Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation.

IF 1.6 4区 医学 Q4 BIOCHEMICAL RESEARCH METHODS
Yi Ding, Shi-Qi Ma, Min Li, Long Chen, Shu-Mei Feng
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引用次数: 0

Abstract

Background: Normal keratinocyte differentiation is important for epidermal wound healing. ΔNp63 is a major gene regulating epidermal formation and differentiation. We identified miRNAs targeting ΔNp63 and studied the association between the miRNAs and DNA methylation in keratinocyte differentiation.

Aims: This study aimed to explore the mechanisms regulating ΔNp63 expression during keratinocyte differentiation.

Methods: Bioinformatics analysis was performed to screen the miRNAs targeting ΔNp63 and uncover potential pathway mechanisms. The differentiation model of keratinocytes was established by CaCl2 treatment. Furthermore, the effects of the miRNA transgenic technique on Δ Np63 and keratinocyte differentiation were studied. In addition, the RNA FISH experiment was conducted to detect the location of different miRNAs. A double luciferase reporter experiment was carried out to verify the potential bindings between the miRNAs and ΔNp63. A rescue experiment was also performed to assess the effects of different miRNAs targeting ΔNp63 on keratinocyte differentiation. We analyzed the methylation patterns of the promoter regions of miRNAs using keratinocytes treated with 5-Aza-2'-deoxycytidine. Finally, we designed a methylation rescue experiment to verify the effects of miRNA promoter methylation on keratinocyte differentiation.

Results: Bioinformatics analysis showed that the miR-125b-5p and miR-199b-5p binding to the ΔNp63 3'UTR region decreased during skin development. Moreover, such binding may downregulate the PI3K/AKT/mTOR pathway. The expression levels of CK10, Inv, TG1, ΔNp63, and PI3K/AKT/mTOR were all significantly increased during keratinocyte differentiation. Both miR- 125b-5p and miR-199b-5p were localized in the cytoplasm. Luciferase assay results showed that both miR-125b-5p and miR-199b-5p can bind to the 3'UTR region of ΔNp63. Overexpression of ΔNp63 can significantly counteract the inhibitory effect of miRNA mimics on keratinocyte differentiation. Moreover, the promoter regions of both miR-125b-5p and miR-199b-5p had methylation sites, and the methylation levels in those promoter regions were significantly increased during keratinocyte differentiation. 5-Aza-2'-Deoxycytidine treatment increased the expression of miR- 125b-5p and miR-199b-5p and inhibited the differentiation of keratinocytes. Finally, miRNA inhibitors reversed the inhibitory effects of 5-Aza-2'-deoxycytidine on keratinocyte differentiation.

Conclusion: Promoter hypermethylation in miR-125b-5p and miR-199b-5p seem to promote keratinocyte differentiation via upregulation of ΔNp63 expression and the activation of the PI3K/AKT/mTOR pathway. The findings of this study are helpful for future research on skin development and clinical scar-free healing.

Promoter Hypermethylation Downregulates MiR-125b-5p and MiR-199b-5p Targeting of ΔNp63, Resulting in PI3K/AKT/mTOR Pathway Activation and Keratinocyte Differentiation.
背景:正常的角质形成细胞分化对表皮伤口愈合非常重要。ΔNp63是调节表皮形成和分化的主要基因。我们发现了靶向ΔNp63的miRNAs,并研究了miRNAs与角质形成细胞分化过程中DNA甲基化之间的关联:方法:通过生物信息学分析筛选靶向ΔNp63的miRNAs,并揭示潜在的通路机制。通过 CaCl2 处理建立了角质形成细胞的分化模型。此外,还研究了 miRNA 转基因技术对 Δ Np63 和角质形成细胞分化的影响。此外,还进行了 RNA FISH 实验,以检测不同 miRNA 的位置。还进行了双荧光素酶报告实验,以验证 miRNA 与 ΔNp63 之间的潜在结合。我们还进行了一项拯救实验,以评估靶向ΔNp63的不同miRNA对角质形成细胞分化的影响。我们用 5-Aza-2'-deoxycytidine 处理的角质形成细胞分析了 miRNA 启动子区域的甲基化模式。最后,我们设计了一个甲基化拯救实验来验证 miRNA 启动子甲基化对角质形成细胞分化的影响:生物信息学分析表明,在皮肤发育过程中,miR-125b-5p 和 miR-199b-5p 与 ΔNp63 3'UTR 区域的结合减少。此外,这种结合可能会下调 PI3K/AKT/mTOR 通路。在角朊细胞分化过程中,CK10、Inv、TG1、ΔNp63 和 PI3K/AKT/mTOR 的表达水平都显著增加。miR- 125b-5p 和 miR-199b-5p 都定位于细胞质中。荧光素酶测定结果显示,miR-125b-5p 和 miR-199b-5p 都能与ΔNp63 的 3'UTR 区域结合。过量表达ΔNp63能显著抵消miRNA模拟物对角质形成细胞分化的抑制作用。此外,miR-125b-5p和miR-199b-5p的启动子区域都有甲基化位点,并且这些启动子区域的甲基化水平在角朊细胞分化过程中显著增加。5-Aza-2'-Deoxycytidine 处理增加了 miR-125b-5p 和 miR-199b-5p 的表达,抑制了角质形成细胞的分化。最后,miRNA抑制剂逆转了5-氮杂-2'-脱氧胞苷对角质形成细胞分化的抑制作用:结论:miR-125b-5p 和 miR-199b-5p 的启动子高甲基化似乎能通过上调 ΔNp63 表达和激活 PI3K/AKT/mTOR 通路促进角质形成细胞的分化。本研究的发现有助于今后对皮肤发育和临床无疤痕愈合的研究。
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来源期刊
CiteScore
3.10
自引率
5.60%
发文量
327
审稿时长
7.5 months
期刊介绍: Combinatorial Chemistry & High Throughput Screening (CCHTS) publishes full length original research articles and reviews/mini-reviews dealing with various topics related to chemical biology (High Throughput Screening, Combinatorial Chemistry, Chemoinformatics, Laboratory Automation and Compound management) in advancing drug discovery research. Original research articles and reviews in the following areas are of special interest to the readers of this journal: Target identification and validation Assay design, development, miniaturization and comparison High throughput/high content/in silico screening and associated technologies Label-free detection technologies and applications Stem cell technologies Biomarkers ADMET/PK/PD methodologies and screening Probe discovery and development, hit to lead optimization Combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) Chemical library design and chemical diversity Chemo/bio-informatics, data mining Compound management Pharmacognosy Natural Products Research (Chemistry, Biology and Pharmacology of Natural Products) Natural Product Analytical Studies Bipharmaceutical studies of Natural products Drug repurposing Data management and statistical analysis Laboratory automation, robotics, microfluidics, signal detection technologies Current & Future Institutional Research Profile Technology transfer, legal and licensing issues Patents.
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