Inhibitory effects of knocking down homeobox C8 on seven factors-induced somatic cell reprogramming

doi:10.3785/j.issn.1008-9209.2021.08.311

Journal of Zhejiang University (Agriculture and Life Sciences)

2022, Vol. 48

Issue (4): 426-433 DOI: 10.3785/j.issn.1008-9209.2021.08.311

Biological sciences & biotechnologies

Inhibitory effects of knocking down homeobox C8 on seven factors-induced somatic cell reprogramming

Yi HUANG^1,²(

),Shicai FANG^1,²,Bo WANG^2,³,Jin MING²,Chen LI²,Duanqing PEI^2,³(

)

^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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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

Received: 31 August 2021 Published: 03 September 2022

CLC:

Q 291

Corresponding Authors: Duanqing PEI E-mail: huang_yi@gibh.ac.cn;pei_duanqing@gibh.ac.cn

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	Yi HUANG
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Cite this article:

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.

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

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

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

Fig. 1 Results of knocking down Hoxc8

Fig. 2 Results of overexpressing Hoxc8A. Images for Oct4-GFP+ generated by overexpressing Hoxc8 and DsRed, respectively after 7 d; B. Numbers of Oct4-GFP+ from Fig. A (compared with the DsRed control, the symbol ns indicates no significant differences at the 0.05 probability level, n=3).

Fig. 3 Gene expression results detected by qRT-PCRA. Effects of knocking down Hoxc8 on the expression of pluripotency marker genes; B. Effects of knocking down Hoxc8 on the expression of mesenchymal-epithelial transition marker genes (Snai1, Twist2 and Cdh2 are mesenchymal cell marker genes, and Cdh1, Cldn3 and Epcam are epithelial cell marker genes); C. Effects of knocking down Hoxc8 on the expression of somatic cell marker genes; D. Effects of knocking down Hoxc8 on the expression of cell proliferation marker genes. shHoxc8 610+698 indicates the simultaneous knockdown of two shRNAs in seven factors’ reprogramming. n=3.

Fig. 4 Results of simultaneous knockdown of Smad6 and Hoxc8A. Knockdown efficiency of shRNA [compared with the shLuciferase control, quadruple asterisks (****) indicate extremely highly significant differences at the 0.000 1 probability level, n=3]; B. Numbers of Oct4-GFP+ when knocking down Smad6 (compared with the shLuciferase control, the symbol ns indicates no significant differences at the 0.05 probability level, n=3); C. Numbers of Oct4-GFP+ after 7 d of simultaneous knockdown of shHoxc8 and shSmad6 (compared with the shLuciferase control, the symbol ns indicates no significant differences at the 0.05 probability level, n=3); D. Results of Dual-Luciferase Reporter Assay System [compared with the pGL3-basic control, double asterisks (**) indicate highly significant differences at the 0.01 probability level, n=3].


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