中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (12): 904-908.doi: 10.3760/cma.j.issn.0412-4030.2017.12.010

• 论著 • 上一篇    下一篇

抑癌基因DKK3对皮肤恶性黑素瘤细胞增殖和凋亡的影响

李晶    余音    刘林红    黎智    刁庆春    王开振    肖亮    刘素桃   

  1. 400011 重庆市中医院皮肤科（李晶、余音、刘林红、黎智、刁庆春、刘素桃），肾病科（王开振）；湖北省十堰市人民医院骨科（肖亮）
  • 收稿日期:2017-05-03 修回日期:2017-06-10 出版日期:2017-12-15 发布日期:2017-11-30
  • 通讯作者: 刘素桃 E-mail:284112611@qq.com

Effects of tumor suppressor gene Dickkopf-3 on proliferation and apoptosis of cutaneous malignant melanoma cells

Li Jing, Yu Yin, Liu Linhong, Li Zhi, Diao Qingchun, Wang Kaizhen, Xiao Liang, Liu Sutao    

  1. Department of Dermatology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400011, China（Li J, Yu Y, Liu LH, Li Z, Diao QC, Liu ST）; Department of Nephrology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400011, China（Wang KZ）; Department of Orthopedics, Shiyan People′s Hospital, Shiyan 442000, HuBei, China （Xiao L）
  • Received:2017-05-03 Revised:2017-06-10 Online:2017-12-15 Published:2017-11-30
  • Contact: Liu Su-tao E-mail:284112611@qq.com

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摘要： 目的 探讨DKK3 在人皮肤恶性黑素瘤细胞及组织中的表达及其对黑素瘤细胞A375增殖与凋亡的影响。方法 通过反转录（RT）?PCR及实时定量PCR分析DKK3 mRNA在人恶性黑素瘤细胞系HM、A375、WM451、WM35、SK?MEL?1、Hs?695T和MDA?MB?435s及38例原发性黑素瘤、4例转移性黑素瘤和20例色素痣组织中的相对表达。分别转染恶性黑素瘤A375细胞pcDNA3.1（+）?Flag?DKK3 质粒（实验组）和pcDNA3.1（+）?Flag?Vector 质粒（对照组），RT?PCR 验证 DKK3 的过表达；通过CCK8法和平板克隆实验分析 DKK3 对A375细胞增殖的影响，流式细胞仪分析 DKK3对A375细胞凋亡的影响，Western印迹检测A375细胞周期和凋亡相关蛋白的表达。结果 DKK3 mRNA 在WM35细胞系表达下调，HM、A375、WM451、SK?MEL?1和Hs?695T细胞系表达缺失，MDA?MB?435s细胞高表达。与色素痣相比，DKK3 mRNA在人恶性黑素瘤组织表达降低（P < 0.001）。与对照组A375细胞相比 （100%），实验组A375细胞克隆形成效率明显受到抑制（23.22% ± 3.55%）；同时转染后24、48、72 h细胞活性受到明显抑制（均P < 0.05）。流式细胞仪检测发现，与对照组A375细胞（G1期，25.38% ± 2.92%）相比，实验组A375细胞明显阻滞于G1期（48.68% ± 3.92%，P < 0.001），细胞凋亡率明显升高（P < 0.001）。与对照组A375细胞相比，实验组周期凋亡相关蛋白p21、Bax、活化的parp和 活化的Caspase?3表达升高，细胞周期蛋白D1和Bcl2表达降低（均P < 0.001）。结论 DKK3在人皮肤恶性黑素瘤组织中表达下调，可能作为潜在的抑癌基因参与皮肤恶性黑素瘤的进展。

Abstract: Li Jing, Yu Yin, Liu Linhong, Li Zhi, Diao Qingchun, Wang Kaizhen, Xiao Liang, Liu Sutao Department of Dermatology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400011, China（Li J, Yu Y, Liu LH, Li Z, Diao QC, Liu ST）; Department of Nephrology, Chongqing Traditional Chinese Medicine Hospital, Chongqing 400011, China（Wang KZ）; Department of Orthopedics, Shiyan People′s Hospital, Shiyan 442000, HuBei, China （Xiao L） Corresponding author: Liu Sutao, Email: 284112611@qq.com 【Abstract】 Objective To investigate the of Dickkopf-3 （DKK3） in human malignant melanoma cell lines and tissues, and to evaluate effects of DKK3 on the proliferation and apoptosis of malignant melanoma cell line A375. Methods Reverse transcription PCR （RT-PCR） and real-time fluores-cence-based quantitative PCR （qRT-PCR） were performed to measure the mRNA of DKK3 in human malignant melanoma cell lines HM, A375, WM451, WM35, SK-MEL-1, Hs-695T and MDA-MB-435s, as well as in 38 primary melanoma tissues, 4 metastatic melanoma tissues and 20 pigmented nevus tissues. Cultured malignant melanoma A375 cells were divided into 2 groups to be transfected with pcDNA3.1（+）-Flag-DKK3 （experiment group） and pcDNA3.1（+）-Flag-Vector （control group） respectively. The over of DKK3 was verified by RT-PCR. Cell counting kit-8 （CCK8） assay and plate colony formation assay were performed to evaluate the proliferative activity of A375 cells, flow cytometry was conducted to detect apoptosis of A375 cells, and Western blot analysis was performed to determine the of cell cycle- and cell apoptosis-related proteins. Results The mRNA of DKK3 was downregulated in WM35 cells, absent in HM cells, A375 cells, WM451 cells, SK-MEL-1 cells and Hs-695T cells, but upregulated in MDA-MB-435s cells. Compared with pigmented nevus tissues, the mRNA of DKK3 was significantly decreased in malignant melanoma tissues （P < 0.001）. Compared with the control group （100%）, cell colony formation was markedly suppressed in the experiment group （23.22% ± 3.55%）, and the proliferative activity of A375 cells was also significantly inhibited in the experiment group 24, 48, 72 hours after the transfection （all P < 0.05）. Flow cytometry showed that compared with the control group, A375 cells were significantly arrested in G1 phase （48.68% ± 3.92% vs. 25.38% ± 2.92%, P < 0.001）, and the apoptosis rate of A375 cells was significantly increased in the experiment group （P < 0.001）. Compared with the control group, the experiment group showed significantly higher of p21, Bax, cleaved-parp and cleaved-casp3, but significantly lower of cyclin D1 and Bcl2 （all P < 0.001）. Conclusion DKK3 is downregulated in human malignant melanoma tissues, so it may serve as a potential tumor suppressor gene involved in the development of cutaneous malignant melanoma.