沉默DNMT3a表达对银屑病样细胞周期进展和细胞增殖的抑制作用及其机制

doi:10.13481/j.1671-587X.20220327

摘要/Abstract

摘要： 目的

探讨沉默DNA甲基化转移酶3a（DNMT3a）表达对银屑病样HaCaT细胞周期进展和细胞增殖的抑制作用，阐明其可能机制。

方法

收集15例寻常性银屑病患者皮损组织（银屑病组）与15名健康者皮肤组织（对照组）。实时荧光定量PCR（RT-qPCR）法检测2组研究对象皮肤组织中DNMT3a mRNA表达水平。siRNA转染技术将沉默DNMT3a表达的si-DNMT3a转染至HaCaT细胞中，RT-qPCR和Western blotting法检测si-DNMT3a的转染效率。采用M5方法诱导HaCaT细胞并建立银屑病样细胞模型，将细胞分为对照组、si-DNMT3a组、si-NC组和HaCaT组。CCK-8法检测各组细胞增殖率，流式细胞术检测不同细胞周期各组细胞百分率，Annexin Ⅴ-FITC实验检测各组细胞凋亡率。MSP实验检测银屑病组和对照组研究对象皮肤组织中LATS1基因启动子区甲基化水平。Western blotting法检测各组细胞中大肿瘤抑制基因1（LATS1）、Yes-相关蛋白1（YAP1）、Cyclin D1、CDK6和cleaved caspase-3蛋白表达水平。

结果

与对照组比较，银屑病组患者皮肤组织中DNMT3a mRNA表达水平明显升高（P<0.01）。与HaCaT组比较，si-NC组HaCaT细胞中DNMT3a mRNA和蛋白表达水平均降低（P<0.05）。与HaCaT组比较，对照组、si-NC组和si-DNMT3a组HaCaT细胞增殖率和S期HaCaT细胞百分率均升高（P<0.05），G₁期细胞百分率和细胞凋亡率均降低（P<0.05）；与对照组和si-NC组比较，si-DNMT3a组HaCaT细胞增殖率和S期HaCaT细胞百分率均降低（P<0.05），细胞凋亡率和G₁期HaCaT细胞百分率升高（P<0.05）。MSP检测，银屑病组患者皮肤组织中LATS1启动子甲基化水平较对照组升高（P<0.01）。Western blotting检测，与HaCaT组比较，对照组、si-NC组和si-DNMT3a组HaCaT细胞中YAP1、Cyclin D1及CDK6蛋白表达水平明显升高（P<0.05），LATS1和cleaved caspase-3蛋白表达水平明显降低（P<0.05）；与对照组和si-NC组比较，si-DNMT3a组HaCaT细胞中YAP1、Cyclin D1和CDK6蛋白表达水平降低（P<0.05），LATS1和cleaved caspase-3蛋白表达水平升高（P<0.05）。

结论

沉默DNMT3a表达可能通过降低LATS1的甲基化水平并下调YAP1的表达，从而抑制银屑病样HaCaT细胞周期进展和细胞增殖，并诱导细胞发生凋亡。

关键词: 银屑病, DNA甲基化转移酶3a, 大肿瘤抑制基因1, Yes-相关蛋白

Abstract: Objective

To investigate the inhibitory effect of silencing the expression of DNA methyltransferase 3A （DNMT3a） on the progression of psoriasis like HaCaT cell cycle and cell proliferation，and to clarify its possible mechanism.

Methods

The skin tissues of 15 patients with psoriasis vulgaris （psoriasis group） and 15 healthy controls （control group） were collected. Real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of DNMT3a mRNA in skin tissue of the subjects in two groups. The Si-DNMT3a silenced by DNMT3a was transfected into the HaCaT cells by siRNA transfection technology. RT-qPCR and Western blotting methods were used to detect the transfection efficiency of si-DNMT3a. The HaCaT cells were induced by M5 method and the psoriasis like cell model was established. The cells were divided into control group， si-DNMT3a group， si-NC group， and HaCaT group. The proliferation rates of the cells in various groups were detected by CCK-8 method， the percentages of the cells at different cell cycles in various groups were detected by flow cytometry， and the apoptotic rates of the cells in various groups were detected by Annexin Ⅴ-FITC test. MSP test was used to detect the methylation levels of LATS1 gene promoter in skin tissue of the subjects in psoriasis group and control group. The expression levels of large tumor suppressor gene 1（LATS1）， Yes associated protein（YAP1）， cyclin D1， CDK6， and cleaved caspase-3 proteins in the HaCaT cells in various groups were detected by Western blotting method.

Results

Compared with control group， the expression level of DNMT3a mRNA in skin tissue of the patients in psoriasis group was significantly increased （P<0.01）. Compared with HaCaT group，the expression levels of DNMT3a mRNA and protein in the HaCaT cells in si-NC group were decreased （P<0.05）. Compared with HaCaT group， the proliferation rates of the HaCaT cells and the percentages of cells at S phase in control group， si -NC group，and si- DNMT3a group were increased （P<0.05）， and the percentages of cells at G₁ phase and the apoptotic rates were significantly decreased （P<0.05）；compared with control group and si-NC group， the proliferation rates of the HaCaT cells and the percentage of cells at S phase in si-DNMT3a group were significantly decreased（P<0.05），and the apoptotic rate and the percentage of cells at G₁ phase were increased （P<0.05）. The MSP results showed that the methylation level of LATS1 promoter in skin tissue of the patients in psoriasis group was higher than that in control group （P<0.01）. The Western blotting results showed that compared with HaCaT group， the expression levels of YAP1， Cyclin D1， and CDK6 proteins in the HaCaT cells in control group， si-NC group，and si-DNMT3a groups were increased （P<0.05）， while the expression levels of LATS1 and cleaved caspase-3 proteins were decreased （P<0.05）； compared with control group and si-NC group， the expression levels of YAP1， cyclin D1， and CDK6 proteins in the HaCaT cells in si- DNMT3a group were decreased （P<0.05）， and the expression levels of LATS1 and cleaved caspase-3 proteins were increased （P<0.05）.

Conclusion

Silencing the DNMT3a expression may down regulate the expression of YAP1 by reducing the methylation level of LATS1， so as to inhibit the progression of psoriasis like HaCaT cell cycle and cell proliferation and induce the apoptosis.

Key words: Psoriasis, DNA methyltransferases 3a, Large tumor suppressor gene 1, Yes-associated protein

中图分类号:

R758.63

潘延斌,苏家光,谭美乐,杨猛,覃文飞,黄榆秀,蒙世豪,黄耀辉,梁坚强,苏雪芳,黄姿婵,李建民. 沉默DNMT3a表达对银屑病样细胞周期进展和细胞增殖的抑制作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(3): 773-782.

Yanbin PAN,Jiaguang SU,Meile TAN,Meng YANG,Wenfei TAN,Yuxiu HUANG,Shihao MENG,Yaohui HUANG,Jianqiang LIANG,Xuefang SU,Zichan HUANG,Jianmin LI. Inhibitory effect of silencing expression of DNMT3a on progression of psoriasis like cell cycle and cell proliferation and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(3): 773-782.

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