WTAP介导的PTEN mRNA m6A甲基化修饰在三七总皂苷抑制血管内膜增生中的作用及机制研究

Intimal hyperplasia, the key event of in-stent restenosis, is a result of vascular smooth muscle cell (VSMC) proliferation and migration. Our studies have found that TPNS inhibited the intimal hyperplasia in balloon catheter-injured rat carotid artery and proliferation, migration of VSMC, increased N6-methyladenosine modification and up-regulated RNA methyltransferase WTAP in vivo and in vitro; Furthermore, gene microarray analysis displayed that PTEN was a candidate target gene of WTAP. We therefore hypothesized that TPNS up-regulated WTAP expression via enhancing the N6-methyladenosine modification of PTEN mRNA and thereby, increased the stability and protein translation efficiency of PTEN mRNA, led to the promotion of PTEN expression, modulation of PI3K-AKT signaling pathway and inhibition of cell proliferation and migration in VSMC and thus, played an inhibitory role in intimal hyperplasia. To test this hypothesis, we will perform drug intervention, construct WTAP and PTEN silenced and overexpressed cell models to investigate the effect and mechanism of WTAP on PTEN N6-methyladenosine modification. Secondly, we plan to establish an animal model of artery restenosis, and verify that whether N6-methyladenosine is involved in the role of TPNS in intimal hyperplasia. Thus, our study will elucidate the hypothesis at molecular, cell, tissue and overall levels and provide a new theoretical basis for TPNS application in the prevention and treatment of in-stent restenosis.

血管平滑肌细胞（VSMC）增殖、迁移所致内膜增生是支架内再狭窄的重要原因。我们的前期研究发现：三七总皂苷（TPNS）可抑制动脉球囊损伤动物模型的血管内膜增生及体外VSMC增殖、迁移，增加RNA m6A甲基化修饰并上调RNA甲基转移酶WTAP；表达谱芯片分析提示PTEN是WTAP候选靶基因。故提出假说：TPNS通过上调WTAP表达，增强PTEN mRNA m6A甲基化修饰，进而增加PTEN mRNA的稳定性及蛋白翻译效率，PTEN表达增加，影响下游PI3K-AKT信号通路，最终抑制VSMC增殖、迁移，发挥抑制内膜增生的作用。拟采用药物干预、WTAP及PTEN沉默、过表达细胞模型，研究WTAP调控PTEN m6A甲基化修饰的作用及机制；动物模型实验验证m6A甲基化在TPNS抑制内膜增生中的作用。从而在分子、细胞、组织和整体水平上论证假说，为TPNS防治支架内再狭窄提供理论依据。

内膜增生是血管平滑肌细胞（VSMC）增殖和迁移的结果，是动脉再狭窄的关键环节。我们既往研究表明，三七总皂苷（TPNS）抑制血管球囊损伤后内膜增生并抑制VSMC增殖迁移。然而，TPNS在内膜增生中的作用机制尚不清楚。本研究中，首先我们发现TPNS逆转了大鼠颈动脉球囊损伤后血管m6A甲基化水平，并抑制了内膜增生。然后，我们检测了动脉组织m6A甲基化酶（METTL3、METTL14和WTAP）和去甲基化酶（FTO、ALKBH5）的表达，发现TPNS上调了大鼠颈动脉球囊损伤后WTAP的表达。此外，我们还证实TPNS通过促进WTAP表达抑制VSMC增殖迁移，而沉默WTAP后该作用消失；同时我们发现，TPNS促进VSMC中p16表达上调，沉默p16会抑制TPNS对VSMC细胞增殖迁移的影响；最后，我们阐明了WTAP通过m6A甲基化修饰调控p16表达进而发挥调控作用。综上，我们研究阐明了TPNS通过WTAP-p16信号轴影响内膜增生，而m6A甲基化修饰在其中发挥关键作用。因此，本研究为评估血管成形术后内膜增生提供了一个潜在的生物标志物，同时也为该疾病的提供了一个新的防治靶点。