上海交通大学学报（医学版）

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2022 , Vol. 42 >Issue 5: 578 - 582

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.05.004

论著 · 基础研究

长链非编码RNA-B230352I09表达改变对H9C2心肌细胞增殖及周期的影响

  • 徐斐翔 ,
  • 汪升 ,
  • 薛明明 ,
  • 童朝阳 ,
  • 陈玉梅
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  • 复旦大学附属中山医院急诊科，上海 200032
徐斐翔（1991—），男，住院医师，学士；电子信箱：xu.feixiang@zs-hospital.sh.cn。
陈玉梅，电子信箱：chen.yumei@zs-hospital.sh.cn。

收稿日期: 2022-02-14

  录用日期: 2022-05-16

  网络出版日期: 2022-05-28

基金资助

国家自然科学基金(81800230)

收起

Effect of altered expression of long non-coding RNA-B230352I09 on proliferation and cycle of H9C2 cardiomyocytes

  • Feixiang XU ,
  • Sheng WANG ,
  • Mingming XUE ,
  • Chaoyang TONG ,
  • Yumei CHEN
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  • Department of Emergency Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
CHEN Yumei, E-mail: chen.yumei@zs-hospital.sh.cn.

Received date: 2022-02-14

  Accepted date: 2022-05-16

  Online published: 2022-05-28

Supported by

National Natural Science Foundation of China(81800230)

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摘要

目的·探讨长链非编码RNA-B230352I09（long non-coding RNA-B230352I09，lncRNA-B230352I09）表达改变对H9C2心肌细胞增殖及周期的影响。方法·构建lncRNA-B230352I09过表达载体（pcDNA-B230352I09）和阴性对照（negative control，NC）载体（pcDNA-NC），借助阳离子脂质体Lipofectamine 3000（Lipo3000）转染液将上述构建的载体转染至H9C2心肌细胞。实验分为空白对照组、阴性对照组、lncRNA-B230352I09过表达组，分别通过实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction，RT-PCR）检测lncRNA-B230352I09的表达量，验证其转染效率；细胞计数试剂盒8（cell counting kit-8，CCK8）检测H9C2心肌细胞在450 nm波长的吸光度（optical density，OD）并绘制生长曲线；5-乙炔基-2'-脱氧尿嘧啶（5-ethynyl-2'-deoxyuridine，EdU）标记增殖期H9C2心肌细胞并通过荧光显微镜观察增殖心肌细胞的数量；流式细胞术分析H9C2心肌细胞在不同细胞周期所占比例；RT-PCR检测H9C2细胞周期相关调控基因的表达情况。结果·与阴性对照组比较，lncRNA-B230352I09过表达组中lncRNA-B230352I09的表达水平显著升高（P＝0.000），提示lncRNA-B230352I09转染H9C2心肌细胞模型构建成功。CCK8实验显示，与阴性对照组相比，lncRNA-B230352I09过表达可显著提高H9C2心肌细胞的OD值，促进H9C2心肌细胞增殖，表现为明显的时间依赖性细胞增殖能力增强（24 h：P=0.000；48 h：P=0.000；72 h：P=0.001）；荧光显微镜观察发现，与阴性对照组相比，lncRNA-B230352I09过表达组中EdU标记阳性的H9C2心肌细胞比例明显增多；流式细胞术分析显示，和阴性对照组比较，lncRNA-B230352I09过表达组中处于S期的H9C2心肌细胞比例明显增加，G1期心肌细胞的比例下降（P＝0.000）；RT-PCR检测结果显示，与阴性对照组比较，lncRNA-B230352I09过表达组中H9C2心肌细胞周期蛋白D1（cyclin D1）和细胞周期蛋白依赖性激酶1（cyclin dependent protein kinase 1，CDK1）的mRNA表达水平显著升高（P＝0.000）。结论·lncRNA-B230352I09通过调节cyclin D1和CDK1促进心肌细胞进入S期，增强H9C2心肌细胞增殖能力。

关键词： H9C2心肌细胞; 长链非编码RNA-B230352I09; 过表达; 细胞增殖; 细胞周期

本文引用格式

徐斐翔 , 汪升 , 薛明明 , 童朝阳 , 陈玉梅 . 长链非编码RNA-B230352I09表达改变对H9C2心肌细胞增殖及周期的影响[J]. 上海交通大学学报（医学版）, 2022 , 42(5) : 578 -582 . DOI: 10.3969/j.issn.1674-8115.2022.05.004

Abstract

Objective

·To investigate the effect of altered expression of long non-coding RNA (lncRNA)-B230352I09 on proliferation and cycle of H9C2 cardiomyocytes.

Methods

·The lncRNA-B230352I09 overexpression vector (pcDNA-B230352I09) and the negative control vector pcDNA-negative control group (NC) were constructed and transfected into H9C2 cardiomyocytes with the Lipofectamine 3000 (Lipo3000) transfection solution. The expression of lncRNA-B230352I09 was measured by real-time fluorescence quantitative polymerase chain reaction (RT-PCR) to verify its transfection efficiency. The H9C2 cardiomyocytes were divided into blank control group, pcDNA-NC group, and lncRNA-B230352I09 overexpression group. Cell counting kit-8 (CCK8) was used to measure the absorbance (optical density, OD) of H9C2 cardiomyocytes at a wavelength of 450 nm and draw a growth curve; 5-ethynyl-2'-deoxyuridine (EdU) was used to label proliferating H9C2 cardiomyocytes, the numbers of which were observed by fluorescence microscopy. Cardiomyocyte cycle was assessed by flow cytometry and cycle-related genes expression was measured by RT-PCR.

Results

·Compared with the pcDNA-NC group, the expression level of lncRNA-B230352I09 was significantly higher in the lncRNA-B230352I09 overexpression group (P＝0.000), suggesting that the lncRNA-B230352I09-transfected H9C2 cardiomyocyte model was successfully constructed. Compared with the pcDNA-NC group, the CCK8 assay showed that lncRNA-B230352I09 overexpression significantly increased the OD of H9C2 cardiomyocytes and promoted the proliferation of H9C2 cardiomyocytes with a significant time-dependent enhancement of cell proliferation (24 h: P=0.000; 48 h: P=0.000; 72 h: P=0.001). Fluorescence microscopy revealed that the proportion of EdU-labeled positive H9C2 cardiomyocytes was significantly increased in the lncRNA-B230352I09 overexpression group compared to the pcDNA-NC group. Flow cytometric analysis showed a significant increase in the proportion of H9C2 cardiomyocytes in S phase and a decrease in G1 phase in the lncRNA-B230352I09 overexpression group compared to the pcDNA-NC group (P＝0.000). RT-PCR showed that the mRNA expression levels of cyclin D1 and cyclin dependent protein kinase 1 (CDK1) in the lncRNA-B230352I09 overexpression group were significantly higher compared with the pcDNA-NC group (P＝0.000).

Conclusion

·lncRNA-B230352I09 can enhance myocardial proliferative capacity by regulating cyclin D1 and CDK1 to promote the entry of cardiomyocytes to into S phase.

Key words： H9C2 cardiomyocyte; lncRNA-B230352I09; overexpression; cell proliferation; cell cycle

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