TNF-α通过影响circHmbox1/microRNA-1247-5p/mRNA调控轴参与绝经后骨质疏松发生的机制研究

The increasing of TNF-αlevel is associated with PMOP in postmenopausal women. Studies showed that TNF-α-induced osteoclastogenesis is a leading cause of bone loss in PMOP, but which mechanisms are not fully elucidated. We previously studies showed that TNF-αcould increase the expression of circHmbox1 in the cytoplasm of osteoclast and promote the production of exosomes-contained circHmbox1 by osteoclasts .Silencing of circHmbox1 could obviously alleviate TNF-α-induced osteoclastogenesis. The exosomes-contained circHmbox1 could inhibited osteoblasts differentiation. Further studies showed that microRNA-1247-5p bonding to circHmbox1 was involved in TNF-α-induced osteoclastogenesis. Thus, we speculated that TNF-αmay increase osteoclastogenesis through circHmbox1/miR-1247-5p/ mRNA axis and indirectly decrease osteoblasts differentiation through affecting circHmbox1/miR-1247-5p/mRNA axis by osteoclasts-derived exosomes-contained circHmbox1.To test this hypothesis, the experiments will be designed in vivo and in vitro. SiRNA and overexpression will be applied to further confirm that circHmbox1 is critical in TNF-α-mediated osteoclastogenesis and osteoblasts differentiation. Furthermore, the exact mechanisms of circHmbox1/miR-1247-5p/mRNA axis involved in TNF-α-mediated osteoclastogenesis and osteoblasts differentiation will be elucidated in this project. Moreover, whether circHmbox1 is a treatment potential target for PMOP will be tested. This study provides an important theoretical basis for clarifying the etiology of PMOP.

TNF-α促进破骨细胞分化是绝经后骨质疏松（PMOP）骨丢失的重要原因。我们前期发现，TNF-α增加了破骨细胞中circHmbox1的表达并促使其分泌富含circHmbox1的外泌体；circHmbox1能够与miR-1247-5p结合参与TNF-α促进破骨细胞形成，而富含circHmbox1的外泌体抑制了成骨细胞分化。因此，推测TNF-α可能通过circHmbox1/miR-1247-5p/mRNA调控轴直接或间接调节破骨细胞及成骨细胞分化。本研究拟通过体内和体外实验，结合分子生物学及生物信息学技术，确定circHmbox1在TNF-α直接或间接调节破骨细胞及成骨细胞分化中的作用；明确TNF-α通过circHmbox1/miR-1247-5p/mRNA调控轴调节破骨细胞及成骨细胞分化的分子机制；验证circHmbox1作为PMOP功能性靶点潜在应用性。本研究有助于阐释PMOP的发病机制。

肿瘤坏死因子-α（TNF-α）水平的升高与绝经后骨质疏松（postmenopausal osteoporosis, PMOP）的发生密切相关，TNF-α引起的破骨细胞过度活跃是PMOP发生的重要机制之一。鉴于TNF-α对破骨细胞调控方式的多样性和调控机制的复杂性，其确切的调控机制仍需进一步探索。目前发现，非编码RNAs (non-codingRNA，ncRNA)的表观遗传学调控在生命基本过程与疾病发生发展中起到重要的调节作用。环状RNA(circRNA)作为一类新近发现的新型非编码RNAs,其生物学功能及其与疾病关系的研究成为生物医学研究的一个前沿热点领域。本课题组通过环状RNA芯片技术及实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)技术筛查，发现去卵巢小鼠的破骨细胞中circHmbox1的表达量明显减少，基因沉默破骨细胞前体细胞中的circHmbox1后，破骨细胞的分化明显增加；过表达circHmbox1缓解了TNF-α对破骨细胞分化的促进作用。炎症微环境下，破骨细胞产生的外泌体中circHmbox1的含量明显降低，而过表达circHmbox1的破骨细胞，其外泌体能够促进成骨细胞的分化。进一步研究发现，circHmbox1能够直接结合miR-1247-5p, 从而调节成骨细胞和破骨细胞分化。通过生物信息学分析和荧光素酶报告基因检测，我们发现miR-1247-5p能够靶向结合到B细胞淋巴瘤6（Bcl6）的3'-UTR区，抑制Bcl6的翻译。这些研究提示，炎症微环境下，circHmbox1主要通过与microRNA-1247-5p结合来抑制RANKL诱导的破骨细胞分化。 TNF-α通过破骨细胞产生低表达circHmbox1的外泌体减少成骨细胞分化。进一步研究显示，microRNA-1247-5p通过靶向Bcl6调节破骨细胞的分化和成骨细胞的分化。我们的研究成果进一步阐明了PMOP的发病机制，尤其是PMOP中的由于TNF-α水平增加导致骨代谢失衡的机制，为PMOP的防治提供了新的思路。