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Lrrc34 Is Highly Expressed in SSCs and Is Necessary for SSC Expansion
In Vitro - 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：
DOI：10.24920/003680
CLC：Received：07 November 2019，
Published Online：31 March 2020，
Published：20 January 2020
- Accepted：
Scan QR Code
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.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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