Expression of Long Noncoding RNA Xist Is Induced by Glucocorticoids

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressive agents. However, their clinical usage is limited by severe multisystemic side effects. Glucocorticoid induced osteoporosis results in significant morbidity and mortality but the cellular and molecular mechanisms underlying GC-induced bone loss are not clear. GC use results in decreased osteoblast differentiation with increased marrow adiposity through effects on bone marrow stem cells. GC effects are transduced through its receptor (GR). To identify novel GR regulated genes, we examined the profile of genes differentially expressed in GR knockout mouse bone marrow mesenchymal stem cells (MSCs) by RNA sequencing (RNA-Seq) analysis. Results showed 15 upregulated genes (3- to 10-fold) and 70 downregulated genes (-2.7- to -10-fold), with the long noncoding RNA X-inactive specific transcript (Xist) downregulated the most. The differential expression of genes measured by RNA-Seq was validated by qRT-PCR analysis of selected genes and the GC/GR signaling-dependent expression of Xist was further demonstrated by GC (dexamethasone) treatment of GR-deficient MSCs in vitro and by GC injection of C57BL/6 mice (males and females) in vivo. Our data revealed that the long noncoding RNA Xist is a GR regulated gene and its expression is induced by GC both in vitro and in vivo. To our knowledge, this is the first evidence showing that Xist is transcriptionally regulated by GC/GR signaling.

糖皮质激素（glucocorticoids, GCs）是一类强效的抗炎及免疫抑制制剂，但其临床应用因严重的多系统性不良反应而受到限制。糖皮质激素诱导的骨质疏松可显著升高患者的发病率与死亡率，但糖皮质激素诱导骨丢失的细胞及分子机制仍未明确。糖皮质激素可通过作用于骨髓间充质干细胞（bone marrow mesenchymal stem cells, MSCs），抑制成骨细胞分化并促进骨髓脂肪生成。糖皮质激素的生物学效应通过其受体（glucocorticoid receptor, GR）介导。为筛选新型GR调控基因，本研究采用RNA测序（RNA sequencing, RNA-Seq）技术，分析了GR敲除小鼠骨髓间充质干细胞中的差异表达基因谱。结果显示，共筛选得到15个上调基因（表达倍数范围为3~10倍）与70个下调基因（表达倍数范围为-2.7~-10倍），其中长链非编码RNA X染色体失活特异性转录本（long noncoding RNA X-inactive specific transcript, Xist）的下调幅度最为显著。本研究通过实时定量反转录聚合酶链反应（quantitative real-time reverse transcription PCR, qRT-PCR）对部分筛选出的基因进行验证，确认了RNA-Seq检测到的基因差异表达情况；同时通过体外以地塞米松（dexamethasone）处理GR缺陷型骨髓间充质干细胞，以及体内向C57BL/6小鼠（雌雄个体）注射糖皮质激素，进一步证实了Xist的表达依赖于GC/GR信号通路。本研究数据表明，长链非编码RNA Xist是一类GR调控基因，其表达在体内外均可被糖皮质激素诱导。据我们所知，本研究首次证实Xist可通过GC/GR信号通路实现转录调控。