NRR:辽宁省人民医院张亮团队揭示环状RNA Rap1b抑制急性缺血性卒中神经元凋亡的机制
撰写:张芳芳、赵琳、陆宇、董欣、张亮
脑卒中是一种常见的神经系统疾病,是全球范围内永久性发病和致残的主要原因之一[1, 2]。脑卒中的2种主要类型是急性缺血性脑卒中(acute ischemic stroke,AIS)和出血性脑卒中。目前美国食品药品监督管理局批准的急性缺血性脑卒中治疗方法只有在发病后4-5 h内静脉注射重组人组织型纤溶酶原激活剂,但由于其治疗时间窗狭窄,且存在颅内出血和血管性水肿的风险,只有约7%的患者符合治疗条件,因此对缺血性卒中的溶栓治疗效果有限[3-7]。急性缺血性脑卒中后,动脉供血受阻的急性缺血初级神经元会迅速死亡,而由其他动脉输入的核心和周围的急性缺血初级神经元则在一定程度上存在延迟凋亡的现象[8, 9]。虽然多种药物在动物模型中表现出明显的神经保护作用,但这些结果并未得到临床试验的证实[10]。鉴于缺血性脑损伤及其全身后果的复杂性,通过靶向多种机制的细胞疗法可能比单一药物治疗更为有效[11, 12]。环状RNA (circular RNA,circRNA)是一类新型非编码RNA,为单链、稳定的RNA,其在3'端和5'端之间存在共价键,可形成无多聚腺苷化尾部的环状结构,因而不受RNA外切酶的影响[13-15]。最近有研究表明,环状RNA高度保守,且在人类细胞中大量表达[13, 16, 17]。最新研究发现,环状RNA的表达具有组织特异性,并受不同的生物学过程调节,如卒中、神经炎症和衰老、心肌肥厚和心力衰竭以及细胞生长[18-23]。微小RNA靶向Rap1b可抑制多种肿瘤类型的细胞迁移、侵袭和转移[24-28]。然而,Rap1b在神经系统疾病中的功能作用尚不清楚,研究者旨在探索circRap1b在急性缺血性脑卒中中的作用。
近期,来自中国辽宁省人民医院张亮团队在《中国神经再生研究(英文版)》(Neural Regeneration Research)上发表了题为“The circular RNA Rap1b promotes Hoxa5 transcription by recruiting Kat7 and leading to increased Fam3a expression, which inhibits neuronal apoptosis in acute ischemic stroke”的研究中发现,circRap1b在急性缺血性脑卒中C57BL/6J小鼠海马组织和神经元HT22细胞中表达下调。过表达circRap1b可抑制急性缺血性脑卒中体外模型HT22细胞的凋亡。而circRap1b/Hoxa5在体外抑制神经元凋亡可能归因于Kat7诱导的Hoxa5启动子区组蛋白H3赖氨酸14乙酰化修饰,这一结果也在体内实验中得到验证。这一研究启示,circRap1b有望成为脑卒中治疗的新靶点。
研究表明,环状RNA可参与调控缺血性脑血管病的发生和进展[23, 29, 30]。circ DLGAP4通过结合miR-143调控HER1表达,影响内皮细胞去分化为间充质细胞,从而减轻脑梗死体积和血脑屏障损伤。环状RNA还能参与调节神经元等多种细胞的凋亡。circ_0000950在阿尔茨海默病发展过程中可通过miR-103负调控神经元凋亡[31]。此外,circ_0000296在慢性脑缺血缺氧海马组织和神经元HT22细胞中表达下调,且过表达circ_0000296能显著抑制慢性脑缺血缺氧诱导的神经元凋亡[32]。张亮等发现,C57BL/6J小鼠和神经元HT22细胞经急性缺血缺氧后,苏木精-伊红染色和TUNEL染色可见神经元凋亡率明显增加(图1A-C)。定量聚合酶链式反应发现,circRap1b在急性缺血缺氧损伤的小鼠海马和HT22细胞中低表达(图1D-G)。而circRap1b过表达显著抑制急性缺血缺氧诱导的HT22神经元凋亡(图1J-K)。
图1 CircRap1b在急性缺血缺氧损伤小鼠海马以及HT22细胞中低表达,且可抑制神经元凋亡(图源:Zhang et al., Neural Regen Res, 2023)
