Transcriptomic Analysis of PTH 1-34 on the Osteogenesis Mechanism of Antler Mesenchymal Stem Cells

doi:10.7668/hbnxb.20194001

Abstract

Abstract:

PTH 1-34 has an osteogenic effect on antler MSCs.The study aimed to explore the possible mechanism of PTH 1-34 promoting osteogenesis in antler MSCs by using the high-throughput non-reference transcriptome sequencing method.The 3rd generation antler MSCs were randomly divided into three groups(control group,induction group,and PTH 1-34 intervention group)with three replicates each.After the identification of markers and calcium nodules,total RNA was extracted using the TRIzol method and these libraries were double-end sequenced using Next-Generation Sequencing,an Illumina-based sequencing platform.Addition of 10 nmol/L PTH 1-34 for osteogenesis induction significantly increased alkaline phosphatase activity and calcium nodule production in antler mesenchymal stem cells.The non-reference transcriptome sequencing was selected due to less than 50% efficiency of gene sequence comparison with deer family published by NCBI.Through differential gene enrichment analysis,a total of 5 737 differentially expressed genes were screened,of which 1 877 were up-regulated and 3 860 were down-regulated;GO enrichment analysis showed that the types of differential genes mainly included cell motility,proliferation and migration,bone development,angiogenesis and morphogenesis related under the action of biological processes;through screening,eight genes related to osteogenesis were found:RUNX2,BGN,ALPL,SPARC,OPN,CPNE1,STRN4,VAPB;two genes related to cellular autophagy:WiPi2,ATG14;four genes related to angiogenesis:UNC5B,SHC1,FLNA,TNFSF15,which were validated using qRT-PCR method,and the results were consistent with the sequencing results;transcription factors with high predicted correlation family was zf-C2H2,and the osteogenic transcription factor SP7 was screened.PTH 1-34 could promote osteogenic differentiation of antler mesenchymal stem cells by upregulating the mRNA expression of RUNX2,SP7,BGN,ALPL,SPARC,OPN,CPNE1,STRN4,VAPB;possibly regulating angiogenesis and cellular autophagy by inhibiting the process of antler ossification.

Key words: Deer antler, Parathyroid hormone 1-34, Mesenchymal stem cell, Osteogenesis induction, Transcriptomics

XING Baorui, ZHAO Haiping, LI Guangyu, MA Zefang, SUN Hongmei, HU Xiao, TAN Zhanqing, LIU Zhen. Transcriptomic Analysis of PTH 1-34 on the Osteogenesis Mechanism of Antler Mesenchymal Stem Cells[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 344-352. doi: 10.7668/hbnxb.20194001.

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Figures/Tables 9

References 47

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样品 Sample	有效条目总数 Clean reads No.	有效数据/bp Clean data	有效条目占比/% Clean reads	有效数据占比/% Clean data
诱导组-1 Induction-1	44 172 308	6 670 018 508	93.45	93.45
诱导组-2 Induction-2	45 872 296	6 926 716 696	93.70	93.70
诱导组-3 Induction-3	45 166 788	6 820 184 988	93.97	93.97
PTH 1-34干预组-1 PTH 1-34 intervention-1	44 298 146	6 689 020 046	94.13	94.13
PTH 1-34干预组-2 PTH 1-34 intervention-2	46 686 304	7 049 63 1904	93.84	93.84
PTH 1-34干预组-3 PTH 1-34 intervention-3	45 340 436	6 846 405 836	93.72	93.72

样品 Sample	有效条目总数 Clean reads No.	有效数据/bp Clean data	有效条目占比/% Clean reads	有效数据占比/% Clean data
诱导组-1 Induction-1	44 172 308	6 670 018 508	93.45	93.45
诱导组-2 Induction-2	45 872 296	6 926 716 696	93.70	93.70
诱导组-3 Induction-3	45 166 788	6 820 184 988	93.97	93.97
PTH 1-34干预组-1 PTH 1-34 intervention-1	44 298 146	6 689 020 046	94.13	94.13
PTH 1-34干预组-2 PTH 1-34 intervention-2	46 686 304	7 049 63 1904	93.84	93.84
PTH 1-34干预组-3 PTH 1-34 intervention-3	45 340 436	6 846 405 836	93.72	93.72

数据库 Database	数目 Number	占比/% Percentage
NR	74 599	32.25
GO	64 944	28.08
KEGG	39 979	17.28
Pfam	18 891	8.17
eggNOG	72 335	31.27
Swissprot	43 865	18.96
所有数据库In all database	13 198	5.71

数据库 Database	数目 Number	占比/% Percentage
NR	74 599	32.25
GO	64 944	28.08
KEGG	39 979	17.28
Pfam	18 891	8.17
eggNOG	72 335	31.27
Swissprot	43 865	18.96
所有数据库In all database	13 198	5.71

基因 Gene	名称 Name	生物功能 Biological function	文献 References
RUNX2	Runt相关转录因子2	成骨细胞分化和骨骼形态发生的转录因子,对成骨细胞成熟和软骨内成骨必不可少	[26⇓—28]
SP7/Osterix	转录因子Sp7	成骨细胞分化必需的转录激活因子	[29—30]
OPN/OCN	骨钙素	是骨矿物代谢的重要指标,与磷灰石和钙紧密结合	[31]
ALPL	碱性磷酸酶	代谢各种磷酸盐化合物,促进羟基磷灰石结晶的形成,在骨骼矿化中起关键作用	[32—33]
SPARC	骨连蛋白	成骨标记物,通过与细胞外基质和细胞因子的相互作用来调节细胞生长,与钙结合来促进成骨	[34]
BGN/SLRR1A	双糖链蛋白聚糖	成骨标记物,在维持骨稳态和骨重塑过程中发挥重要作用	[35]
CPNE1	钙离子依赖性的磷脂结合蛋白1	钙依赖性磷脂结合蛋白,在钙介导的细胞内过程中起作用	[36]
STRN4	钙调蛋白结合蛋白4	以钙依赖性的方式结合钙调素,可能起支架或信号蛋白的作用	[37]
VAPB	囊泡相关膜蛋白相关蛋白B/C	参与细胞钙稳态调节	[38]
WiPi2	WD重复结构域磷酸肌醇互作蛋白2	自噬机制的组成部分,控制细胞内主要降解过程,在此过程中,细胞质物质被包装成自噬体并递送到溶酶体进行降解	[39—40]
ATG14	自噬相关蛋白14	促进自噬体-溶酶体复合物的形成	[41—42]
UNC5B	网状蛋白受体UNC5B	在血管生成过程中作为网蛋白受体负调控血管分支	[43]
SHC1	SHC转化蛋白1	参与血管生成素受体TEK/TIE2下游信号传导,在内皮细胞迁移和芽生血管生成中发挥调节作用。促进破骨细胞的生成	[44—45]
FLNA	细丝蛋白A	在血管、心脏和大脑器官的发育过程中,在细胞和细胞的接触和黏附连接中起作用	[46]
TNFSF15	肿瘤坏死因子配体超家族成员15	介导NF-kappa-B的激活,抑制血管内皮生长和血管生成	[47]

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