先天性肾上腺发育不良症基因-1对垂体瘤细胞自噬和增殖的调控作用

The role of dosage-sensitive sex reversal-adrenal hypoplasia congenita region on the X chromosome 1 in autophagy and proliferation on pituitary adenoma cells

目的:探讨先天性肾上腺发育不良症基因-1(DAX-1)对垂体瘤(MMQ)细胞自噬和增殖的作用。方法:利用基因芯片技术筛选垂体瘤与正常垂体之间的差异基因群,并通过PubMed数据库基因组分析侵袭性垂体瘤和非侵袭性垂体瘤之间DAX-1的表达差异;将siRNA转染至MMQ细胞敲低DAX-1的表达(DAX-1 KD组),DAX-1过表达质粒转染至MMQ细胞提高DAX-1的表达(DAX-1 OE组),正常对照组(NC组),采用CCK-8和平板克隆实验检测DAX-1对MMQ细胞增殖的影响;通过实时定量聚合酶链反应(RT-qPCR)检测自噬相关基因ATG5、ATG7和ATG12的表达;通过蛋白质印迹(Western blot)法和免疫荧光法检测DAX-1对自噬标记蛋白LC3的表达影响,通过裸鼠移植瘤实验观察敲减DAX-1对MMQ细胞生长和自噬的影响。结果:基因芯片结果提示,和正常垂体组比,垂体肿瘤中DAX-1基因表达显著下调,并且DAX-1在侵袭性垂体瘤中比非侵袭性垂体瘤表达低(P<0.05);与NC组MMQ细胞比,DAX-1 KD组MMQ细胞增殖水平升高,而DAX-1 OE组MMQ细胞增殖受抑且卡麦角林和溴隐亭的药物敏感性增高(均P<0.05);与NC组MMQ细胞比,DAX-1 KD组MMQ细胞自噬水平降低(P<0.05);裸鼠移植瘤实验显示,与NC组MMQ细胞移植瘤比,DAX-1 KD组移植肿瘤生长速度更快(P<0.05)。结论:DAX-1在垂体瘤中表达降低,且DAX-1表达降低后促进垂体瘤发展,DAX-1可能是垂体瘤治疗的潜在靶点。

Objective:To investigate the role of the dosage-sensitive sex reversal-adrenal hypoplasia congenita region on the X chromosome 1(DAX-1)in autophagy and proliferation of pituitary tumor cells.Methods:The differential gene group between pituitary tumors and normal pituitaries was screened by Microarray and the expression difference of DAX-1 between invasive pituitary tumor and non-invasive pituitary tumor was analyzed by Pubmed database.The expression of DAX-1 pair was knocked down by small interfering RNAs(siRNA),and the effect of DAX-1 on MMQ cells proliferation was detected by CCK-8 and colony formation assays.The expression of autophagy related genes ATG5,ATG7 and ATG12 was detected by real-time quantitative polymerase chain reaction(RT-qPCR).Western blot and immunofiuorescence experiments were performed to detect the effect of DAX-1 on the expression of LC3.Finally,xenograft experiments in nude mice was made to observe the effect of DAX-1 knockdown on the growth and autophagy of pituitary tumor cells.Results:Compared with normal pituitary tumors,the DAX-1 gene expression was significantly down-regulated in pituitary tumors,and was lower in invasive and non-invasive pituitary tumors(P<0.05).Knockdown DAX-1 could promote MMQ cells proliferation,while overexpression of DAX-1 inhibited proliferation of MMQ cells and promoted drug sensitivity of cabergoline and bromocriptine(P<0.05).In addition,knocking-down DAX-1 could inhibit the autophagy level in MMQ cells.It was proved that knocking down DAX-1 promoted tumor growth in vivo(P<0.05).Conclusion:The DAX-1 expression is decreased in pituitary tumors,and the decreased DAX-1 promotes the development of pituitary tumors.DAX-1 may be a potential therapeutic target for pituitary tumors.