[1]廖 烘,刘 伟,龙运峰.LncRNA NEAT1 通过调节miR-125b-5p/IGFBP5 轴对血管瘤内皮细胞增殖、凋亡、迁移的实验研究[J].现代检验医学杂志,2023,38(03):65-71.[doi:10.3969/j.issn.1671-7414.2023.03.012]
　LIAO Hong,LIU Wei,LONG Yun-feng.Experimental Study on LncRNA NEAT1 on Proliferation, Apoptosis and Migration of Hemangioma Endothelial Cells by Regulating miR-125b-5p/IGFBP5 Axis[J].Journal of Modern Laboratory Medicine,2023,38(03):65-71.[doi:10.3969/j.issn.1671-7414.2023.03.012]
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LncRNA NEAT1 通过调节miR-125b-5p/IGFBP5 轴对血管瘤内皮细胞增殖、凋亡、迁移的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年03期
页码:
65-71
栏目:
论著
出版日期:
2023-05-15

文章信息/Info

Title:
Experimental Study on LncRNA NEAT1 on Proliferation, Apoptosis and Migration of Hemangioma Endothelial Cells by Regulating miR-125b-5p/IGFBP5 Axis
文章编号:
1671-7414（2023）03-065-07
作者:
廖 烘; 刘 伟; 龙运峰
（邵阳学院附属第 一医院儿科，湖南邵阳 417000）
Author(s):
LIAO Hong ;  LIU Wei ;  LONG Yun-feng
（Department of Pediatrics, the First Affiliated Hospital of Shaoyang University, Hunan Shaoyang 417000，China）
关键词:
长链非编码RNA 核富集转录本1; 微小核糖核酸-125b-5p; 胰岛素样生长因子结合蛋白5; 血管瘤; 凋亡
分类号:
R543；R392.11
DOI:
10.3969/j.issn.1671-7414.2023.03.012
文献标志码:
A
摘要:
目的 探讨长链非编码RNA（long non-coding RNA，LncRNA）核富集转录本1（nuclear enriched abundanttranscript 1，NEAT1）通过调节微小核糖核酸（micro RNAs，miR）-125b-5p/ 胰岛素样生长因子结合蛋白5（insulinlikegrowth factor binding protein 5，IGFBP5）轴对血管瘤内皮细胞增殖、凋亡和迁移的影响。方法 实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）、蛋白免疫印迹（Western blot）分别检测血管瘤组织（2016 年3 月～ 2019 年3 月收集，n=18）、瘤旁组织样本（2016 年3 月～ 2019 年3 月收集，n=18）以及人脐静脉内皮细胞HUVES，人血管瘤内皮细胞HemECs，HDEC 中NEAT1，miR-125b-5p 及IGFBP5 蛋白表达。构建沉默NEAT1，同时沉默NEAT1 和miR-125b-5p 的HemECs 细胞系，通过细胞活力检测试剂盒（cell counting kit-8，CCK-8）、台盼蓝染色、流式细胞术、划痕愈合实验、Western blot 分别观察NEAT1 和miR-125b-5p 对HemECs 细胞增殖、凋亡、迁移及IGFBP5，增殖细胞核抗原（proliferating cell nuclear antigen，PCNA）、B 细胞淋巴瘤/ 白血病-2（B cell lymphoma/lewkmia-2，Bcl-2）和基质金属蛋白酶-9（matrix metalloproteinase-9，MMP-9）蛋白表达的影响；双荧光素酶报告基因实验检测NEAT1 与miR-125b-5p，miR-125b-5p 与IGFBP5 的关系。结果 与瘤旁组织比较，血管瘤组织中NEAT1（2.87±0.22 vs 1.00±0.00），IGFBP5 蛋白（1.45±0.14 vs 0.27±0.02）表达水平升高，miR-125b-5p（0.24±0.02 vs 1.00±0.00）表达水平降低，差异具有统计学意义（t ＝ 35.400~161.220，均P <0.05）；与HUVES 细胞比较，HemECs，HDEC 细胞中NEAT1（2.76±0.24，1.78±0.13 vs 1.00±0.00），IGFBP5 蛋白（1.31±0.15，0.78±0.06 vs 0.24±0.02）表达升高，miR-125b-5p 表达（0.19±0.02，0.45±0.04 vs 1.00±0.00）降低，差异具有统计学意义（t ＝ 17.320~99.204，14.697~33.680，均P<0.05），且HemECs细胞中NEAT1 和IGFBP5 蛋白表达量最高，miR-125b-5p 表达量最低，因此，选取HemECs 细胞为研究对象；与si-NC组比较，si-NEAT1组NEAT1（0.32±0.02 vs 1.01±0.12）表达、A值（0.45±0.04 vs 1.13±0.11）、细胞生长率（32.28%±2.79%vs 99.41%±0.22%）、划痕愈合率（20.33%±1.23% vs 49.24%±2.43%） 及IGFBP5（0.41±0.04 vs 1.31±0.20），PCNA（0.36±0.04 vs 1.27±0.14），Bcl-2（0.48±0.04 vs 1.39±0.16）和MMP-9（0.21±0.02 vs 1.09±0.10）蛋白表达降低，miR-125b-5p（1.87±0.15 vs 1.02±0.10）表达、细胞凋亡率（45.58%±3.34% vs 12.36%±1.07%）升高，差异具有统计学意义（t ＝ 10.809~58.755，均P <0.05）；下调miR-125b-5p 减弱了沉默NEAT1 对HemECs 细胞增殖、迁移的抑制及对细胞凋亡的促进作用（t=9.218~15.010，均P <0.05）；NEAT1 与miR-125b-5p，miR-125b-5p 与IGFBP5 存在靶向调控关系。结论 沉默NEAT1 通过上调miR-125b-5p 来抑制IGFBP5 表达，从而抑制HemECs 细胞增殖、迁移，并促进细胞凋亡。
Abstract:
