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浙江大学学报（农业与生命科学版）
浙江大学学报（农业与生命科学版）  2022, Vol. 48 Issue (4): 426-433    DOI: 10.3785/j.issn.1008-9209.2021.08.311
生物科学与技术     
敲降同源异型盒C8对7因子诱导的体细胞重编程的抑制作用
黄依1,2(),方仕才1,2,王波2,3,明金2,李陈2,裴端卿2,3()
1.广州医科大学-中国科学院广州生物医药与健康研究院联合生命科学学院，广州 511436
2.中国科学院广州生物医药与健康研究院，广州 510530
3.生物岛实验室（广州再生医学与健康广东省实验室），广州 510530
Inhibitory effects of knocking down homeobox C8 on seven factors-induced somatic cell reprogramming
Yi HUANG1,2(),Shicai FANG1,2,Bo WANG2,3,Jin MING2,Chen LI2,Duanqing PEI2,3()
1.Joint School of Life Sciences, Guangzhou Medical University and Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 511436, China
2.Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
3.Bio-land Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510530, China
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摘要：

为探究7因子（Jdp2-Jhdm1b-Mkk6-Glis1-Nanog-Esrrb-Sall4）诱导的体细胞重编程机制，通过正向与反向遗传学方法、定量分析重编程关键分子事件及同时敲降同源异型盒C8（homeobox C8, Hoxc8）和SMAD家族成员6（SMAD family member 6, Smad6）等方式解析Hoxc8在细胞多能性网络重建过程中的相关作用。结果表明：敲降Hoxc8可显著抑制7因子诱导的体细胞重编程，但过表达Hoxc8对其无影响。敲降Hoxc8既不影响重编程中多能性标记基因的上调与体细胞标记基因的下调，也不影响间质-上皮转化（mesenchymal-epithelial transition, MET）标记基因与细胞增殖标记基因的表达。同时敲降Hoxc8与Smad6可使单独敲降Hoxc8导致的重编程效率降低情况得到恢复。综上所述，Hoxc8在7因子诱导的体细胞重编程中具有重要作用，为进一步揭示Hoxc8调控细胞命运转变的机制提供了参考。
关键词： 体细胞重编程;  诱导多能干细胞;  同源异型盒C8;  SMAD家族成员6    
Abstract:

To explore the mechanism of seven factors (Jdp2-Jhdm1b-Mkk6-Glis1-Nanog-Esrrb-Sall4)-induced somatic cell reprogramming, we analyzed the related role of homeobox C8 (Hoxc8) in the process of pluripotency network reconstruction through forward and reverse genetics, quantitative analysis of the classic reprogramming process, and simultaneous knockdown of Hoxc8 and SMAD family member 6 (Smad6). The results showed that knockdown of Hoxc8 could significantly inhibit seven factors-induced somatic cell reprogramming, but overexpression of Hoxc8 had no effects. Furthermore, knockdown of Hoxc8 neither impeded the up-regulation of pluripotency marker genes and down-regulation of somatic cell marker genes nor impeded the expressions of mesenchymal-epithelial transition (MET) marker genes and cell proliferation marker genes. It was also found that simultaneous knockdown of Hoxc8 and Smad6 could rescue the inhibitory effects caused by knocking down Hoxc8 alone. In conclusion, these results suggest that Hoxc8 plays a pivotal role in somatic cell reprogramming, which provides a reference for further revealing the mechanism of Hoxc8 regulating cell fate transition.
Key words: somatic cell reprogramming    induced pluripotent stem cells    homeobox C8    SMAD family member 6
收稿日期: 2021-08-31 出版日期: 2022-09-03
CLC:  Q 291  
基金资助: 国家自然科学基金项目(3200050044);广东省自然科学基金面上项目(2019A1515012032)
通讯作者: 裴端卿     E-mail: huang_yi@gibh.ac.cn;pei_duanqing@gibh.ac.cn
作者简介: 黄依（https://orcid.org/0000-0002-6819-6957），E-mail：huang_yi@gibh.ac.cn
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引用本文:

黄依,方仕才,王波,明金,李陈,裴端卿. 敲降同源异型盒C8对7因子诱导的体细胞重编程的抑制作用[J]. 浙江大学学报（农业与生命科学版）, 2022, 48(4): 426-433.

Yi HUANG,Shicai FANG,Bo WANG,Jin MING,Chen LI,Duanqing PEI. Inhibitory effects of knocking down homeobox C8 on seven factors-induced somatic cell reprogramming. Journal of Zhejiang University (Agriculture and Life Sciences), 2022, 48(4): 426-433.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2021.08.311    或    https://www.zjujournals.com/agr/CN/Y2022/V48/I4/426

图1  敲降 Hoxc8 的结果
图2  过表达 Hoxc8 的结果A. 过表达Hoxc8和DsRed 7 d后Oct4-GFP阳性克隆图；B.图A中Oct4-GFP阳性克隆数目统计结果（与DsRed对照相比，ns表示在P＜0.05水平差异无统计学意义，n=3）。
图3  基因表达结果的qRT-PCR检测A.敲降Hoxc8对多能性标记基因表达的影响；B.敲降Hoxc8对间质-上皮转化标记基因表达的影响（Snai1、Twist2、Cdh2为间质细胞标记基因，Cdh1、Cldn3、Epcam为上皮细胞标记基因）；C.敲降Hoxc8对体细胞标记基因表达的影响；D.敲降Hoxc8对细胞增殖标记基因表达的影响。shHoxc8 610+698表示在7因子重编程中利用2条shRNA同时进行敲降。n=3。
图4  同时敲降 Smad6 和 Hoxc8 的结果A. shRNA敲降效率检测（与shLuciferase对照相比，****表示在P＜0.000 1水平差异有极高度统计学意义，n=3）；B.敲降Smad6时Oct4-GFP阳性克隆数目统计结果（与shLuciferase对照相比，ns表示在P＜0.05水平差异无统计学意义，n=3）；C.同时敲降shHoxc8和shSmad6 7 d后Oct4-GFP阳性克隆数目统计结果（与shLuciferase对照相比，ns表示在P＜0.05水平差异无统计学意义，n=3）；D.双萤光素酶报告基因系统检测结果（与pGL3-basic对照相比，**表示在P＜0.01水平差异有高度统计学意义，n=3）。
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