中华皮肤科杂志 ›› 2022, Vol. 55 ›› Issue (2): 102-109.doi: 10.35541/cjd.20210363

• 论著 • 上一篇    下一篇

溶血磷脂酸受体6在蕈样肉芽肿向大细胞转变中的意义及其对皮肤T细胞淋巴瘤细胞增殖和凋亡的影响

林羽洁    刘凤洁    高玉梅    刘向君    许卜方    李映依    赖盼    陈卓婧    孙婧茹    涂平    汪旸   

  1. 北京大学第一医院皮肤性病科  北京市皮肤病分子诊断重点实验室  国家皮肤与免疫疾病临床医学研究中心，北京  100034
  • 收稿日期:2021-05-06 修回日期:2021-11-25 发布日期:2022-01-27
  • 通讯作者: 汪旸 E-mail:yangwang_dr@bjmu.edu.cn

Significance of lysophosphatidic acid receptor 6 in the large-cell transformation of mycosis fungoides and its effect on the proliferation and apoptosis of cutaneous T-cell lymphoma cells

Lin Yuchieh, Liu Fengjie, Gao Yumei, Liu Xiangjun, Xu Bufang, Li Yingyi, Lai Pan, Chen Zhuojing, Sun Jingru, Tu Ping, Wang Yang   

  1. Department of Dermatology and Venereology, Peking University First Hospital, Beijing Key Laboratory of Molecular Diagnosis on Dermatoses, National Clinical Research Center for Skin and Immune Diseases, Beijing 100034, China
  • Received:2021-05-06 Revised:2021-11-25 Published:2022-01-27
  • Contact: Wang Yang E-mail:yangwang_dr@bjmu.edu.cn

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摘要： 【摘要】 目的 探讨皮肤T细胞淋巴瘤（CTCL）蕈样肉芽肿（MF）患者中溶血磷脂酸受体6（LPAR6）的表达水平及其对CTCL疾病发展及预后的影响与可能机制。方法 收集北京大学第一医院皮肤性病科2011—2020年间确诊的110例MF患者，包括49例发现队列的24例大细胞转变（LCT）及25例非大细胞转变（NLCT），61例验证队列的24例LCT及37例NLCT。提取患者组织总RNA分别采用RNA测序及RT-PCR检测LPAR6的表达，比较LPAR6在LCT与NLCT患者间的表达差异及对生存预后的影响。构建2种CTCL细胞系MyLa、Sz4过表达LPAR6细胞模型，以正常表达细胞为对照，检测过表达LPAR6对MyLa、Sz4细胞增殖活力的影响；通过加入LPAR6相关配体溶血磷脂酸（LPA）、2S-OMPT、三磷酸腺苷（ATP）、腺苷（ADO），采用细胞增殖实验检测试剂盒及流式细胞仪检测激活LPAR6对过表达LPAR6 MyLa、Sz4细胞增殖活力与凋亡的影响。采用log-rank检验进行预后分析，两组间数据的比较采用t检验或Mann-Whitney U检验。结果 RNA测序结果显示，发现队列中LPAR6是LCT中显著性低表达的基因之一；验证队列中，LCT组的LPAR6表达水平［M（Q1，Q3）为204.90（81.90，512.70）］低于NLCT组［809.40（417.50，1 829.20），U = 242.00，P = 0.002］；两队列中，低表达LPAR6均与不良预后的发生风险显著相关（P < 0.01）。细胞增殖实验结果显示，在实验的0、24、48、72 h，过表达组MyLa、Sz4细胞与对照组细胞的细胞增殖活力差异均无统计学意义（均P > 0.05）；分别采用LPA、2S-OMPT、ATP、ADO激活MyLa细胞LPAR6后48 h，过表达组MyLa细胞的增殖活力（1.38 ± 0.01、1.04 ± 0.01、1.09 ± 0.03、1.23 ± 0.01）均低于对照组（1.73 ± 0.04、1.23 ± 0.01、1.24 ± 0.01、1.42 ± 0.03，t值分别为30.33、18.38、4.78、5.75，均P < 0.05），细胞凋亡率（17.93% ± 0.88%、17.75% ± 0.35%、23.97% ± 0.57%、31.44% ± 0.34%）均高于对照组（3.98% ± 0.03%、7.81% ± 0.59%、11.95% ± 0.85%、12.02% ± 0.48%，t值分别为15.93、14.49、11.74、33.01，均P < 0.05）；分别采用2S-OMPT、ADO激活Sz4细胞LPAR6后48 h，Sz4细胞增殖活力2S-OMPT对照组为1.48 ± 0.01、过表达组为1.29 ± 0.04；ADO对照组为1.51 ± 0.02、过表达组为1.27 ± 0.01，过表达组均低于对照组（P < 0.05），细胞凋亡率均高于对照组（2S-OMPT对照组29.35% ± 0.55%、过表达组41.70% ± 0.70%；ADO对照组24.60% ± 1.00%、过表达组37.05% ± 0.15%，均P < 0.05）。结论 LPAR6在LCT患者中低表达并与不良预后的发生风险显著相关，体外激活LPAR6能够抑制CTCL细胞的增殖，促进细胞凋亡，LCT患者LPAR6表达水平降低可能是疾病进展的重要机制之一。