在circRap1b过表达引起的差异表达蛋白中,转录因子Hoxa5表达上调最明显(图2)。Hox基因家族编码的转录因子有助于决定神经元的命运[18]。具体来说,Hoxa5最近被证明参与了小鼠大脑中具有自主功能的核细胞[33]和后迷走神经运动神经元[29]的轴突生长。Hernández等[34]提出,Hoxa5是人脂肪来源干细胞分化为神经元的重要基因,因此是探索神经疾病细胞治疗策略的潜在候选基因。研究显示,Hoxa5在急性缺血缺氧小鼠海马和HT22细胞中低表达(图3A-E)。流式细胞仪检测发现过表达Hoxa5显著抑制了急性缺血缺氧诱导的神经元凋亡,而敲低Hoxa5后HT22细胞的凋亡率明显恢复(图3F和G)。研究显示,过表达增生性瘢痕或瘢痕疙瘩来源的成纤维细胞中的HOXA5减弱了细胞增殖、迁移和胶原合成,而加速了细胞凋亡[35]。沉默HOXA5基因表达可诱导Jurkat细胞凋亡和细胞周期阻滞[36]。且HOXA5直接作用于维甲酸受体的下游,参与维甲酸诱导的细胞凋亡和生长抑制[37]。实验显示,Hoxa13在慢性脑缺血缺氧诱导的海马和HT22细胞中表达下调,而过表达Hoxa13减弱了慢性脑缺血缺氧诱导的海马神经元HT22的凋亡[38]。同时小鼠Hoxa13突变体通过下调Bmp7表达抑制尿路上皮细胞凋亡[39]。
图2 在circRap1b过表达引起的差异表达蛋白中,转录因子Hoxa5表达上调最明显(图源:Zhang et al., Neural Regen Res, 2023)
图3 Hoxa5在急性缺血缺氧海马和HT22细胞中低表达,并可抑制神经元的凋亡(图源:Zhang et al., Neural Regen Res, 2023)
组蛋白修饰主要包括甲基化、乙酰化、磷酸化、泛素化以及类泛素化[40]。组蛋白乙酰化受组蛋白乙酰基转移酶和组蛋白去乙酰化酶的动态调控。既往研究表明,Kat7倾向于催化组蛋白H4的赖氨酸5和赖氨酸12以及组蛋白H3的赖氨酸14的乙酰化[41, 42]。有研究发现,过表达circFoxo3在体外抑制氧糖剥夺并诱导心肌细胞自噬、凋亡、炎症和损伤。过表达circFoxo3可通过减少KAT7、H3K14ac和RNA Poly II在HMGB1启动子中的富集而抑制HMGB1表达,从而抑制自噬,减轻心肌缺血再灌注损伤[43]。在哺乳动物细胞中过表达CDK11(P58)可增强Kat7对游离组蛋白的乙酰转移酶活性。CDK11(P58)是一种Kat7的调节蛋白,可在体内外与Kat7相互作用,在细胞周期进程中发挥重要作用,并与凋亡密切相关[44]。张亮等过表达circRap1b后,Hoxa5 mRNA和蛋白表达显著增加(图4A)。荧光原位杂交技术结果显示,circRap1b存在于细胞核(图4B),与Kat7相互作用共存于Hoxa5的启动子区(图4C-E)。H3K14ac在Hoxa5启动子区富集,主要富集与-1000-0 bp(图4F和G)。在急性缺血缺氧HT22细胞敲低Kat7可逆转circRap1b过表达对H3K14ac富集和Hoxa5 mRNA和蛋白表达的影响(图4H-L)。进一步RNA下拉和染色质免疫共沉淀结果同样证实H3K14ac可与circRap1b相互作用,且circRap1b存在于Hoxa5启动子区(图4M-O)。因此实验结果说明,circRap1b可招募Kat7诱导Hoxa5启动子区H3K14ac修饰来激活其转录。既往也有研究显示,circMRPS35可通过募集KAT7升高FOXO1和FOXO3a启动子区H4K5ac水平,从而抑制胃癌进展[45]。与本次研究的结果相似,lncRNA-MRCCAT1可通过结合EZH2来诱导NPR3启动子区域的H3K27me3来介导NPR3转录[46]。将MALAT1与PRC2结合,分离EZH2与HIV-1 LTR启动子的结合,去除H3K27me3,可逆转人类免疫缺陷病毒转录的表观遗传沉默[47]。
图4 circRap1b可通过招募乙酰基转移酶Kat7诱导Hoxa5启动子区H3K14ac修饰,促进Hoxa5的转录(图源:Zhang et al., Neural Regen Res, 2023)
