摘要 目的探究青蒿琥酯对人大肠癌CCL229细胞恶性生物学行为抑制作用的机制。方法采用MTT法检测青蒿琥酯对CCL229细胞活力的影响;通过流式细胞术检测青蒿琥酯对CCL229细胞凋亡的影响;采用Transwell法检测青蒿琥酯对CCL229细胞侵袭能力的影响;采用克隆形成实验检测青蒿琥酯对CCL229细胞集落形成能力的影响;采用Western blotting法检测青蒿琥酯对CCL229细胞内自噬特异性蛋白如自噬效应蛋白(Beclin1)、轻链3-Ⅰ/Ⅱ蛋白(light chain 3-Ⅰ/Ⅱ,LC3-Ⅰ/Ⅱ)、自噬相关蛋白5(autophagy related protein 5,Atg5)、Atg5-Atg12复合物以及上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白如紧密连接蛋白(ZO-1)、上皮钙黏蛋白(epithelial cadherin,E-cadherin)、神经钙黏蛋白(neuronal cadherin,N-cadherin)、锌指转录因子(Slug)和第10号染色体同源缺失性磷酸酶-张力蛋白(phosphatase and tensin homolog deleted on chromosome ten,PTEN)表达的影响;采用qRT-PCR法检测青蒿琥酯对CCL229细胞内mi R-21 m RNA表达的影响;通过双荧光素酶报告基因实验验证miR-21与PTEN的靶向关系;考察过表达或抑制miR-21与PTEN对青蒿琥酯抑制CCL229细胞恶行生物学行为的影响。结果青蒿琥酯显著降低CCL229细胞存活率(P<0.05、0.01、0.001),显著促进CCL229细胞凋亡(P<0.001),显著抑制CCL229细胞侵袭和克隆形成能力(P<0.001),显著上调CCL229细胞内Atg5-Atg12复合物、Atg5、Beclin1、LC3-Ⅰ/Ⅱ、ZO-1、E-cadherin表达水平(P<0.05、0.01),显著下调N-cadherin和Slug蛋白表达水平(P<0.05)。CCL229细胞内miR-21 mRNA高表达(P<0.01),青蒿琥酯显著抑制CCL229细胞内miR-21 mRNA表达水平(P<0.05)。过表达miR-21显著抑制青蒿琥酯对CCL229细胞的促凋亡作用(P<0.001),显著减弱青蒿琥酯对CCL229细胞侵袭和克隆形成能力的抑制作用(P<0.01),显著抑制青蒿琥酯对CCL229细胞Atg5-Atg12复合物、Atg5、Beclin1、LC3-Ⅰ/Ⅱ、ZO-1、E-cadherin表达水平的上调作 Objective To explore the mechanism of inhibitory effect of artesunate on malignant biological behavior of human colorectal cancer CCL229 cells. Methods MTT method was used to detect the effect of artesunate on viability of CCL229 cells;Flow cytometry was used to detect the effect of artesunate on apoptosis of CCL229 cells;Transwell method was used to detect the effect of artesunate on invasion of CCL229 cells;Clone formation experiment was used to detect the effect of artesunate on colony forming ability of CCL229 cells;Western blotting was used to detect the effects of artesunate on expressions of intracellular autophagy-specific proteins such as autophagy effector protein(Beclin1), light chain 3-Ⅰ/Ⅱ protein(LC3-Ⅰ/Ⅱ), autophagy related protein 5(Atg5), Atg5-Atg12 complex, and epithelial-mesenchymal transition(EMT) related proteins such as tight junction protein(ZO-1), epithelial cadherin(E-cadherin), neuronal cadherin(N-cadherin), zinc finger transcription factor(Slug) and phosphatase and tensin homolog deleted on chromosome ten(PTEN) in CCL229 cells;qRT-PCR was used to detect the effect of artesunate on expression of miR-21 mRNA in CCL229 cells;The dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-21 and PTEN;Effect of overexpression or inhibition of mi R-21 and PTEN on biological behavior of artesunate in inhibiting CCL229 cells was investigated. Results Artesunate significantly inhibited the survival rate of CCL229 cells(P < 0.05, 0.01, 0.001), promoted the apoptosis of CCL229 cells(P < 0.001), inhibited CCL229 cell invasion and clone formation ability(P < 0.001), up-regulated the expressions of Atg5-Atg12 complex, Atg5, Beclin1, LC3-Ⅰ/Ⅱ, ZO-1, E-cadherin in CCL229 cells(P < 0.05, 0.01), and significantly down-regulated the expressions of N-cadherin and Slug(P < 0.05). The expression of miR-21 mRNA in CCL229 cells was high(P < 0.01), artesunate significantly inhibited the expression of miR-21 mRNA in CCL229 cells(P < 0.05). Overexpression of miR-21
机构地区 驻马店市中心医院消化内科
出处 《中草药》 CAS CSCD 北大核心 2021年第8期2331-2342,共12页 Chinese Traditional and Herbal Drugs
基金 河南省高等学校重点科研计划项目(20A320085)。
关键词 大肠癌 青蒿琥酯 MIRNA 增殖 侵袭 上皮间质转化 自噬 凋亡 colorectal cancer artesunate miRNA proliferation invasion epithelial-mesenchymal transition autophagy apoptosis
