RelA转录激活Linc00665调控血管平滑肌细胞异常增殖的作用与机制研究

Vascular smooth muscle cells(VSMC) excessive proliferation and restenosis are based on abnormal cell cycle. We detected upregulated expression of Linc00665 and CCND in stenosis vessel, while Let-7a with the same MRE element was downregulated. Bioinformatic analysis showed that Let-7a sites on the 3’UTR of CCND, which suggested that Linc00665 inhibits CCND regulation of VSMC cell cycle as a ceRNA. We also predicted potential binding sites between inflammatory microenvironment transcription factor RelA and Linc00665 promoter region; Additionally, RelA and Linc00665 expression has positive correlation. All the evidences above lead to RelA-Linc00665-Let-7a-CCND mediated cell cycle regulates VSMC proliferation. In the project, both cell and transgenic mice will be used to explore the hypotheses: the pattern of RelA transcription activated Linc00665; Linc00665 endogenously competes Let-7a as ceRNA through MRE elements; Let-7a targets CCND-3’UTR. The program will be able to reveal molecular mechanism of long non-coding RNA regulating VSMC cell cycle mediated by inflammatory microenvironment, also can provide a novel cutting point in VSMC abnormal proliferation adjustment network.

细胞周期异常介导VSMC过度增殖及血管狭窄。课题组在狭窄血管中检测到Linc00665和CCND高表达，而存在相同MRE元件的Let-7a却低表达，并预测到Let-7a与CCND-3'UTR特异结合，提示Linc00665通过ceRNA拮抗Let-7a对CCND介导的VSMC周期调控;同时预测到介导炎性微环境的转录因子RelA与Linc00665启动子存在结合位点、且表达正相关；表明VSMC增殖与RelA-Linc00665-Let-7a-CCND途径介导细胞周期相关。本项目拟在细胞及转基因鼠水平明确：RelA转录激活Linc00665作用方式；Linc00665通过MRE元件介导ceRNA方式内源性竞争Let-7a作用；Let-7a与CCND-3'UTR靶向结合与抑制；阐明炎性微环境介导非编码RNA调控VSMC细胞周期分子机制。本研究将为探讨VSMC异常增殖的新型调控网络提供新思路。

细胞周期异常介导VSMC过度增殖及血管狭窄。课题组前期研究发现细胞周期蛋白 D（Cyclin D, CCND）与细胞 G1/S 期转换有关并且能够调控VSMC的增殖。同时课题组在狭窄血管中检测到长链非编码RNA Linc00665高表达，而存在相同MRE元件的Let-7a却低表达，并预测到Let-7a与CCND-3'UTR特异结合，提 示Linc00665通过ceRNA拮抗Let-7a对CCND介导的VSMC周期调控;同时预测到介导炎性微环境的转录因子RelA与Linc00665启动子存在结合位点、且表达正相关；表明VSMC增殖与RelA-Linc00665-Let-7a-CCND途径介导细胞周期相关。本研究同时考察了内源性调控Linc00665及Let-7a的具体机制，通过生物信息学初步预测，荧光素酶报告分析验证RelA可以与Linc00665启动子区直接结合，Linc00665与Let-7a存在结合位点，激活RelA可以使Linc00665的表达增加、Let-7a的表达降低、CCND1的表达增加；转染Let-7a mimic 能够反转过表达linc00665-207导致的CCND1表达上调和促增殖作用，RIP实验及荧光素酶报告分析验证linc00665-207和Let-7a存在直接的结合关系及结合位点。综上，我们的研究结果表明在尼古丁等刺激因素的作用下，RelA 表达上调和或激活水平提高，linc00665-207 被转录激活，进而胞浆中的 Let-7a更多的被 linc00665-207 吸附，而 CCND1 得以免于遭受Let-7a介导的降解，最终促进细胞周期由 G1 期进入 S 期引起HA-VSMC 过度增殖。该研究将为血管狭窄的诊断和治疗提供新的靶点。