[关键词]
[摘要]
目的 研究紫草素对人骨肉瘤U-2 OS细胞存活的抑制和对内质网应激蛋白的调控机制。方法 采用CCK-8法检测0.156 25~4 μmol/L紫草素对人骨肉瘤U-2 OS细胞抑制率;取对数生长期U-2 OS细胞,分别给予0~4μmol/L紫草素诱导U-2 OS细胞凋亡,24 h后用流式细胞术检测细胞凋亡率;通过Western blotting检测紫草素对内质网应激蛋白ATF6、IRE1α、ATF4、GRP78的表达调控作用;将混有质粒或小干扰RNA的Lipofectamine 2000试剂处理U-2 OS细胞6 h换液,加入紫草素处理,检测ATF4或GRP78对紫草素处理的细胞活力的影响;将ATF4或空白质粒(3 μg)、GRP78报告质粒(3 μg)和海肾内参质粒(4 μg)均匀混合,用Lipofectamine 2000试剂对人骨肉瘤U-2 OS细胞进行转染,6 h换液,加入0.8 μmol/L紫草素继续孵育24 h,实验结果用各孔海肾荧光做参比进行校正,检测紫草素对ATF4介导的GRP78转录的调控作用。结果 紫草素显著抑制人骨肉瘤细胞的活力并能浓度相关性促进细胞凋亡;Western blotting结果显示,紫草素能特异性增加ATF4和GRP78蛋白的表达;过表达ATF4能显著抑制U-2 OS细胞活力(P<0.01);敲低ATF4能部分逆转紫草素对细胞活力的抑制(P<0.01);过表达GRP78并不影响紫草素对细胞存活的抑制效果,但敲低GRP78可以显著增加紫草素对U-2 OS细胞活力的抑制(P<0.01);报告基因结果显示紫草素能增加ATF4对GRP78基因的转录调控。结论 紫草素能显著抑制人骨肉瘤U-2 OS细胞活力,诱导内质网应激蛋白的表达,敲低GRP78能进一步增加紫草素的细胞毒性。
[Key word]
[Abstract]
Objective To study the inhibition of shikonin on osteosarcoma U-2 OS cells and regulate the levels of endoplasmic reticulum stress protein in U-2 OS cells. Methods CCK-8 assay was used to detect U-2 OS cell viability treated with shikonin (0.156 25-4 μmol/L). Flow cytometry analysis was used to measure cell apoptosis induced with shikonin 0-4 μmol/L during 24 h. The levels of endoplasmic reticulum stress proteins ATF6, IRE1α, ATF4, and GRP78 stimulated with shikonin were determined by Western blotting method. The U-2OS cells were treated with Lipofectamine 2000 reagent mixed with plasmid or small interfering RNA for 6 h. Shikonin was added to treat U-2OS cells to detect the effect of ATF4 or GRP78 on the cell viability of shikonin-treated cells. ATF4 or blank plasmid (3 μg), GRP78 reporter plasmid (3 μg) and sea kidney reference plasmid (4 μg) were homogeneously mixed, and human osteosarcoma U-2OS cells were transfected with Lipofectamine 2000 reagent. After dyeing for 6 h, adding 0.8 μmol/L shikonin to incubate for 24 h. The results were calibrated with fluorescence of each porous sea kidney as a reference to detect the regulatory effect of shikonin on ATF4-mediated GRP78 transcription. Results Shikonin showed inhibitory effect on U-2 OS cells activities, and induced cell apoptosis in a concentration-dependent manner in U-2 OS cells. Western blotting results showed that ATF4 and GRP78 proteins levels were markedly increase treated with shikonin. Overexpression of ATF4 could significantly inhibit the viability of U-2OS cells (P<0.01). Knockdown of ATF4 could partially reverse the inhibition of shikonin on cell viability (P<0.01). Overexpression of GRP78 did not affect the inhibition of shikonin on cell survival, but knockdown of GRP78 could significantly increase inhibitory effects of shikonin on U-2OS cells. Reporter gene results showed that shikonin could increase transcriptional regulation of GRP78 gene by ATF4. Conclusion Shikonin can significantly inhibit the viability of osteosarcoma U-2 OS cells, induce the expression of endoplasmic reticulum stress protein, and further increase the cytotoxicity of shikonin by knockdown of GRP78.
[中图分类号]
[基金项目]
江苏省中医药管理局科技项目(YB2017046)