miR-214在绝经后骨质疏松中的作用及其机制研究

新Postmenopausal osteoporosis (PMOP) is defined as a progressive skeletal disorder, which results in women's fractures, longstanding pain and reduced quality of life. However, the underlying mechanism is unclear. Our previous studies have shown that the level of serum miR-214 was significantly higher in the PMOP compared to the control groups. Also, the results of bioinformatics analysis and luciferase reporter assay showed that PTEN as candidate target of miR-214 was identified. Furthermore, the expression of PTEN was down-regulated in osteoporotic mouse model. These results suggested that the mechanism of miR-214 inducing PMOP may be related to PTEN. In this present study, we will assess the biological characteristics of osteoporosis and investigate the relationship of miR-214 and osteoporosis-related index in OVX-induced osteoporotic mouse model（C57BL/6） using overexpression or silencing of miR-214; We will observe the effect of miR-214 on osteoblast proliferation, apoptosis and differentiation at the cellular level using overexpression or silencing techniques; Regarding the PTEN-PI3K-AKT signaling pathway as the cutting point, we detect the relationship of miR-214 and bone histomorphometric parameters and expression level of PTEN,PI3K and AKT to determine the precise regulation mechanism of miR-214 and PTEN-PI3K-AKT signaling pathway in the development of PMOP by gene overexpression or gene silencing. To the best of our knowledge, there is no study on the mechanism of miR-214 targeting PTEN in the development of PMOP. Therefore, the results gained from the proposed studies will help to understand the molecular mechanism of miR-214 and PMOP pathogenesis and provide a novel clue and potential intervention target for the prevention of PMOP.

绝经后骨质疏松(PMOP)及其相关疾病严重危害妇女健康，其发病机制不明。前期结果：miR-214在 PMOP血清水平显著上调，生物信息学预测发现PTEN是miR-214下游靶基因，PTEN在PMOP起关键作用，AKT失活是通过上调PTEN的表达而实现的，我们推测：miR-214通过调控PTEN靶点影响AKT从而影响骨的形成，提出 "miR-214-PTEN-PI3K-AKT-PMOP"假说。本研究拟采用miR-214过表达、表达沉默技术，检测OVX鼠骨质疏松相关指标，并做骨组织形态计量学分析，研究miR-214与PTEN-PI3K-AKT通路各个因子的变化；结合反义寡核苷酸技术和过表达技术，评判miR-214发生骨质疏松的机制，采用荧光素酶报告基因技术，揭示miR-124对PTEN分子调控机制。本课题以miR-214对PTEN转录后调控为切入点，有源头创新性，可为PMOP防治提供新靶点。

骨质疏松症和低骨量与骨折的高风险有关，每天约有25,000例骨质疏松性骨折发生，这一发病率高于全世界心脏病和中风的总发病率。因此，骨质疏松症和骨质减少症是一个重要的全球公共卫生问题，与受影响个人特别是老年人生活质量的持续下降有关。绝经后骨质疏松（postmenopausal osteoporosis，PMOP）是一种与雌激素减少相关的全身性代谢性骨病，其特征是骨量减少，骨微观结构受损，骨骼脆性增加，导致骨折发生率增加，严重影响绝经后妇女的健康和生活质量。MicroRNA（miRNA）是一类由内源性基因编码的非编码单链小RNA，约有22个核苷酸，通过碱基互补配对与靶基因的3'UTR结合，从而负性调控靶基因的转录和翻译。以往的研究表明miRNAs可以通过调节成骨细胞和破骨细胞的分化和活性在绝经后骨质疏松的发生发展中发挥重要作用。在本课题的资助下，以 PMOP 和绝经后骨量正常妇女为研究对象，检测 miRNA 表达谱的差异，发现miR-21、miR-122、miR-214、miR-218、miR-150、miR-155、miR-99a及miR-618的表达与正常对照相比表达有差异。其中miR-214表达比正常对照显著升高，miR-218和miR-618比正常对照显著降低。我们不仅在成骨细胞和动物水平探讨了miR-214 调控FGFR1/FGF信号通路致骨质疏松发生的机制，而且探讨了miR-218和miR-618在破骨细胞分化过程中的作用，进一步研究发现，1、miR-214 通过抑制FGFR1的表达负性调控成骨细胞的分化，功能性抑制miR-214可以加速MMSCs的成骨分化。2、miR-218通过靶向TNFR1调节破骨细胞的分化。3、miRNA-618通过TLR4/MyD88/NF-κB 信号通路调节破骨分化。本研究的科学意义是，鉴定出miR-218和miR-618与TLR-4的靶向关系。我们的研究结果提示miR-218和miR-618下降促使破骨形成是通过激活 TLR4/MyD88/NF-κB信号通路实现的，靶向miR-214 、miR-218及miR-618可能是绝经后骨质疏松症的潜在治疗选择。