LncRNA-AK139328调控自噬在糖尿病心肌缺血再灌注损伤中的作用及机制探讨

Myocardial ischemia-reperfusion injury (MIRI) severely reduces the beneficial effects of reperfusion therapy for acute myocardial infarction. Studies have demonstrated that MIRI is significantly increased in diabetics, but the mechanism remains unclear. Our preliminary study observed an elevated expression level of advanced glycation end products (AGEs) in the myocardium of Type2 db/db diabetic mice with MIRI, accompanied with increased autophagy. Our data also showed that AGEs aggravate MIRI in diabetics through the enhancement of autophagy. Furthermore, we screened the long non-coding RNA (lncRNA) expression profile using microarray analysis in myocardial tissue after MIRI. The results showed that the highly expressed lncRNA is AK139328. Bioinformatics analysis revealed that lncRNA-AK139328 could recognize and bind with microRNA-204. In addition, the expression of microRNA204 was significantly decreased in the myocardium of Type2 db/db diabetic mice with MIRI. It has been proved that microRNA204 played an important role in negative regulating autophagy through LC3-II protein during MIRI. Therefore, we hypothesized that lncRNA-AK139328 may act as endogenous sponge RNA to interact with and inhibit the expression of miR204, which may involve in regulation of autophagy by AGEs and aggravate diabetic MIRI. Luciferase activity assays and RNA pull-down were used in the current study. We will investigate the role and mechanism of AK139328-miR204-autophagy pathway in diabetic MIRI. Our research may provide rationales and novel approaches to attenuate MIRI in diabetic patients.

心肌缺血再灌注损伤（MIRI）严重制约急性心肌梗死再灌注治疗的获益。糖尿病加重MIRI程度，但机制不明。我们前期研究发现：糖尿病小鼠MIRI时，①心肌晚期糖基化终产物（AGEs）增加且通过增强自噬加重MIRI；②芯片筛选提示心肌长链非编码RNA-AK139328（lncRNA-AK139328）表达上调最显著；③生物信息学预测lncRNA-AK139328能与microRNA204（miR204）高强度结合；④心肌miR204表达下降。已知miR204可负性调控自噬。因此推测AGEs通过上调心肌lncRNA-AK139328表达，结合并抑制miR204而增强自噬，从而加重糖尿病MIRI。本项目拟采用荧光素酶报告基因、RNA pull-down等技术，结合体内外实验探讨AK139328-miR204-自噬通路在糖尿病MIRI中的作用及机制。本研究可能为减轻糖尿病MIRI提供新思路。

急性心肌梗死（AMI）是人类健康的主要杀手之一，尽早进行血运重建，恢复心肌灌注是治疗AMI的重要手段。然而，部分患者在接受再灌注治疗后反而出现心脏损伤和功能障碍加重的现象，这种称之为“心肌缺血再灌注损伤（MIRI）”的矛盾现象严重制约了再灌注治疗的受益。值得注意的是，在糖尿病状态下，AMI患者更容易发生MIRI，且程度更重，不良事件概率更高，但潜在机制尚不清楚。长链非编码RNA（lncRNA）是一类新发现的、长度超过200nt的RNA转录本，参与基因转录调控和表观遗传学修饰等诸多生物学过程，在AMI、心衰等心血管疾病中扮演着关键角色。本研究中，我们分别在野生型小鼠和糖尿病小鼠构建了MIRI模型，采用Microarray芯片技术筛选了各组小鼠心肌组织中差异性表达的lncRNA，并通过荧光定量PCR验证，发现lncRNA AK139328在糖尿病小鼠MIRI中显著上调。随后我们构建了能够特异性抑制小鼠lncRNA AK139328表达的shRNA序列，通过半自动生化分析仪检测血清中心肌坏死标记物水平，HE染色观察心肌损伤程度，Evans Blue/TTC染色观察心肌梗死面积，Western Blot检测自噬相关蛋白的表达，TUNEL染色观察心肌细胞凋亡，分光光度法检测心肌组织中Caspase-3的活性，Masson染色观察心肌组织纤维化，心脏B超观察心脏功能。结果显示lncRNA AK139328被敲低后，糖尿病小鼠血清中CK、CK-MB和LDH水平下降，心肌损伤程度减轻，心肌梗死面积缩小，自噬相关蛋白ATG5、ATG7、LC3-II/I水平下降而p62水平上升，心肌细胞凋亡减弱，心肌纤维化和心脏功能改善。另外，我们运用生物学信息分析和荧光素酶报告基因检测，发现lncRNA AK139328上存在miR-204结合位点并可负向调控miR-204表达。MiR-204已被证实可抑制自噬，参与MIRI。我们通过离体实验发现高糖条件下，下调miR-204可明显逆转lncRNA AK139328 shRNA对心肌细胞自噬和缺氧/复氧损伤的抑制效应。因此，lncRNA AK139328极有可能通过miR-204调节自噬，参与糖尿病MIRI。这有助于揭示糖尿病加重MIRI的内在分子机制，为干预糖尿病心血管并发症提供新的靶点。