Chiba Noriyuki, Furukawa Ken-Ichi, Takayama Shohei, Asari Toru, Chin Shunfu, Harada Yoshifumi, Kumagai Gentaro, Wada Kanichiro, Tanaka Toshihiro, Ono Atsushi, Motomura Shigeru, Murakami Manabu, Ishibashi Yasuyuki
Department of Pharmacology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan; Department of Orthopaedic Surgery, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Department of Pharmacology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
J Pharmacol Sci. 2015 Apr;127(4):467-73. doi: 10.1016/j.jphs.2015.03.008. Epub 2015 Apr 1.
Mesenchymal stem cells (MSCs) isolated from spinal ligaments with ectopic ossification have a propensity toward the osteogenic lineage. To explore epigenetic control of the osteogenic features of MSCs, we treated MSCs obtained from the spinal ligaments of ossification of yellow ligament (OYL) patients and non-OYL patients with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AdC). We compared the non-OYL groups (untreated and treated with 5AdC) with the OYL groups (untreated and treated with 5AdC) by genome-wide microarray analysis. Next, we used methylated DNA immunoprecipitation combined with quantitative real-time PCR to assess gene methylation. Ninety-eight genes showed expression significantly increased by 5AdC treatment in MSCs from non-OYL patients but not from OYL patients. In contrast, only two genes, GDNF and WNT5A, showed significantly higher expression in OYL MSCs compared with non-OYL MSCs without 5AdC treatment. Both genes were hypermethylated in non-OYL MSCs but not in OYL MSCs. Small interfering RNA targeted to each gene decreased expression of the target gene and also several osteogenic genes. Both small interfering RNAs also suppressed the activity of alkaline phosphatase, a typical marker of osteogenesis. These results suggest that the osteogenic features of MSCs from OYL patients are promoted by unmethylated WNT5A and GDNF genes.
从伴有异位骨化的脊柱韧带中分离出的间充质干细胞（MSCs）具有向成骨谱系分化的倾向。为了探究MSCs成骨特性的表观遗传调控，我们用DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5AdC）处理了从黄韧带骨化（OYL）患者和非OYL患者的脊柱韧带中获取的MSCs。我们通过全基因组微阵列分析，将非OYL组（未处理组和5AdC处理组）与OYL组（未处理组和5AdC处理组）进行了比较。接下来，我们使用甲基化DNA免疫沉淀结合定量实时PCR来评估基因甲基化。98个基因在非OYL患者的MSCs中经5AdC处理后表达显著增加，但在OYL患者的MSCs中未增加。相反，与未用5AdC处理的非OYL MSCs相比，只有两个基因GDNF和WNT5A在OYL MSCs中表达显著更高。这两个基因在非OYL MSCs中高度甲基化，但在OYL MSCs中未甲基化。靶向每个基因的小干扰RNA降低了靶基因以及几个成骨基因的表达。两种小干扰RNA也抑制了碱性磷酸酶的活性，碱性磷酸酶是成骨的典型标志物。这些结果表明，未甲基化的WNT5A和GDNF基因促进了OYL患者MSCs的成骨特性。
