Upregulation of miR-133a-3p enhances Bufothionine-induced gastric cancer cell death by modulating IGF1R/PI3K/Akt signal pathway mediated ER stress
Upregulation of miR-133a-3p enhances Bufothionine-induced gastric cancer cell death by modulating IGF1R/PI3K/Akt signal pathway mediated ER stress
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DOI：
10.1016/j.lfs.2020.118180
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发表时间：
2020-10-15
期刊：
影响因子：
6.1
通讯作者：
Hu, Xiu-Mei
中科院分区：
文献类型：
--
作者：
Hu, Zhi-Hao;Wang, Guo-Jun;Hu, Xiu-Mei
目的：材料与方法：采用CCK-8法和集落形成法检测细胞增殖，并与对照组比较。流式细胞术（FCM）和TUNEL法检测细胞凋亡率。用DCFH-DA探针测定细胞内ROS。使用qRT-PCR测定miRNA水平。进行蛋白质印迹以探测蛋白质。采用双荧光素酶报告基因系统验证miR-133 a-3 p和IGF 1 R mRNA 3 'UTR区域的结合位点。免疫组化法检测肿瘤组织中Ki-67的表达，结果表明：蟾蜍噻呋能抑制胃癌细胞的存活，触发内质网应激，促进ROS的产生，内质网应激抑制剂Salburinal（Sal）和ROS清除剂NAC（NAC）均能阻断蟾蜍噻呋诱导的胃癌细胞死亡。此外，蟾蜍噻呋菌素上调miR-133 a-3 p，蟾蜍噻呋菌素诱导的细胞死亡被miR-133 a-3 p过表达增强，而被miR-133 a-3 p敲低减轻。此外，miR-133 a-3 p通过海绵状的IGF 1 R来灭活PI 3 K/Akt信号通路，并且蟾蜍噻托溴铵通过上调miR-133 a-3 p来抑制胰岛素样生长因子1受体（IGF 1 R）和灭活PI 3 K/Akt级联。值得注意的是，过表达miR-133 a-3 p对蟾蜍硫氨酸诱导的GC细胞死亡的促进作用被过表达IGF 1 R消除，并被PI 3 K/Akt级联抑制剂（LY 294002）加重。
Aims: Bufothionine had been used for gastric cancer (GC) treatment, and this study managed to uncover the underlying mechanisms.Materials and methods: Cell proliferation was determined by CCK-8 assay and colony formation assay. Flow cytometry (FCM) and TUNEL assay were used to measure cell apoptosis ratio. Intracellular ROS was measured by DCFH-DA probes. qRT-PCR was used to determine miRNAs levels. Western Blot was performed to probe proteins. Dual-luciferase reporter gene system was employed to validate the binding sites of miR-133a-3p and 3'UTR regions of IGF1R mRNA. Immunohistochemistry (IHC) was used to determine the expressions of Ki-67 in mice tumor tissues.Key findings: Bufothionine inhibited cell viability, triggered ER stress and promoted ROS production in GC cells, and both ER stress inhibitor Salburinal (Sal) and ROS scavenger (NAC) abrogated Bufothionine induced GC cell death. Besides, miR-133a-3p was upregulated by Bufothionine, and Bufothionine-induced cell death was enhanced by miR-133a-3p overexpression while alleviated by miR-133a-3p knockdown. Furthermore, miR-133a-3p inactivated PI3K/Akt signal pathway by sponging IGF1R, and Bufothionine inhibited insulin-like growth factor 1 receptor (IGF1R) and inactivated PI3K/Akt cascade by upregulating miR-133a-3p. Notably, the promoting effects of overexpressed miR-133a-3p on Bufothionine-induced GC cell death were abrogated by over-expressing IGF1R, and aggravated by the PI3K/Akt cascade inhibitor (LY294002).Significance: Bufothionine promoted GC cell death by triggering miR-133a-3p/IGF1R/PI3K/Akt axis mediated ER stress and ROS production.