关键词: 淋巴瘤, T细胞, 皮肤, 蕈样真菌病, 受体, 溶血磷脂酸, 细胞凋亡, 不良预后, 大细胞转变

Abstract: 【Abstract】 Objective To determine lysophosphatidic acid receptor 6 （LPAR6）expression in patients with mycosis fungoides （MF）, a variant of cutaneous T-cell lymphoma（CTCL）, and to investigate its role and mechanism of action in the development and prognosis of CTCL. Methods A total of 110 patients with confirmed MF were collected from Department of Dermatology, Peking University First Hospital from 2011 to 2020, including 24 with large-cell transformation （LCT） and 25 with non-large cell transformation （NLCT） in the discovery cohort, and 24 with LCT and 37 with NLCT in the validation cohort. RNA sequencing and RT-PCR were conducted to determine the LPAR6 expression in patients in the discovery cohort and validation cohort respectively. LPAR6 expression was compared between patients with LCT and those with NLCT, and its effect on the prognosis of patients was evaluated. Two LPAR6-overexpressing CTCL cell lines MyLa and Sz4 were constructed to evaluate the effect of LPAR6 overexpression on proliferative activity of MyLa and Sz4 cells, with the cells normally expressing LPAR6 as the control group; after the treatment with LPAR6-related ligand lysophosphatidic acid （LPA）, 2S-OMPT, adenosine triphosphate （ATP） or adenosine （ADO）, the effects of LPAR6 activation on the proliferative activity and apoptosis of LPAR6-overexpressing MyLa and Sz4 cells were evaluated by the MTS method and flow cytometry respectively. Log-rank test was used for prognostic analysis, and t test or Mann-Whitney U test was used for comparisons between two groups. Results As RNA sequencing showed, LPAR6 was one of the significantly underexpressed genes in the LCT group in the discovery cohort; in the validation cohort, LPAR6 expression （median ［Q1, Q3］） was significantly lower in the LCT group （204.90［81.90, 512.70］） than in the NLCT group（809.40［417.50, 1 829.20］, U = 242.00, P = 0.002）; in the two cohorts, the underexpression of LPAR6 was significantly associated with increased risk of poor prognosis （both P < 0.01）. Cell proliferation assay showed no significant difference in the proliferative activity of MyLa or Sz4 cells between the LPAR6 overexpression group and control group at 0, 24, 48 and 72 hours during the experiment （all P > 0.05）; 48 hours after activation of LPAR6 by LPA, 2S-OMPT, ATP and ADO in MyLa cells, the LPAR6 overexpression group showed significantly decreased cellular proliferative activity （1.38 ± 0.01, 1.04 ± 0.01, 1.09 ± 0.03, 1.23 ± 0.01, respectively） compared the control group （1.73 ± 0.04, 1.23 ± 0.01, 1.24 ± 0.01, 1.42 ± 0.03, t = 30.33, 18.38, 4.78, 5.75, respectively, all P < 0.05）, but significantly increased cell apoptosis rate （17.93% ± 0.88%, 17.75% ± 0.35%, 23.97% ± 0.57% , 31.44% ± 0.34%, respectively） compared the control group （3.98% ± 0.03%, 7.81% ± 0.59%, 11.95% ± 0.85%, 12.02% ± 0.48%, t = 15.93, 14.49, 11.74, 33.01, respectively, all P < 0.05）; 48 hours after activation of LPAR6 by 2S-OMPT and ADO in Sz4 cells, compared with the control group, the LPAR6 overexpression group also showed significantly decreased cellular proliferative activity （2S-OMPT: 1.29 ± 0.04 vs. 1.48 ± 0.01; ADO: 1.27 ± 0.01 vs. 1.51 ± 0.02; both P < 0.05）, but significantly increased cell apoptosis rate （2S-OMPT: 41.70% ± 0.70% vs. 29.35% ± 0.55%; ADO: 37.05% ± 0.15% vs. 24.60% ± 1.00%; both P < 0.05）. Conclusions LPAR6 was underexpressed in the patients with LCT, and its underexpression was significantly associated with increased risk of poor prognosis. In vitro activation of LPAR6 could inhibit the proliferation of CTCL cells and promote their apoptosis, suggesting that the decrease of LPAR6 expression may be one of the important mechanisms underlying disease progression in patients with LCT.

Key words: Lymphoma, T-cell, cutaneous, Mycosis fungoides, Receptors, lysophosphatidic acid, Apoptosis, Poor prognosis, Large cell transformation