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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 investigate the effect of Ghrelin silencing on cell apoptosis and its mechanism in small cell lung cancer (SCLC), H446 cells and ghrelin. Methods: Ghrelin levels were detected by immunohistochemistry in SCLC and adjacent normal tissues. The levels of Ghrelin in H446 cells were detected by RT-PCR and Western blot. The levels of Ghrelin were detected by ELISA in cell culture supernatants. Ghrelin was silenced by siRNA interference method, silencing effect was verified by RT-PCR, flow cytometry was used to detect apoptosis, active-caspase-3 and Bcl-2 levels were detected by Western blot, miR-21 was detected by RT-PCR and MTT assay Cell viability was measured; liposomal transfection method was used to achieve miR-21 overexpression. Results: The expression of Ghrelin in SCLC tissues was significantly higher than that in adjacent normal tissues. The expression of Ghrelin in H446 cells and its culture supernatant was significantly higher than that in BEAS-2B cells. After silencing Ghrelin, the apoptosis rate of H446 cells was significantly higher than that of other groups, accompanied by an increase of active-caspase-3 and a decrease of Bcl-2 levels and a decrease of miR-21 levels. MiR-21 levels were the lowest in Ghrelin gene / GHS-R co-suppressive cells after GST-R antagonist or antibody treatment, indicating that Ghrelin regulates miR-21 levels by recognizing GHS-R receptors. In addition, miR-21 Overexpression could reverse the increase of apoptosis induced by Ghrelin silencing and the expression of apoptosis related proteins. Conclusion: The expression of Ghrelin in SCLC is significantly increased, and Ghrelin silencing can promote the apoptosis of SCLC by inhibiting the expression of miR-21.