lncRNA CRNDE调控miR-29a-3p对LPS诱导的人脐静脉内皮细胞增殖与凋亡影响的分子机制研究-现代医学

lncRNA CRNDE调控miR-29a-3p对LPS诱导的人脐静脉内皮细胞增殖与凋亡影响的分子机制研究

单位：青海大学附属医院急诊中心ICU, 青海西宁 810000

分类号：R329.25;R459.7

出版年·卷·期（页码）：2021·49·第九期（1080-1086）

摘要：

目的：探讨长链非编码RNA （lncRNA）结直肠肿瘤差异表达基因（CRNDE）对脂多糖（LPS）诱导的人脐静脉内皮细胞（HUVECs）增殖和凋亡的影响及其可能的作用机制。方法：将HUVECs分为对照组（细胞正常培养）、LPS组（细胞用1.0μg·ml^-1的LPS诱导24 h）、si-NC+LPS组（用1.0 μg·ml^-1 LPS诱导转染si-NC的细胞24 h）、si-CRNDE+LPS组（用1.0 μg·ml^-1 LPS诱导转染si-CRNDE的细胞24 h）、miR-NC+LPS组（用1.0 μg·ml^-1 LPS诱导转染miR-NC的细胞24 h）、miR-29a-3p+LPS组（用1.0 μg·ml^-1LPS诱导转染miR-29a-3p mimics的细胞24 h）、anti-miR-NC+si-CRNDE+LPS组（用1.0 μg·ml^-1 LPS诱导共转染anti-miR-NC与si-CRNDE的细胞24 h）和anti-miR-29a-3p+si-CRNDE+LPS组（用1.0 μg·ml^-1 LPS诱导共转染anti-miR-29a-3p与si-CRNDE的细胞24 h）。实时定量聚合酶链反应（RT-qPCR）检测细胞中CRNDE和miR-29a-3p表达，细胞计数试剂盒-8（CCK-8）、流式细胞仪和蛋白质印迹法分别检测细胞增殖、凋亡及细胞中细胞周期蛋白D1（CyclinD1）和活化的半胱氨酸天冬氨酸蛋白酶-3（Cleaved-caspase-3）表达。双荧光素酶报告基因实验验证CRNDE与miR-29a-3p调控关系。结果：与对照组比较，LPS组HUVECs中CRNDE表达升高（P<0.05），而miR-29a-3p表达降低（P<0.05）。与si-NC+LPS组比较，si-CRNDE+LPS组HUVECs存活率和CyclinD1表达升高（P<0.05），细胞凋亡率和Cleaved-caspase-3表达降低（P<0.05）。与miR-NC+LPS组比较，miR-29a-3p+LPS组HUVECs存活率和CyclinD1表达升高（P<0.05），细胞凋亡率和Cleaved-caspase-3表达降低（P<0.05）。CRNDE靶向负调控miR-29a-3p。与anti-miR-NC+si-CRNDE+LPS组比较，anti-miR-29a-3p+si-CRNDE+LPS组HUVECs存活率和CyclinD1表达降低（P<0.05），细胞凋亡率和Cleaved-caspase-3表达升高（P<0.05）。结论：低表达CRNDE可抑制LPS处理的内皮细胞增殖，并诱导细胞凋亡，其作用机制与负调控miR-29a-3p表达有关。

Objective: To investigate the effect of long non-coding RNA(lncRNA) colorectal neoplasia differentially expressed(CRNDE) on LPS-induced proliferation and apoptosis of human umbilical vein endothelial cells(HUVECs) and possible mechanisms. Methods: HUVECs were divided into control group (cells were cultured normally), LPS group (cells were induced with 1.0 μg·ml^-1 LPS for 24 h), si-NC+LPS group (cells transfected with si-NC were induced with 1.0 μg·ml^-1 LPS 24 h), si-CRNDE+LPS group (cells transfected with si-CRNDE were induced with 1.0 μg·ml^-1 LPS for 24 h), miR-NC+LPS group (cells transfected with miR-NC were induced with 1.0 μg·ml^-1 LPS for 24 h), miR-29a-3p+LPS group (cells transfected with miR-29a-3p mimics were induced with 1.0 μg/mL LPS for 24 h), anti-miR-NC+si-CRNDE+LPS group (cells co-transfected with anti-miR-NC and si-CRNDE were induced with 1.0 μg·ml^-1 LPS for 24 h) and anti-miR-29a-3p+si-CRNDE+LPS group (cells co-transfected with anti-miR-29a-3p and si-CRNDE were induced with 1.0 μg·ml^-1 LPS for 24 h). RT-qPCR was used to detect the expression levels of CRNDE and miR-29a-3p in the cells; CCK-8, flow cytometry and Western blot method were used to detect cell proliferation, apoptosis, and the protein expression of CyclinD1 and Cleaved-caspase-3. Double luciferase reporter gene experiments verified the regulatory relationship between CRNDE and miR-29a-3p. Results: Compared with the control group, the expression of CRNDE in HUVECs in the LPS group increased (P<0.05), but the expression of miR-29a-3p decreased(P<0.05). Compared with the si-NC+LPS group, the survival rate of HUVECs and the protein expression of CyclinD1 in the si-CRNDE+LPS group increased (P<0.05), while the rate of apoptosis and and the protein expression of Cleaved-caspase-3 decreased (P<0.05). Compared with miR-NC+LPS group, the survival rate of HUVECs and the protein expression of CyclinD1 in the miR-29a-3p+LPS group increased (P<0.05), while the rate of apoptosis and and the protein expression of Cleaved-caspase-3 decreased (P<0.05). CRNDE negatively regulated the expression of miR-29a-3p. Compared with the anti-miR-NC+si-CRNDE+LPS group, the survival rate of HUVECs and the protein expression of CyclinD1 in the anti-miR-29a-3p+si-CRNDE+LPS group decreased (P<0.05), but the apoptosis rate and the protein expression of Cleaved-caspase-3 increased(P<0.05). Conclusion: Low expression of CRNDE can inhibit LPS-induced endothelial cell proliferation and induce apoptosis, and its mechanism is related to the negative regulation of miR-29a-3p expression.

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