平滑肌特异性表达的长链非编码RNA - LncVSM诱发高血压血管平滑肌表型转换的机制

In previous work, we identified a vascular smooth muscle cell (VSMC) specific long non-coding RNA (LncRNA) and named "LncVSM". We also confirmed that LncVSM expression was increased in hypertensive patient's plasma. LncVSM promoted VSMC proliferation and migration induced by angiotensin II or PDGF-BB by binding myosin heavy chain 11 (MYH11) and fibronectin-1 (FN1). LncVSM transgenic rat spontaneously developed hypertension at 10 weeks after born. Based on these findings, our project firstly wants to clear whether LncVSM inhibits VSMC contractile phenotype but increases synthetic phenotype in vitro and in vivo. Using domain specific chromatin isolation by RNA purification (dChIRP) and RNA immunoprecipitation assay, we confirmed that LncVSM directly bound to MYH11 and FN1. Proteomics (LncVSM pulldown) analysis showed LncVSM might bind other contractile protein such as MYH9, MYH10, smooth muscle enteric actin γ (1 and 2); and 26S proteasome regulatory subunit 11 (PSMD11). Thereof, secondly, the present project aims to investigate whether LncVSM regulates the ubiqitination of contractile proteins and PSMD11 activity to modulate proteins degradation and stability. In our preliminary data, knockdown MYH11 and FN1 increased LncVSM expression; accordingly, knockdown LncVSM upregulated MYH11 and FN1 mRNA and protein levels, which suggesting there is a feedback regulation between MYH11 and LncVSM gene transcription. So our project may use dChIRP and high-throughput sequencing to find the main target of genomic sequences which interaction with LncVSM and confirm them in VSMC. According to the three aspects of the project, we aim to demonstrate our hypothesis: LncVSM play an essential role in pathogenesis of hypertension by modulation VSMC phenotype. Our project also find a novel biological function of non-coding RNA, that LncRNA might act as a cofactor of mechanical elements to drive VSMCs motility.

前期工作发现一条在平滑肌细胞(VSMC)特异表达的长链非编码RNA并命名为LncVSM，在高血压病人血浆中高表达，并直接结合肌球蛋白重链(MYH)及纤连蛋白促进VSMC增殖、迁移；且LncVSM转基因大鼠自发高血压。在此基础上，首先从细胞及组织水平验证LncVSM抑制VSMC收缩表型、促进其分泌表型；进而鉴定LncVSM直接结合的MYH、平滑肌特异的γ肌动蛋白、26S蛋白酶体调节亚基PSMD11，观察收缩蛋白的泛素化、蛋白稳定性以及PSMD11活性，探讨LncVSM促进收缩蛋白的降解机制；最后，在预实验发现LncVSM与MYH11在转录水平有相互调节的基础上，拟采用RNA-pulldown DNA并进行高通量测序技术并分析两者在转录水平调节的靶点并加以验证，探讨LncVSM调节收缩蛋白基因表达的分子机制。通过以上研究，阐明LncVSM调节平滑肌细胞表型转换参与高血压发病的新机制。

在本项目资助下，我们首先确定了长链非编码RNA—AK098656为非编码RNA，并确定其是平滑肌特异表达的长链非编码RNA。应用RNAscope技术证明其主要表达于细胞浆中，因此推测其功能可能与蛋白相互作用调控蛋白质功能。应用CHIRP技术，我们发现并证明了AK098656可直接结合MYH11以及Fibronectin-1，同时还可结合蛋白酶体非-ATP依赖的调节亚基11，因而促进MYH11蛋白降解，促进平滑肌细胞由由收缩型向合成型转换。在AK098656转基因大鼠模型，我们发现血压显著增加，血管舒张功能障碍，阻力血管中膜增厚。此项目的工作证明了一条新的人血管平滑肌特异表达的长链非编码RNA，可以直接结合收缩蛋白促进其降解而诱导平滑肌细胞表型转换，造成阻力血管狭窄而诱发高血压。.为进一步开展转化研究，我们还制备了平滑肌细胞特异转基因小鼠，也证明其血压显著增加，而给予其相互作用miRNA，可以显著降低血压。同时我们还发现转基因小鼠平滑肌细胞ROS产生增加，并初步证实AK098656可以直接结合NAD而影响线粒体功能。.在本项目资助下，共发表SCI论著9篇，申请专利1项。完成了项目设定目标和研究内容。