低,表明Ghrelin是通过识别GHS-R受体来调控miR-21水平的;此外,miR-21过表达能够逆转Ghrelin沉默诱导的细胞凋亡率增加及凋亡相关蛋白表达的变化。结论:Ghrelin在SCLC中表达显著升高,且Ghrelin沉默能够通过抑制miR-21表达促进SCLC细胞凋亡。Objective： To exploring the expression of Ghrelin in SCLC tissues and cells influence and mechanism of Ghrelin gene knock-down on cell apoptosis. Methods： Levels （ H446 ） , and the of Ghrelin in SCLC tissues and para-carcinoma tissues were determined by immunohistochemical methods, while the levels of Ghrelin in H446 cells were determined by Western blots, and in culture supernatant were measured by ELISA. Ghrelin gene was silenced by siRNA interference, and the effectiveness of gene silence was verified by RT-PCR. Cell apoptosis was detected by Flow cytometry. The expression of active-caspase 3 and Bcl-2 was detected by Western blots. The level of miR-21 was detected by RT-PCR. Cell viability was detected by MTF assay. The overexpression of miR-21 was realized by cell transfection. Results： The expression of Ghrelin was significantly higher in SCLC tissues than that in para-carcinoma tissues. Similarly, Ghrelin level was markedly higher in H446 cell and cell culture supernatant than that in BEAS-2B cells. After silencing of Ghrelin gene, the total apoptosis rate and active-caspase 3 level of H446 cells were significantly higher than that of other groups, while Bcl-2 and miR-21 level of H446 cells were markedly lower than that of other groups. Meanwhile, cell viability was dramatically declined after silencing Ghrelin. After treatment by GHS-R antagonist or antibody, miR-21 level reduced to the lowest in Ghrelin gene and GHS-R co-suppressed H446 cells, which indicated that the regulating effect of Ghrelin on miR-21 was fulfilled by recognizing GHS-R receptor. Furthermore, in Ghrelin-silenced H446 cell, the increased cell apoptosis rate induced by Ghrelin deficiency could be reversed by overexpressed miR-21, as well as apoptosis-related proteins. Conclusion： Expression of Ghrelin was markedly increased in SCLC. And silencing of Ghrelin gene can induce the apoptosis of SCLC cell by decreasing miR-21.

关 键 词：GHRELIN 小细胞肺癌 H446细胞 细胞凋亡 MICRORNA-21 

分 类 号：R734.2[医药卫生—肿瘤]