miRNA-196a-5p调控Smad4表达对胃癌干细胞特性的影响

Regulation of Smad4 by miRNA-196a-5p influences gastric cancer stem cell characteristics

背景:miRNA-196a是新近发现的与乳腺癌、宫颈癌等多种肿瘤相关的microRNAs生物靶分子。研究证实,miRNA-196a-5p在胃癌细胞株和胃癌组织中均表达增加,然而,关于miRNA-196a-5p在胃癌干细胞中的功能和机制研究甚少。目的:分析miRNA-196a-5p在胃癌干细胞侵袭中的作用及机制。方法:流式细胞术分选胃癌BGC-823细胞株中的CD44^+细胞(即胃癌干细胞),qRT-PCR检测miRNA-196a-5p表达,Transwell实验检测细胞侵袭能力;参照Lipofectamine 2000转染试剂说明书的操作步骤,将miRNA-196a-5p inhibitor(即miRNA-196a-5p INH)转染至细胞中;双荧光素酶报告基因实验验证miRNA-196a-5p对Smad4基因的调控作用;Western blotting实验检测上皮-间质转化相关蛋白(E-cadherin、N-cadherin和Vimentin)和Smad4蛋白表达。结果与结论:(1)miRNA-196a-5p在胃癌干细胞中表达增加(P<0.05);(2)抑制miRNA-196a-5p表达后,能够降低胃癌干细胞的侵袭能力(P<0.05),增加E-cadherin蛋白表达(P<0.05),降低N-cadherin和Vimentin蛋白表达(P均<0.05),增加Smad4蛋白表达(P<0.05);(3)结果表明,miRNA-196a-5p通过调控Smad4表达在胃癌干细胞上皮-间质转化和侵袭过程中发挥重要作用,miRNA-196a-5p具有作为胃癌治疗靶标的潜能。

BACKGROUND： miRNA-196a is a recently discovered microRNAs biotarget molecule associated with various tumors such as breast cancer and cervical cancer. Studies have confirmed that miRNA-196a-5p is upregulated in both gastric cancer cell line and gastric cancer tissues. However, little is known about the function and mechanism of miRNA-196a-5p in gastric cancer stem cells.OBJECTIVE： To analyze the function of miRNA-196a-5p in the invasion of gastric cancer stem cells and to investigate the possible mechanism.METHODS： CD44＋ gastric cancer stem cells were sorted from the gastric cancer BGC-823 cell line using flow cytometry. The expression of miRNA-196a-5p was detected by quantitative real-time PCR （qRT-PCR） and the cell invasion was determined by Transwell assay. With reference to the specification of Lipofectamine 2000m kit, miRNA-196a-5p inhibitor （miRNA-196a-5p INH） was transferred into the sorted cells. Regulation of Smad4 gene by the miRNA-196a-5p was validated by dual-luciferase reporter gene assay. Expression of epithelial-mesenchymal transition-associated proteins （E-cadherin, N-cadherin and Vimentin） and Smad4 protein was evaluated by western blot assay.RESULTS AND CONCLUSION： The miRNA-196a-5p was up-regulated in the gastric cancer stem cells （P 〈 0.05）. Inhibiting the miRNA-196a-5p expression could reduce the invasion ability of gastric cancer stem cells （P 〈 0.05）, increase the E-cadherin protein expression （P 〈 0.05）, decrease the N-cadherin and Vimentin protein expression （P 〈 0.05）, and enhance the Smad4 protein expression （P 〈 0.05）. Therefore, miR-196a-5p has a key role in epithelial-mesenchymal transition and invasion by targeting Smad4 in the gastric cancer stem cells. miR-196a-5p may serve as a potential target for gastric cancer therapy.