[关键词]
[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miR-21 mRNA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表达水平的上调作用(P<0.05、0.01),显著抑制青蒿琥酯对CCL229细胞N-cadherin和Slug蛋白表达水平的下调作用(P<0.01);抑制miR-21则作用相反。PTEN是miR-21的下游靶基因,过表达miR-21显著抑制CCL229细胞内PTEN蛋白表达水平(P<0.01),抑制miR-21显著上调PTEN蛋白表达水平(P<0.01),过表达miR-21显著抑制野生型PTEN(WT-PTEN)质粒的荧光素酶活性(P<0.01)。过表达PTEN与抑制miR-21表达对CCL229细胞作用一致,同时过表达miR-21和PTEN不会影响青蒿琥酯对CCL229细胞恶性生物学行为的抑制作用。结论 青蒿琥酯能够通过调控miR-21和PTEN表达诱导CCL229细胞凋亡和自噬,并抑制细胞增殖和侵袭。
[Key word]
[Abstract]
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 miR-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 significantly inhibited the pro-apoptotic effect of artesunate on CCL229 cells (P < 0.001), weakened the inhibitory effect of artesunate on CCL229 cell invasion and clonal formation (P < 0.01), inhibited the up-regulation of artesunate on expressions of Atg5-Atg12 complex, Atg5, Beclin1, LC3-Ⅰ/Ⅱ, ZO-1, and E-cadherin in CCL229 cells (P < 0.05, 0.01), and significantly inhibited the down-regulation of artesunate on expressions of N-cadherin and Slug protein in CCL229 cells (P < 0.01); The inhibition of miR-21 expression had the opposite effect. PTEN was the downstream target gene of miR-21. Overexpression of miR-21 significantly inhibited the expression of PTEN in CCL229 cells (P < 0.01), the inhibited expression of miR-21 significantly upregulated the expression of PTEN (P < 0.01), overexpression of miR-21 significantly inhibited the luciferase activity of wild-type PTEN (WT-PTEN) plasmid (P < 0.01). Overexpression of PTEN had the same effect with inhibition of miR-21 expression on CCL229 cells, while overexpression of miR-21 and PTEN did not affect the inhibitory effect of artesunate on malignant biological behavior of CCL229 cells. Conclusion Artesunate can induce apoptosis, autophagy and inhibit cell proliferation and invasion of CCL229 cells by regulating the expression of miR-21 and PTEN.
[中图分类号]
R285.5
[基金项目]
河南省高等学校重点科研计划项目(20A320085)