Objective To investigate the impacts of long non-coding RNA nuclear enriched abundant transcript 1 (LncRNA NEAT1) on the proliferation, apoptosis, and proliferation of hemangioendothelial cells by regulating the micro RNAs（miRNA， miR）-125b-5p/insulin-like growth factor binding protein 5 (IGFBP5) axis. Methods Quantitative real-time PCR(qRT-PCR) and western blot were performed to detect the protein expressions of NEAT1, miR-125b-5p and IGFBP5 in hemangioma tissue (collected from March 2016 to March 2019, n=18), paratumor tissue samples (collected from March 2016 to March 2019, n=18),human umbilical vein endothelial cells HUVES, human hemangioma endothelial cells HemECs, and HDEC, respectively. HemECs cell lines that silence NEAT1 and simultaneously silence NEAT1 and miR-125b-5p were constructed, the effects of NEAT1 and miR-125b-5p on the proliferation, apoptosis and migration of HemECs cells and the expression of IGFBP5, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), matrix metalloproteinase-9 (MMP-9) proteins were observed by cell counting kit-8（CCK-8）method, trypan blue staining, flow cytometry, scratch healing test and western blot, respectively. Dual-luciferase reporter assays were performed to examine the relationship between NEAT1 and miR-125b- 5p, and between miR-125b-5p and IGFBP5. Results The expression levels of NEAT1 (2.87±0.22 vs 1.00±0.00) and IGFBP5 (1.45±0.14 vs 0.27±0.02) in hemangioma tissues were higher than those in paratumoral tissues, the expression level of miR- 125b-5p was decreased (0.24±0.02 vs 1.00±0.00), and the difference was statistically significant (t = 35.400~161.220, all P <0.05). Compared with HUVES cells, the expression of NEAT1 (2.76±0.24，1.78±0.13 vs 1.00±0.00), IGFBP5 protein (1.31±0.15 ，0.78±0.06 vs 0.24±0.02) in HemECs and HDEC cells increased, the expression of miR-125b-5p (0.19 ± 0.02， 0.45±0.04 vs 1.00±0.00) was decreased, and the differences were statistically significant (t=17.320~99.204, 14.697~33.680, all P <0.05). The expression of NEAT1 and IGFBP5 protein was the highest in HemECs cells, and the expression of miR-125b-5p was the lowest. Therefore, HemECs cells were selected as the research object. Compared with the si-NC group, the expression of NEAT1 (0.32±0.02 vs 1.01±0.12), A value (0.45±0.04 vs 1.13±0.11), cell growth rate (32.28%±2.79% vs 99.41%±0.22%), scratch healing rate (20.33%±1.23% vs 49.24% ± 2.43%), IGFBP5 (0.41 ± 0.04 vs 1.31 ± 0.20), PCNA (0.36 ± 0.04 vs 1.27 ± 0.14), Bcl-2 (0.48 ± 0.04 vs 1.39 ± 0.16) and MMP-9 protein (0.21 ± 0.02 vs 1.09 ± 0.10) in the si-NEAT1 group reduced. The expression of miR-125b-5p (1.87 ± 0.15 vs 1.02 ± 0.10) and the rate of apoptosis (45.58%±3.34% vs 12.36%± 1.07%) were significantly increased (t=10.809~58.755, all P<0.05). Downregulation of miR-125b-5p attenuated the inhibition of NEAT1 silencing on the proliferation and migration of HemECs cells and the promotion of apoptosis (t=9.218~15.010, all P<0.05). there was a targeted regulatory relationship between NEAT1 and miR-125b-5p, and between miR-125b-5p and IGFBP5. Conclusion Silencing NEAT1 inhibits the expression of IGFBP5 by up-regulating miR-125b-5p, thereby inhibiting proliferation and migration of HemECs cells, and promoting cell apoptosis.

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备注/Memo

备注/Memo:
作者简介：廖烘（1985-），男，本科，主治医师，研究方向：儿科疾病基础研究，E-mail：a18207350536@126.com。
更新日期/Last Update: 2023-05-15