中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (10): 858-863.doi: 10.35541/cjd.20220030

• 论著 • 上一篇    下一篇

阿扎胞苷对黑素瘤A375细胞HOXA9基因表达及生物学行为的影响

李婷婷    赵娟    王鹏    刘冬梅    康晓静   

  1. 新疆维吾尔自治区人民医院皮肤性病科  新疆皮肤病临床医学研究中心  新疆皮肤病研究重点实验室（XJYS1707），乌鲁木齐  830001
  • 收稿日期:2022-01-14 修回日期:2022-06-29 发布日期:2022-10-08
  • 通讯作者: 康晓静 E-mail:kangxiaojing163@163.com
  • 基金资助:
    新疆维吾尔自治区自然科学基金（2021D01C201）；新疆维吾尔自治区人民医院院内项目（20200104）

Effect of azacitidine on HOXA9 gene expression and biological behaviors of A375 melanoma cells

Li Tingting, Zhao Juan, Wang Peng, Liu Dongmei, Kang Xiaojing   

  1. Department of Dermatology and Venereology, People′s Hospital of Xinjiang Uygur Autonomous Region, Xinjiang Clinical Research Center for Dermatologic Diseases, Xinjiang Key Laboratory of Dermatology Research（XJYS1707）, Urumqi 830001, China
  • Received:2022-01-14 Revised:2022-06-29 Published:2022-10-08
  • Contact: Kang Xiaojing E-mail:kangxiaojing163@163.com
  • Supported by:
    Natural Science Foundation of Xinjiang Uygur Autonomous Region（2021D01C201）; Intramural Project of People′s Hospital of Xinjiang Uygur Autonomous Region （20200104）

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摘要： 【摘要】 目的 研究甲基转移酶抑制剂阿扎胞苷（5-azaC）对黑素瘤A375细胞中同源框A9（HOXA9）基因表达及细胞增殖、侵袭和迁移能力的影响。方法 1、5、10、20 μmol/L 5-azaC分别作用于体外培养的A375细胞，以未经药物干预的常规培养A375细胞作为对照组，甲基化特异性PCR检测不同浓度5-azaC处理后HOXA9基因启动子区甲基化状态，筛选5-azaC最佳浓度进行后续实验。细胞计数试剂盒（CCK8）检测5-azaC对A375细胞增殖能力的影响，Transwell及细胞划痕实验分析5-azaC对A375细胞侵袭及迁移能力的影响，实时荧光定量PCR及Western印迹检测5-azaC作用后A375细胞中HOXA9 mRNA及蛋白表达水平。两组间比较采用独立样本t检验。结果 对照组A375细胞中HOXA9基因启动子区表现为甲基化状态，经5-azaC处理后，A375细胞中HOXA9基因启动子区甲基化与非甲基化状态并存，且随5-azaC处理浓度的增加，HOXA9基因去甲基化程度越高，因此选择20 μmol/L 5-azaC浓度处理A375细胞72 h作为5-azaC处理组，用于后续实验。与对照组相比，5-azaC处理组A375细胞增殖能力显著降低（72.46% ± 2.19%比100%，t = 28.09，P < 0.001），侵袭细胞数量显著减少［（242.70 ± 29.19）个比（466.00 ± 22.65）个，t = 10.47，P < 0.001］，迁移能力减弱（27.56% ± 2.74% 比35.69% ± 2.50%，t = 3.79，P = 0.019），HOXA9 mRNA表达量升高（1.73 ± 0.28 比1.01 ± 0.15，t = 3.93，P = 0.017），HOXA9蛋白表达量增多（0.62 ± 0.03比0.50 ± 0.01，t = 3.82，P = 0.019）。结论 5-azaC能够降低黑素瘤细胞株A375的增殖、侵袭、迁移能力，且该过程可能机制之一是5-azaC反转HOXA9基因启动子区甲基化状态，激活基因表达，从而参与调控黑素瘤细胞生物学行为。

关键词: 黑色素瘤, DNA甲基化, 去甲基化, 同源框A9, 阿扎胞苷, A375细胞

Abstract: 【Abstract】 Objective To investigate the effect of the methyltransferase inhibitor azacitidine （5-azaC） on the expression of homeobox A9 （HOXA9） gene in, as well as proliferation, invasion and migration of A375 cells. Methods In vitro cultured A375 cells were treated with 5-azaC at various concentrations of 1, 5, 10 and 20 μmol/L, while routinely cultured A375 cells receiving no drug intervention served as control group. Methylation-specific PCR was performed to analyze methylation status of the HOXA9 gene promoter region after the treatment with different concentrations of 5-azaC, in order to screen the optimal concentration of 5-azaC for following experiments. Cell counting kit-8 （CCK8） assay was conducted to evaluate the proliferation of A375 cells, Transwell and wound healing assays were performed to estimate the invasion and migration of A375 cells, and real-time fluorescence-based quantitative PCR （qRT-PCR） and Western blot analysis were conducted to determine the mRNA and protein expression of HOXA9 in A375 cells after 5-azaC treatment. Two-independent-sample t test was used for comparisons between two groups. Results Methylation was observed in the HOXA9 gene promoter region in A375 cells in the control group. After 5-azaC treatment, methylated and unmethylated states coexisted in the HOXA9 gene promoter region in A375 cells, and the higher the concentration of 5-azaC, the higher the degree of demethylation of the HOXA9 gene. Therefore, 20 μmol/L 5-azaC was selected to treat A375 cells for 72 hours, which served as 5-azaC treatment group in subsequent experiments. Compared with the control group, the 5-azaC treatment group showed significantly decreased cellular proliferative ability（72.46% ± 2.19% vs. 100%, t = 28.09, P < 0.001）, significantly decreased number of invasive cells （242.70 ± 29.19 vs. 466.00 ± 22.65, t = 10.47, P < 0.001）, significantly decreased migratory ability （27.56% ± 2.74% vs. 35.69% ± 2.50%, t = 3.79, P = 0.019）, significantly increased HOXA9 mRNA expression （1.73 ± 0.28 vs. 1.01 ± 0.15, t = 3.93, P = 0.017）, and significantly increased HOXA9 protein expression （0.62 ± 0.03 vs. 0.50 ± 0.01, t = 3.82, P = 0.019）. Conclusion 5-azaC can inhibit the proliferative, invasive and migratory ability of A375 melanoma cells, and one of the possible mechanisms underlying this process may be that 5-azaC reverses the methylation in the HOXA9 gene promoter region, activates HOXA9 gene expression, and participates in the regulation of biological behaviors of melanoma cells.

Key words: Melanoma, DNA methylation, Hypomethylation, HOXA9, Azacitidine, A375 cells