摘要
目的 :研究miR-145对胃癌耐顺铂细胞株SGC7901/DDP顺铂耐药的影响。方法 :应用QRT-PCR检测miR-145在胃癌组织、细胞中的表达水平;MTT法和细胞克隆形成实验检测细胞活性与增殖能力;荧光素酶实验验证miR-145的靶基因;Western blot、免疫组化和免疫荧光实验检测相关蛋白表达;流式细胞术检测耐药细胞对顺铂诱导凋亡的影响。结果 :miR-145在胃癌组织、各种胃癌细胞株中呈低表达;在胃癌耐顺铂细胞株SGC7901/DDP中,miR-145呈低表达,IGF1R与IRS1呈高表达;上调miR-145增强SGC7901/DDP细胞对顺铂的敏感性;荧光素酶报告实验证实IGF1R与IRS1为miR-145的靶基因;上调miR-145显著降低靶蛋白表达,抑制SGC7901/DDP细胞增殖,促进顺铂诱导的凋亡。结论 :上调miR-145通过靶向IGF1R与IRS1逆转胃癌细胞对顺铂的耐药性。
Objective:To investigate the possible role of miR-145 in the formation of cisplatin resistance in human gastric cancer cell line. Methods:Expression of miR-145 was assayed by quantitative real-time PCR. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays were used to detect cell viability and the drug-resistance phenotype changes of?cancer? cells associated with up-regulation or down-regulation of miR-145. Dual-luciferase activity assay was used to testify the target genes of miR-145. Protein expressions were measured by western blot,immunohistochemistry or Immunofluorescence staining. Flow cytometry was used to detect CDDP induced apoptosis. Results:We found that miR-145 was significantly down-regulated in both gastric cancer tissues and various gastric cancer cell lines. In addition,it was down-regulated in cisplatin-resistant gastric cancer cell line SGC7901 / cisplatin(DDP) and the down-regulation of miR-145 was concurrent with the up-regulation of IGF1 R and IRS1,compared with the parental SGC7901 cell line,respectively. In vitro drug sensitivity assay demonstrated that over-expression of miR-145 sensitized SGC7901 / DDP cells to cisplatin. The luciferase activity of the above proteins 3'-untranslated region-based reporters constructed respectively in SGC7901 / DDP cells suggested that IGF1 R and IRS1 were the direct target genes of miR-145. Enforced miR-145 expression reduced its target proteins level,inhibited SGC7901 / DDP cells proliferation and enhanced SGC7901 / DDP cells to DDP-induced apoptosis. Conclusion:Our findings suggested that hsa-miR-145 could modulate cisplatin resistance of human gastric cancer cell line at least in part by targeting IGF1 R and IRS1.
出处
《南京医科大学学报(自然科学版)》
CAS
CSCD
北大核心
2016年第2期144-150,共7页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(81171908
81201705)