胃饥饿素对结肠癌细胞增殖和侵袭的影响及机制探讨

Regulation of Ghrelin in proliferation and invasion of colon carcinoma

摘要:

目的探讨胃饥饿素（Ghrelin）对结肠癌细胞增殖、侵袭的影响及作用机制。方法收集90例行手术切除的结肠癌（CRC）组织及癌旁组织，免疫组织化学染色检测组织Ghrelin表达，分析Ghrelin表达与结肠癌患者临床资料、生存预后的关系。按照转染类型将细胞分为3组：空白对照组（WT）、阴性对照组（NC）和sh-Ghrelin转染组。采用短发夹RNA（shRNA）技术抑制结肠癌HT29、SW480 细胞Ghrelin 表达，CCK-8 法检测细胞增殖能力，流式细胞术检测细胞周期及早期凋亡的情况，划痕实验检测细胞迁移能力，Transwell 实验检测细胞侵袭能力，Western blot 检测磷脂酰肌醇3- 激酶（PI3K）、蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）表达水平。结果结肠癌组织Ghrelin高表达率为64.4%（58/90），癌旁组织Ghrelin 高表达率为27.8%（25/90），两者差异有统计学意义（χ2=24.347， P=0.001）。肿瘤直径≥5 cm 组Ghrelin 高表达率高于肿瘤直径<5 cm 组，Ⅲ、Ⅳ期组Ghrelin 高表达率高于Ⅰ、Ⅱ组，淋巴结转移组Ghrelin高表达率高于无淋巴结转移组，组间比较差异有统计学意义（P <0.05）；Ghrelin 表达与性别、年龄及肿瘤分级无关（P >0.05）。生存曲线分析显示，Ghrelin高表达组总生存期和无病生存率均低于Ghrelin低表达组（P <0.05）。在结肠癌HT29和SW480 细胞中，sh-Ghrelin组细胞增殖能力低于NC 组和WT 组，处于S 期比例、早期凋亡比例高于NC 组和WT 组（P <0.05）。处于G0/G1期比例低于NC组和WT 组，差异有统计学意义（P <0.05）。sh-Ghrelin组细胞迁移距离、侵袭细胞数少于NC 组和WT 组（P <0.05）。Western blot检测结果显示，在HT29、SW480细胞中，sh-Ghrelin 组PI3K、p-Akt及p-mTOR蛋白相对表达量低于NC 组和WT组（P <0.05），NC 组、WT 组间PI3K、Akt、p-Akt、mTOR 及p-mTOR 蛋白相对表达量比较差异无统计学意义（P >0.05）。结论结肠癌组织Ghrelin 表达上调，并与结肠癌患者生存预后有关。干扰Ghrelin 表达能够抑制结肠癌细胞增殖、侵袭，其作用机制可能与抑制PI3K-Akt-mTOR信号通路有关。

Abstract:

Objective To investigate the effect of Ghrelin on cellular proliferation and invasion of colon carcinoma and potential mechanisms. Methods A total of 90 patients diagnosed with colon carcinoma were included in this study, and cancer as well as paracancerous normal tissue was collected. Expression of Ghrelin was measured by Immunohistochemical staining assay; Co -relationship analysis between ghrelin expression and clinical manifestation of patients including survival rate was performed. Expression of Ghrelin in colon carcinoma cell line HT29 and SW480 was genetically inhibited by short hairpin RNA (shRNA).Cellular proliferation, cell cycle as well as apoptosis, migration, and cell invasion were determined by CCK-8 assay, Flow cytometry, Wound heal assay, and Transwell assay, respectively. Expression levels of PI3K, Akt,p-Akt, mTOR, p-mTOR protein were measured by Western blot. Results Expression of Ghrelin in cancer tissue, group with tumor diameter ≥5 cm, and group with tumor under Ⅲ-Ⅳphase was upregulated when compared with that in paracancerous tissue, group with tumor diameter < 5 cm group, group with tumor under metastasis-free phase, respectively (P < 0.05). No correlation between Ghrelin expression and gender, age and tumor grade respectively was observed ( P> 0.05). Overall survival and disease-free survival in group with highly expressed Ghrelin was significantly lower than that in group with lowly expressed Ghrelin (P < 0.05).Proliferation, migration and invasion capacity of HT29 and SW480 cells decreased while ratio of cells in S phase and early apoptosis increased significantly in sh-Ghrelin group when compared with those in NC group and WT group (P < 0.05). In HT29 and SW480 cells expression levels of PI3K, p-Akt, p-mTOR protein were dramatically down-regulated in sh-Ghrelin group when compared with those in NC group and WT group (P <0.05). No statistical difference in the expression of PI3K, Akt, p-Akt, mTOR, p-mTOR protein in NC group and WT group was observed (P > 0.05). Conclusions The expression of Ghrelin is up-regulated in colon carcinoma tissue, which is closely associated with disease prognosis. Beneficial effect of Ghrelin is potentially mediated through inhibition of PI3K-Akt-mTOR signaling pathway.