序列相似家族3A (Family with sequence similarity 3A,FAM3A)是一种线粒体蛋白,可在细胞适应应激和细胞存活中发挥重要作用,并参与神经元凋亡的调节[38, 48]。据报道,序列相似家族3A是内质网应激诱导的HT22细胞死亡的重要调节因子[49]。序列相似家族3A可通过激活PI3K/Akt通路保护HT22细胞免受过氧化氢诱导的氧化应激[50]。序列相似家族3A还在慢性脑缺血缺氧诱导的海马和神经元中表达下调,而过表达序列相似家族3A则可抑制慢性脑缺血缺氧诱导的海马神经元凋亡[38]。张亮等发现序列相似家族3A在急性缺血性脑卒中诱导的海马和HT22中低表达,而过表达序列相似家族3A则显著抑制损伤HT22细胞的凋亡,且敲低序列相似家族3A则促进损伤HT22细胞的凋亡(图5)。据报道,序列相似家族3A可通过激活促生存PI3K/AKT/mTOR通路抑制白细胞介素1β诱导的软骨细胞凋亡[48]。序列相似家族3A可正向调节内皮细胞的缺血后血管生成[51]。序列相似家族3A可减轻新生小鼠的缺氧缺血性脑损伤[52]。根据JASPAR生物信息学数据库,在人序列相似家族3A的启动子区域-3000-0 bp发现了44个HOXA5的推测结合位点,且在小鼠序列相似家族3A的启动子区域-3000-0 bp发现了4个HOXA5的推测结合位点。进一步采用染色质免疫共沉淀和荧光素酶报告基因系统验证Hoxa5可结合序列相似家族3A的启动子区域,促进其转录和蛋白表达(图5),从而调控海马神经元的凋亡。Hoxa13还可通过转录激活序列相似家族3A蛋白的表达,抑制慢性脑缺血缺氧诱导的海马神经元凋亡[38]。Wu等[53]发现Hoxa13通过靶向BMP7启动子区域促进胃癌中的转录表达。Hoxa13靶向Aldh1a2启动子并上调其表达来调节肢体形态发生[54]。
图5 Hoxa5可通过转录激活序列相似家族3A的表达调控神经元凋亡(图源:Zhang et al., Neural Regen Res, 2023)
为进一步阐明circRap1b和Hoxa5共调控急性缺血损伤HT22细胞凋亡的机制,张亮等转染了circRap1b过表达以及Hoxa5的小干扰RNA,流式细胞仪检测发现,过表达circRap1b可抑制细胞凋亡,而敲低Hoxa5则促进细胞凋亡,且敲低Hoxa5可逆转单独circRap1b过表达对细胞凋亡的抑制作用(图6 A和B)。过表达circRap1b后,Hoxa5和序列相似家族3A的mRNA和蛋白表达水平均上调,而敲低Hoxa5则可逆转过表达circRap1b对序列相似家族3A 蛋白的影响。提示circRap1b可通过Hoxa5调控序列相似家族3A的表达(图6C-H)。最后体内实验验证了过表达circRap1b或Hoxa5后,小鼠海马神经元凋亡明显减少,且同时过表达circRap1b和Hoxa5的凋亡率最低(图7A-D)。
图6 circRap1b可通过Hoxa5调控序列相似家族3A的表达,调节神经元的凋亡(图源:Zhang et al., Neural Regen Res, 2023)
图7 circRap1b和Hoxa5可在体内抑制神经元的凋亡(图源:Zhang et al., Neural Regen Res, 2023)
综上所述,circRap1b可作为模块化支架在Hoxa5的启动子区域招募Kat7,从而提高H3K14ac修饰水平,激活Hoxa5的转录和表达,最终抑制海马神经元的凋亡。目前已有研究利用慢病毒或腺病毒载体在体内过表达天然的保护性环状RNA。这些RNA可编码带有外显子的微盒、内源性剪接供体和受体位点,支持RNA反向折叠的侧翼内含子的反向重复(Holdt等人,2018)。如微量注射circDLGAP4慢病毒可显著改善缺血性卒中预后(Bai et al., 2018)。图8为circRap1b / Hoxa5 /序列相似家族3A轴调控神经元凋亡的机制示意图。研究可能为开发急性缺血性脑卒中的治疗方法和拓宽治疗靶点提供新思路,但研究也存在局限性:首先,该研究只关注雄性大鼠。为了有更多的临床意义,未来的研究应该考虑使用雌性大鼠。其次,尚无转化医学研究。需要加强转化医学研究,开拓脑卒中治疗的新方式。
图8 circRap1b/Hoxa5/序列相似家族3A轴调控神经元凋亡(图源:Zhang et al., Neural Regen Res, 2023)
原文链接:https://doi.org/10.4103/1673-5374.369115
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张芳芳,女,硕士,辽宁省人民医院康复医学科技师,主要从事神经系统疾病的基础研究,发表并参与SCI论文4篇,其中一作影响因子达11.452。
赵琳,女,辽宁省人民医院康复医学科门诊护士长,长期从事神经系统疾病的康复及研究,曾发表核心期刊论文数篇。
通讯作者:张亮,男,硕士,辽宁省人民医院康复医学科主任,主要从事神经系统疾病的康复及研究,曾发表核心期刊论文数篇。
研究获得辽宁省自然科学基金资助(2021-MS-061)。