莱菔硫烷下调miR-200c启动子甲基化水平抑制肺腺癌干细胞的干性特征

梁广霞; 覃喜团; 谢维; 谢有科

doi:10.3971/j.issn.1000-8578.2021.21.0375

莱菔硫烷下调miR-200c启动子甲基化水平抑制肺腺癌干细胞的干性特征

Sulforaphane Suppressed Stemness Characters of Lung Adenocarcinoma Stem Cells by Down-regulating Methylation of miR-200c Promoter

摘要

摘要:
目的探讨莱菔硫烷（SFN）对肺癌干细胞增殖与自我更新的影响及其调控机制。
方法 MTT法检测SFN对肺腺癌细胞株H460和A549细胞增殖的影响；肿瘤球形成实验、蛋白印迹法等检测SFN处理前后肺腺癌细胞肿瘤球形成能力、干性相关基因（如β-catenin、Klf4、c-myc）表达水平等；NGS测序法分析SFN对肿瘤细胞miRNAs表达谱的影响；qRT-PCR验证SFN对重点miRNAs转录水平的改变；蛋白印迹法检测SFN对肿瘤细胞DNA甲基化转移酶（DNMTs）表达的影响；构建miR-200c启动子-GFP质粒，细胞免疫荧光、甲基化PCR法和DNA测序法检测SFN对肿瘤球和miRNA启动子甲基化水平的影响。
结果 SFN（5.0 μmol/L）处理肺腺癌细胞肿瘤球后miRNAs表达谱发生显著变化，其中miRNA-200c增高最为明显。与对照组比较，SFN-S组H460和A549细胞β-catenin、Klf4、c-myc和Vimentin等蛋白表达下降，DNMT1和DNMT3a蛋白表达水平也明显降低。与对照组比较，SFN-S组稳定表达pEGFP-R200c质粒的H460和A549细胞肿瘤球直径显著减少，而肿瘤球荧光强度增加，GFP蛋白表达上调。上述表达pEGFP-R200c质粒细胞株中miR-200c启动子区域存在9个CpG丰富区域位点，其甲基化水平在对照组、SFN-S组和5-Aza-dC组分别为88.9%、44.4%、38.8%。
结论 SFN可能经由表观遗传学水平下调miR-200c启动子甲基化水平，促进miR-200c的转录，从而调控肺癌干细胞的干性相关基因表达。

Abstract:
Objective To investigate the effect of sulforaphane (SFN) on the proliferation and self-renewal of lung cancer stem cells and its regulatory mechanism.
Methods MTT method was used to detect the effect of SFN on the proliferation of lung adenocarcinoma cell lines H460 and A549; tumor sphere formation experiment was used to detect the ability of tumor sphere formation; Western blot was applied to explore the expression of stemness-related proteins (such as β-catenin, Klf4, c-myc) in lung adenocarcinoma cells before and after SFN treatment; NGS sequencing was used to analyze the effect of SFN on the expression profile of tumor cell miRNAs. qRT-PCR verified the changes in the transcription level of key miRNAs by SFN. Western blot was used to detect the effect of SFN on the expression of DNMTs in tumor cells. We constructed miR-200c promoter-GFP plasmid, and applied IF, methylation PCR and DNA sequencing methods to detect the effect of SFN on the methylation level of tumor spheres and miRNA promoter.
Results The miRNAs expression profile of lung adenocarcinoma tumor spheres changed significantly after SFN (5.0μmol/L) treatment, and miRNA-200c increased the most. Compared with the control group, the expression of β-catenin, Klf4, c-myc and Vimentin genes in H460 and A549 cells of SFN-S group decreased, and the protein expression levels of DNMT1 and DNMT3a were also significantly decreased. Compared with the control group, H460 and A549 cells stably expressing pEGFP-R200c plasmid in SFN-S group significantly reduced tumor sphere diameter, while tumor sphere fluorescence intensity increased, and GFP protein expression was up-regulated. There were 9 CpG-rich regions in the miR-200c promoter region in the above-mentioned pEGFP-R200c plasmid cell line, and the methylation levels were 88.9%, 44.4% and 38.8% in the control group, SFN-S group and 5-Aza-dC group, respectively.
Conclusion SFN may downregulate the expression of stem-related genes in lung cancer stem cells by epigenetically decreasing the methylation level of miR-200c promoter and promoting the transcription of miR-200c.

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