Apbb1 基因对心肌细胞增殖影响的探究
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1.山西医科大学基础医学院 生物化学与分子生物学教研室,太原 030000;2.河北燕达陆道培医院检验科, 河北 廊坊 065201;3.中国医学科学院,北京协和医学院,国家心血管疾病中心,动物实验中心, 心血管植入材料临床前研究评价北京市重点实验室,北京 102300

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R-33


The effect of Apbb1 gene on cardiomyocyte proliferation
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1.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030000, China. 2. Department of Laboratorial Medicine, Hebei Yanda LU Dao-Pei Hospital, Langfang 065201. 3. Beijing Key Laboratory of Preclinical Research and Evaluation for Cardiovascular Implant Materials, Animal Experimental Centre, Fuwai Hospital, National Centre for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 102300

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    摘要:

    目的 探究 β 淀粉样蛋白前体蛋白结合蛋白 B-1(Apbb1)基因对 H9c2 心肌细胞增殖的影响。 方法 利用小干扰RNA 与 pcDNA3. 1+质粒对 H9c2 心肌细胞系进行敲低与过表达 Apbb1 基因处理,通过 CCK8 实验、 qPCR 实验以及细胞免疫荧光实验检测细胞增殖情况,评价 Apbb1 基因对心肌细胞增殖的作用;利用在线数据库 String 分析 Apbb1 蛋白的互作蛋白,探究其可能发挥作用的方式。 实验分为 4 组:敲低对照组、敲低 Apbb1 组、过表达对照组和过表达 Apbb1 组。 结果 相较于敲低对照组,敲低 Apbb1Apbb1 基因表达下降 52%(P<0.01,n= 3) ,细胞增殖在 48 h 时下降 44%(P<0.01,n= 3) ,但是在 72 h 时细胞增殖增加 86%(P<0.0001,n= 3) ;细胞周期相关基因 Ccnb1Ccna2 mRNA 表达水平分别升高 80%(P<0.0001,n=3)和 76%(P<0.0001,n= 3) ;细胞免疫荧光实验发现 PH3 与 Aurora B 阳性细胞分别增加 4. 33%(P<0.01,n= 3)和 4. 67%(P<0.05,n= 3) 。 相较于 过表达对照组,过表达 Apbb1Apbb1 基因表达上调 119 倍(P<0.001,n= 3) ,细胞增殖在 48 h 与 72 h 分别降低 1. 23 倍(P<0.0001,n= 3)和 1. 04 倍(P<0.0001,n= 3) ;细胞增殖标志分子 Ki67 和细胞周期相关基因 Ccnb1Ccna2 mRNA 表达水平分别降低 25%(P< 0.01,n= 3) 、72% ( P< 0.001,n= 3) 和 38% ( P< 0.001,n= 3) ;PH3 与 Aurora B 阳性细胞分别降低 3. 7% ( P< 0.01,n= 3) 和 4. 36% ( P< 0.05,n= 3) 。 蛋白互作网络分析结果表明 KAT5、APP 与 APLP2 是 Apbb1 互作最为显著的蛋白。 结论 敲低 Apbb1 基因促进 H9c2 心肌细胞增殖,过表达 Apbb1 基因抑制 H9c2 心肌细胞增殖,Apbb1 可能通过影响细胞周期 G2 / M 期影响心肌细胞增殖,KAT5 蛋白可能与 Apbb1 蛋白互作影响心肌细胞增殖。

    Abstract:

    Objective To investigate the effect of the amyloid beta precursor protein binding protein B1 (Apbb1) gene on H9c2 cardiomyocyte proliferation and its potential mechanism. Methods H9c2 cardiomyocytes were treated with small interfering RNA and pcDNA3. 1+ plasmid for knockdown and overexpression of the Apbb1 gene, and cell proliferation was detected by CCK8, qPCR and immunofluorescence assay, to evaluate the effect of this gene on cardiomyocyte proliferation. Next, the proteins interacting with the Apbb1 protein were analyzed using the online database String to explore how Apbb1 might function. In the experiment, four groups were established: knockdown control group, Apbb1- knockdown group, overexpression control group and Apbb1-overexpression group. Results Compared with that in the knockdown control group, expression of the Apbb1 gene in the Apbb1-knockdown group decreased by 52% (P<0.05, n= 3) and cell proliferation decreased by 44% at 48 h (P<0.01, n= 3), but cell proliferation increased by 86% at 72 h (P< 0.0001, n= 3). The expression levels of Ccnb1 and Ccna2 mRNA were increased by 80% (P<0.001, n= 3) and 76% (P<0.001, n= 3), respectively. Immunofluorescence assay showed that PH3- and Aurora B-positive cells increased by 4. 33% (P< 0.01, n= 3) and 4. 67% (P< 0.05, n= 3). Compared with that in the overexpression control group, the expression of the Apbb1 gene was upregulated 119-fold in the Apbb1-overexpression group (P<0.0001, n= 3), and cell proliferation decreased by 1. 23 times at 48 h (P< 0.0001, n= 3) and 1. 04 times at 72 h ( P< 0.0001, n= 3). The expression levels of Ki67, a marker of cell proliferation, and Ccnb1 and Ccna2 mRNA encoded by cell cycle-related genes were decreased by 25% ( P< 0.01, n= 3), 72% ( P< 0.001, n= 3), and 38% ( P< 0.001, n= 3), respectively. Meanwhile, PH3- and Aurora B-positive cells decreased by 3. 7% (P< 0.01, n= 3) and 4. 36% (P< 0.05, n= 3), respectively. The result of protein interaction network analysis showed that KAT5, APP and APLP2 were the proteins that most significantly interacted with Apbb1. Conclusions Knockdown of the Apbb1 gene promotes the proliferation of H9c2 cardiomyocytes, while its overexpression inhibits their proliferation. Apbb1 may affect the proliferation of cardiomyocytes by influencing the G2 / M phase of the cell cycle and KAT5 may interact with Apbb1 protein to affect cardiomyocyte proliferation .

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刘 俊,孙 佳,刘维静,郝琰琰,廉 虹,王玉瑶.Apbb1 基因对心肌细胞增殖影响的探究[J].中国比较医学杂志,2022,32(5):29~38.

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  • 收稿日期:2021-11-18
  • 在线发布日期: 2022-07-05
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