Lrrc34在精原干细胞中高表达,且为体外精原干细胞增殖的必需因子

欧金环; 李怡然; 王志鹏; 靳程; 李凯; 卢艳; 邹定峰; 李鹏宇; 李梦真; 缪时英; 王琳芳; 宋伟

doi:10.24920/003680

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Lrrc34在精原干细胞中高表达,且为体外精原干细胞增殖的必需因子

中国医学科学杂志（英文版） 2020年35卷第1期页码：20-30

Original Article | Updated：2024-04-10

- Lrrc34在精原干细胞中高表达,且为体外精原干细胞增殖的必需因子
- Affiliations：
  
  Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
- Author bio：
  
  * Tel: 86-13810749579, E-mail: songwei@ibms.pumc.edu.cn
- Funds：
  
  This work was supported by grants from the National Key Research and Development Program of China(2018YFC1003500);CAMS Innovation Fund for Medical Sciences(2017-I2M-3-009);CAMS Innovation Fund for Medical Sciences(2016-12M-1-001);CAMS Innovation Fund for Medical Sciences(2017-I2M-1-004);National Natural Science Foundation of China(81672472);National Natural Science Foundation of China(31970794);State Key Laboratory Special Fund from the Ministry of Science of China(2060204)
- DOI：10.24920/003680
  中图分类号：
- 收稿日期：2019-11-07，
  
  网络出版日期：2020-03-31，
  
  纸质出版日期：2020-01-20
- Accepted：
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欧金环, 李怡然, 王志鹏, 等. Lrrc34在精原干细胞中高表达,且为体外精原干细胞增殖的必需因子[J]. 中国医学科学杂志（英文版）, 2020,35(1):20-30.

Ou Jinhuan, Li Yiran, Wang Zhipeng, et al. Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro[J]. Chinese medical sciences journal, 2020, 35(1): 20-30.
欧金环, 李怡然, 王志鹏, 等. Lrrc34在精原干细胞中高表达,且为体外精原干细胞增殖的必需因子[J]. 中国医学科学杂志（英文版）, 2020,35(1):20-30. DOI： 10.24920/003680.

Ou Jinhuan, Li Yiran, Wang Zhipeng, et al. Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion In Vitro[J]. Chinese medical sciences journal, 2020, 35(1): 20-30. DOI： 10.24920/003680.

摘要

目的

探讨维持精原干细胞及其干细胞特性的关键基因及亮氨酸重复序列（LRR）家族成员Lrrc34在小鼠精原干细胞中的功能。

方法

应用差异表达基因（DEG）分析、基因本体（GO）分析和京都基因和基因组百科全书（KEGG）分析等生物信息学方法寻找潜在的多能性相关基因。分别采用逆转录聚合酶链式反应（RT-PCR）、免疫荧光（IF）检测Lrrc34在小鼠精原干细胞中的mRNA和蛋白质表达水平。采用RNA干扰实验研究Lrrc34敲低后对体外培养精原干细胞的瞬时效应。

结果

我们分别在ID4-EGFP

(G) 和 ID4-EGFP

/TSPAN8

High

(TH)、G和ID4-EGFP

/TSPAN8

Low

(TL)、TH 和 TL组发现了8、8、11个差异表达基因

并在这些差异表达基因组成的相互作用网络中发现了11个显著的相互作用模块。第五个相互作用模块中与纤维母细胞生长因子2存在相互作用的差异表达基因Lrrc34被选择作为多能性相关候选基因。免疫荧光实验显示Lrrc34特异表达于被LIN28A标记的未分化精原细胞的部分亚群中

RT-PCR证明了Lrrc34高表达于出生后7天和成年小鼠的精原干细胞中。Lrrc34的瞬时敲低导致体外培养的精原干细胞克隆减小

且其转录组和凋亡信号通路发生显著变化。

结论

lrrc34在小鼠精原干细胞中高表达;lrrc34是体外培养的精原干细胞增殖的必需因子

通过其转录组和信号通路调节精原干细胞体外扩增。

Abstract

Objective

To discover critical genes contributing to the stemness and maintenance of spermatogonial stem cells (SSCs) and provide new insights into the function of the leucine-rich repeat (LRR) family member Lrrc34 (leucine-rich repeat-containing 34) in SSCs from mice.

Methods

Bioinformatic methods

including differentially expressed gene (DEG)

gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses

were used to uncover latent pluripotency-related genes. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence analyses were utilized to verify the mRNA and protein expression levels

respectively. RNA interference of Lrrc34 using siRNA was performed to detect its transient impact on SSCs.

Results

Eight DEGs between ID4-EGFP

(G) and ID4-EGFP

/TSPAN8

High

(TH)

eight DEGs between G and ID4-EGFP

/TSPAN8

Low

(TL) and eleven DEGs between TH and TL were discovered

and eleven protein-protein interaction (PPI) modules were found to be significant in the PPI network of DEGs. One of the DEGs

Lrrc34

was selected as a potential pluripotency-related gene due to its differential expression among ID4-EGFP

spermatogonia subsets and its interaction with fibroblast growth factor 2 in the fifth module. Immunofluorescence experiments exhibited specific expression of Lrrc34 in a subpopulation of undifferentiated spermatogonia marked by LIN28A

and RT-PCR experiments confirmed the high expression of Lrrc34 in SSCs from P7 and adult mice. The transient knockdown of Lrrc34 in SSCs resulted in reduced colony sizes and significant changes in the transcriptome and apoptotic pathways.

Conclusion

Lrrc34 is highly expressed in mouse SSCs and is required for SSC proliferation

in vitro

through effects on transcriptome and signaling transduction pathways.

关键词

Keywords

references

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