Abstract：
BACKGROUND: Osteoporosis (OP) is a prevalent disease associated with age, and one of the primary pathologies is the defect of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This study aimed to elucidate whether Nuclear Receptor Binding SET Domain Protein 2 (NSD2) transcriptionally regulates osteogenic differentiation of BMSCs in osteoporosis.
METHODS: Identification of human BMSCs (hBMSCs) in vitro was measured by flow cytometry. Osteogenesis of hBMSCs in vitro was measured by Alizarin Red and Alkaline Phosphatase staining. The protein levels of H3K36me1/2/3, NSD2, and Hoxa2 were measured by western blotting. The mRNA levels of NSD2, Runx2, and BSP were measured by qPCR. The role of NSD2 in the osteogenic differentiation of BMSCs was further identified by silencing NSD2 via shRNA or overexpression of NSD2 via lentivirus transfection. The interactions of NSD2, H3K36me2 and Hoxa2 were identified via chromatin immunoprecipitation (ChIP). Luciferase reporting analysis was employed to confirm that NSD2 regulated the transcriptional activity of Hoxa2. Ovariectomized (OVX) was performed on mice to construct osteoporosis (OP) model. Subsequently, the bone mass was assessed by micro computed tomography (micro-CT) scan.
RESULTS: During the osteogenesis of OP-derived hBMSCs, the levels of NSD2 and H3K36me2 significantly increased in 14 days of osteogenic induction. Inhibition of NSD2 via shRNA increased the RUNX2 and BSP expression of hBMSCs, while overexpression of NSD2 decreased RUNX2 and BSP expression of hBMSCs. ChIP analysis indicated NSD2-mediated H3K36me2 reduced the osteogenic differentiation of hBMSCs by regulating the osteogenic inhibitor Hoxa2. Accordingly, inhibition of NSD2 in vivo via tail vein injection of LV-shNSD2 lentivirus greatly alleviated OVX-induced osteoporosis in mice.
CONCLUSIONS: We demonstrated that NSD2 inhibited the osteogenic differentiation in hBMSCs by transcriptionally downregulating Hoxa2 via H3K36me2 dimethylation. Inhibition of NSD2 effectively attenuated bone loss in murine osteoporosis and NSD2 is a promising target for clinical treatment of osteoporosis.
METHODS: Identification of human BMSCs (hBMSCs) in vitro was measured by flow cytometry. Osteogenesis of hBMSCs in vitro was measured by Alizarin Red and Alkaline Phosphatase staining. The protein levels of H3K36me1/2/3, NSD2, and Hoxa2 were measured by western blotting. The mRNA levels of NSD2, Runx2, and BSP were measured by qPCR. The role of NSD2 in the osteogenic differentiation of BMSCs was further identified by silencing NSD2 via shRNA or overexpression of NSD2 via lentivirus transfection. The interactions of NSD2, H3K36me2 and Hoxa2 were identified via chromatin immunoprecipitation (ChIP). Luciferase reporting analysis was employed to confirm that NSD2 regulated the transcriptional activity of Hoxa2. Ovariectomized (OVX) was performed on mice to construct osteoporosis (OP) model. Subsequently, the bone mass was assessed by micro computed tomography (micro-CT) scan.
RESULTS: During the osteogenesis of OP-derived hBMSCs, the levels of NSD2 and H3K36me2 significantly increased in 14 days of osteogenic induction. Inhibition of NSD2 via shRNA increased the RUNX2 and BSP expression of hBMSCs, while overexpression of NSD2 decreased RUNX2 and BSP expression of hBMSCs. ChIP analysis indicated NSD2-mediated H3K36me2 reduced the osteogenic differentiation of hBMSCs by regulating the osteogenic inhibitor Hoxa2. Accordingly, inhibition of NSD2 in vivo via tail vein injection of LV-shNSD2 lentivirus greatly alleviated OVX-induced osteoporosis in mice.
