基于长链非编码RNA uc.431+介导Clcf1/Jak2/Stat3 信号通路的绝经后骨质疏松症肾阴虚证分子机制研究

Our previous studies showed that the Clcf1/Jak2/stat3 signal pathway plays important roles in kidney-yin deficiency syndrome of postmenopausal osteoporosis (PMOP). Lnc uc.431+ and its target gene Clcf1 were down-regulated in kidney-yin deficiency syndrome of PMOP. We put forward the hypothesis that the imbalance of Clcf1/Jak2/stat3 signaling pathway mediated by down regulation of Lnc uc.431+ is one of the molecular mechanism of kidney-yin deficiency syndrome inPMOP . Firstly, This study intends to veryfy the correlation between Lnc RNA and kidney-yin deficiency syndrome of PMOP. RIP experiment was used to verify the interaction between Lnc uc.431+ and Clcf1; Secondly, Thp1 cell lines with over expression or knockdown of Lnc uc.431+ were screened to analyze the effects on the Clcf1/Jak2/Stat3 signaling pathway , also cell proliferation and cell cycle; Thirdly, RIP experiment was used to verify the upstream transcription factor NFATC2 regulation on Lnc uc.431+; Lnc uc.431+-protein interaction experiments were performed to identify the other interacting proteins of Lnc uc.431+. Fourly, CRISPR/cas9 technique was used to knockout Lnc uc.431+ in mice, observe the effect of deletionf of Lnc uc.431+ on Clcf1/Jak2/Stat3 signal pathway in peripheral blood. We investigate the molecular mechanism of kidney-yin deficiency syndrome of PMOP from long chain non-coding RNA level.

课题组研究表明Clcf1/Jak2/stat3是绝经后骨质疏松症（PMOP）肾阴虚证的重要信号通路，且Lnc uc.431+及靶基因Clcf1表达下调，因此提出“假说”：Lnc uc.431+下调导致Clcf1/Jak2/Stat3通路失调是PMOP肾阴虚证的分子机制之一。本研究拟①临床进一步验证Lnc RNA与PMOP肾阴虚证的相关性；RIP验证与Clcf1的相互作用；②过表达及敲低THP1细胞株Lnc uc.431+，分析对Clcf1/Jak2/Stat3信号通路及细胞增殖、周期的影响；③CRISPR/cas9技术敲除小鼠Lnc uc.431+，观察对小鼠外周血Clcf1/Jak2/Stat3的影响；④RIP实验验证调控Lnc uc.431+的转录因子NFATC2；RNA-蛋白相互作用组学实验鉴定与Lnc uc.431+互作蛋白；从长链非编码RNA水平探讨PMOP肾阴虚证的分子机制。

绝经后骨质疏松症主要是由多因素引起的年龄相关性慢性退行性病变。主要是由于雌激素水平下降造成骨代谢失衡，骨微结构破坏，骨量减少，最终导致骨脆性增加和骨折危险性增加为特征的全身性骨骼疾病。随着人均寿命的增加和老龄化的加剧，骨质疏松症的发病率正逐年攀升，骨质疏松防治形势日趋严峻。中医药治疗骨质疏松症（OP）具有较好的疗效，探讨其治疗机制具有重要意义。本研究围绕课题组提出的“Lnc uc.431+下调导致Clcf1/Jak2/Stat3通路失调是PMOP肾阴虚证的分子机制之一”假说，通过临床试验、基因敲除动物实验和细胞实验，结合前期的研究数据，探讨PMOP 肾阴虚证与长链非编码RNA及其参与的Jak2/Stat3信号通路的关系。结果：①Lnc uc. 431+在PMOP 肾阴虚证组、肾阳虚证组、无肾虚证组存在差异表达，在肾阴虚证组表达水平最低，与对照组、肾阳虚证组、无肾虚证组相比均有统计学意义。②Lnc uc. 431+定位在细胞核及细胞浆；Lnc uc. 431+过表达能够上调Jak2、STAT3 基因表达水平和 p-JAK2、p-STAT3 水平，转录组测序表明过表达Lnc uc. 431+后差异基因参与JAK/STAT等信号通路；siRNA敲低后，Jak2、STAT3基因表达水平下调，说明 Lnc uc. 431+与 Jak2/STAT3信号通路有密切联系。LncRNA-蛋白相互作用组学研究表明，与Lnc uc. 431+互作蛋白有DDOST、DMBT1、MUC5AC、LTF、RPL23等10个蛋白；在THP1细胞株尚未观察到Lnc uc. 431+与CLCF1存在直接相互作用。③CRISPR-Cas9全身性敲除Lnc uc. 431+使 2 月龄Lnc uc. 431+KO雌性小鼠的体重显著降低，骨密度也具有下降趋势；Micro-CT 检测12月龄KO小鼠股骨的骨密度显著下降，差异具有统计学意义。Lnc uc. 431+KO雌性小鼠外周血单核细胞JAK2、STAT3基因表达水平显著降低。本研究对于从长链非编码RNA角度探讨中医证的分子机制是重要的尝试，对对防治骨质疏松和推动中医药走向国际都具有积极作用。