Ni Shu-Yuan, Xu Wen-Ting, Liao Guang-Yuan, Wang Yin-Ling, Li Jing
Department of Intensive Care Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, Guangdong Province, People's Republic of China.
Inflammation. 2021 Aug;44(4):1452-1463. doi: 10.1007/s10753-021-01431-0. Epub 2021 Mar 4.
Sepsis is one of the primary causes of death in intensive care units. Recently, increasing evidence has identified lncRNA HOTAIR is involved in septic cardiomyopathy. However, the potential mechanism underlying HOTAIR on septic cardiomyopathy is still unknown. H9C2 cells were treated with lipopolysaccharide (LPS) after transfection with sh-HOTAIR, sh-Lin28, pcDNA3.1-HOTAIR, and pcDNA3.1-PDCD4. qRT-PCR was used to examine the level of HOTAIR, Lin28, PDCD4, and sepsis-related inflammatory cytokines. Flow cytometric analysis was applied to detect cell apoptosis. The interaction between Lin28 and HOTAIR or PDCD4 was verified by RNA pull-down and RIP assay. HOTAIR levels were interfered by AAV9-sh-HOTAIR in LPS-induced septic cardiomyopathy mice. ELISA analysis was used to evaluate TNF-α, IL-6, and IL-1β level. Western blot was used to detect the expression of LIN28 and PDCD4 in mouse cardiomyocytes. Echocardiography was used to evaluate the cardiac function. In our study, knockdown of HOTAIR inhibited LPS-induced inflammation and H9C2 cells apoptosis. HOTAIR promoted LPS-induced inflammatory response and apoptosis of H9C2 cells by enhancing PDCD4 stability. RNA pull-down and RIP assay exhibited that Lin28, a highly conserved RNA-binding protein, was combined with HOTAIR and PDCD4. The in vivo experiments verified that the HOTAIR knockdown alleviated the cardiac function injury and secretion of inflammatory factors caused by sepsis. In conclusion, our findings supported that the HOTAIR/Lin28/PDCD4 axis serves as a critical regulator of sepsis, which may open a new direction for the development of sepsis therapeutic.
脓毒症是重症监护病房患者主要死亡原因之一。最近，越来越多的证据表明长链非编码RNA HOTAIR参与了脓毒症性心肌病。然而，HOTAIR在脓毒症性心肌病中发挥作用的潜在机制仍不清楚。用sh-HOTAIR、sh-Lin28、pcDNA3.1-HOTAIR和pcDNA3.1-PDCD4转染H9C2细胞后，再用脂多糖（LPS）处理。采用qRT-PCR检测HOTAIR、Lin28、PDCD4及脓毒症相关炎性细胞因子水平。应用流式细胞术分析检测细胞凋亡情况。通过RNA下拉实验和RNA免疫沉淀实验验证Lin28与HOTAIR或PDCD4之间的相互作用。在LPS诱导的脓毒症性心肌病小鼠中，用腺相关病毒9型-sh-HOTAIR干扰HOTAIR水平。采用ELISA分析评估肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和白细胞介素-1β（IL-1β）水平。采用蛋白质免疫印迹法检测小鼠心肌细胞中LIN28和PDCD4的表达。采用超声心动图评估心脏功能。在本研究中，敲低HOTAIR可抑制LPS诱导的炎症反应和H9C2细胞凋亡。HOTAIR通过增强PDCD4的稳定性促进LPS诱导的H9C2细胞炎症反应和凋亡。RNA下拉实验和RNA免疫沉淀实验表明，高度保守的RNA结合蛋白Lin28与HOTAIR和PDCD4结合。体内实验证实，敲低HOTAIR可减轻脓毒症所致的心脏功能损伤和炎性因子分泌。综上所述，我们的研究结果支持HOTAIR/Lin28/PDCD4轴是脓毒症的关键调节因子，这可能为脓毒症治疗的发展开辟新方向。