CONCLUSIONS: We demonstrated that NSD2 inhibited the osteogenic differentiation in hBMSCs by transcriptionally downregulating Hoxa2 via H3K36me2 dimethylation. Inhibition of NSD2 effectively attenuated bone loss in murine osteoporosis and NSD2 is a promising target for clinical treatment of osteoporosis.
摘要：
背景：骨质疏松症（OP）是一种与年龄相关的普遍疾病，骨髓间充质干细胞（BMSCs）成骨分化缺陷是其主要病理之一。本研究旨在阐明核受体结合SET结构域蛋白2（NSD2）是否转录调节骨质疏松症中BMSCs的成骨分化。
方法:通过流式细胞术测量体外人BMSCs(hBMSCs)的鉴定。通过茜素红和碱性磷酸酶染色测量hBMSCs在体外的成骨作用。通过蛋白质印迹法测量H3K36me1/2/3、NSD2和Hoxa2的蛋白质水平。通过qPCR测量NSD2、Runx2和BSP的mRNA水平。通过经由shRNA沉默NSD2或经由慢病毒转染过表达NSD2进一步鉴定NSD2在BMSCs的成骨分化中的作用。通过染色质免疫沉淀（ChIP）鉴定了NSD2，H3K36me2和Hoxa2的相互作用。采用荧光素酶报告分析来确认NSD2调节Hoxa2的转录活性。对小鼠进行卵巢切除（OVX）以构建骨质疏松症（OP）模型。随后，通过显微计算机断层扫描（micro-CT）扫描评估骨质量。
结果：在OP来源的hBMSCs成骨过程中，在成骨诱导的14天，NSD2和H3K36me2的水平显着增加。通过shRNA抑制NSD2增加hBMSCs的RUNX2和BSP表达,而NSD2的过表达降低了hBMSCs的RUNX2和BSP表达。ChIP分析表明，NSD2介导的H3K36me2通过调节成骨抑制剂Hoxa2降低了hBMSCs的成骨分化。因此，通过尾静脉注射LV-shNSD2慢病毒在体内抑制NSD2可以大大减轻OVX诱导的小鼠骨质疏松症。
结论：我们证明NSD2通过H3K36me2二甲基化转录下调Hoxa2抑制hBMSCs的成骨分化。抑制NSD2可有效减轻小鼠骨质疏松症的骨丢失，NSD2是临床治疗骨质疏松症的有希望的靶标。
方法:通过流式细胞术测量体外人BMSCs(hBMSCs)的鉴定。通过茜素红和碱性磷酸酶染色测量hBMSCs在体外的成骨作用。通过蛋白质印迹法测量H3K36me1/2/3、NSD2和Hoxa2的蛋白质水平。通过qPCR测量NSD2、Runx2和BSP的mRNA水平。通过经由shRNA沉默NSD2或经由慢病毒转染过表达NSD2进一步鉴定NSD2在BMSCs的成骨分化中的作用。通过染色质免疫沉淀（ChIP）鉴定了NSD2，H3K36me2和Hoxa2的相互作用。采用荧光素酶报告分析来确认NSD2调节Hoxa2的转录活性。对小鼠进行卵巢切除（OVX）以构建骨质疏松症（OP）模型。随后，通过显微计算机断层扫描（micro-CT）扫描评估骨质量。
结果：在OP来源的hBMSCs成骨过程中，在成骨诱导的14天，NSD2和H3K36me2的水平显着增加。通过shRNA抑制NSD2增加hBMSCs的RUNX2和BSP表达,而NSD2的过表达降低了hBMSCs的RUNX2和BSP表达。ChIP分析表明，NSD2介导的H3K36me2通过调节成骨抑制剂Hoxa2降低了hBMSCs的成骨分化。因此，通过尾静脉注射LV-shNSD2慢病毒在体内抑制NSD2可以大大减轻OVX诱导的小鼠骨质疏松症。
结论：我们证明NSD2通过H3K36me2二甲基化转录下调Hoxa2抑制hBMSCs的成骨分化。抑制NSD2可有效减轻小鼠骨质疏松症的骨丢失，NSD2是临床治疗骨质疏松症的有希望的靶标。
