Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (19): 3471-3484.doi: 10.3864/j.issn.0578-1752.2019.19.016

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Construction of Co-expression Network of lncRNA and mRNA Related to Hair Follicle Development of Subo Merino Sheep

CHEN HuaFeng1,2,TIAN KeChuan2(),HUANG XiXia1(),Ablat Sulayman1,HE JunMin3,TIAN YueZhen2,XU XinMing2,FU XueFeng2,ZHAO BingRu4,ZHU Hua1,Hanikezi Tulafu2   

  1. 1 College of Animal Science, Xinjiang Agricultural University, Urumqi 830052
    2 Institute of Animal Science, Xinjiang Academy of Animal Science, Urumqi 830011
    3 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070
    4 College of Animal Science and Technology, China Agricultural University, Beijing 100083
  • Received:2018-12-19 Accepted:2019-07-03 Online:2019-10-01 Published:2019-10-11
  • Contact: KeChuan TIAN,XiXia HUANG E-mail:tiankechuan@163.com;au-huangxixia@163.com

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Abstract:

【Objective】 With the development and improvement of high-throughput sequencing technology, massive transcriptome sequencing data has emerged, and more and more methods of gene network have been used in research; the hair follicles development of fine wool sheep is regulated by multiple genes and lncRNAs. It is not enough to study a certain molecule alone to discover its regulatory mechanism. The aim of this study was to construct a co-expression network of lncRNAs and mRNAs related to hair follicle development, and to explore potential candidate genes for hair follicle development. 【Method】 The study was performed on fetal skin tissues of the 65th, 85th, 105th, and 135th days and lambs skin tissues of the 7th and 30th days from Subo Merino sheep, with 3 biological replicates in each period, a total of 18 samples were sequenced by transcriptome to obtain lncRNAs and mRNAs expression profiles of 6 different developmental stages, and the differential expressed lncRNAs and mRNAs in adjacent periods was screened to construct a co-expression module by Weighted Gene Co-expression Network Analysis (WGCNA) method, and GO (Gene Ontology) and KEGG pathway enrichment analysis of DAVID online tool was used to find hair follicle related modules. Finally, the high-interconnect lncRNAs and mRNAs were screened from the target module for network visualization using Cytoscape software.【Result】 From the expression profile data, 9070 differentially expressed lncRNAs and mRNAs were screened, and 11 modules were obtained by WGCNA method. The DAVID enrichment analysis revealed that the genes in the honeydew1 module, paleturquoise module, and skyblue2 module were involved in biological processes such as skin development, hair follicle development, hair follicle morphogenesis, negative regulation of Wnt signaling pathway, and cell adhesion, et al; and the signaling pathways involved in hair follicle development such as Wnt signaling pathway, TGF-β signaling pathway, Hedgehog signaling pathway, tight junction, MAPK signaling pathway, ECM-receptor interaction, et al. The sub-networks of lncRNAs and mRNAs with high connectivity were screened in these 3 modules, and the hair follicle development related genes including TGFB2, CTSB, SFN, SPINT1, FAM83G, GSDMA, MPZL3, VIM and CRABP1 were obtained, and possible target genes of 24 lncRNAs including ENSOART00000029117, TCONS_00489976, TCONS_00376759 were predicted.【Conclusion】 In this study, we first constructed a co-expression network of lncRNAs and mRNAs related to the hair follicles development of Subo merino sheep using WGCNA method, identified 3 co-expression modules related to hair follicle development, and found several potential candidate genes related to hair follicle development, and predicted possible target genes of 24 lncRNAs.

Key words: Subo Merino sheep, WGCNA, mRNA, lncRNA, hair follicle development

Table 1

Statistics of differential expression of lncRNAs and mRNAs"

D85
vs.
D65		 D105
vs.
D85		 D135
vs.
D105		 A7
vs.
D135		 A30
vs.
A7		 去重复
Deduplication
差异表达lncRNA数 No. DE lncRNAs 35 55 46 43 25 140
差异表达已知mRNA数 No. DE known mRNAs 1562 2302 1520 699 166 4372
差异表达novel mRNA数 No. DE novel mRNAs 1762 2066 611 842 28 4558

Fig. 1

Screening of soft thresholds"

Fig. 2

Gene clustering tree and module construction"

Table 2

Number of genes and lncRNAs in co-expressing network module"

mRNA 新mRNA Novel mRNA lncRNA 总计 Total
bisque4 55 8 2 65
brown4 66 2 0 68
cyan 309 120 6 435
darkolivegreen 24 217 1 242
darkorange 206 1402 13 1621
greenyellow 380 151 6 537
grey 224 206 33 463
honeydew1 665 1188 14 1867
paleturquoise 1483 265 38 1786
skyblue2 890 980 23 1893
steelblue 70 19 4 93

Fig. 3

GO enrichment bubble diagram of target module"

Table 3

Part GO enrichment results of the target modules"

模块
Module		 ID GO条目
GO Term		 基因数
No. genes		 P值
P value		 基因
Genes
honeydew1 GO:0090090 经典Wnt信号通路负调控
Negative regulation of canonical Wnt signaling pathway		 12 4.26E-06 WNT5A, CTHRC1, SOX10, NOG, SOST, SFRP2, DRAXIN, SOX2, LIMD1, LRP4, MLLT3, GLI1
GO:0043588 皮肤发育 Skin development 6 5.74E-04 FRAS1, TFAP2B, PTCH2, LTB, COL5A2, COL5A1
GO:0050680 上皮细胞增殖负调控
Negative regulation of epithelial cell proliferation		 6 3.77E-03 WNT5A, HPN, SFRP2, SOX2, PTCH1, TGFB2
GO:0001942 毛囊发育 Hair follicle development 5 7.28E-03 INHBA, EDAR, ALX4, LRP4, TGFB2
GO:0030514 BMP信号通路负调控
Negative regulation of BMP signaling pathway		 5 1.28E-02 WNT5A, NOG, SOST, BMPER, SFRP2
GO:0007155 细胞粘附
Cell adhesion		 8 2.24E-02 LAMA1, HES5, TLN2, ITGA11, CNTN2, CNTN4, THBS2, COL5A1
paleturquoise GO:0043588 皮肤发育
Skin development		 6 2.68E-02 KRT9, COL3A1, ITGB4, ADAMTS2, ASPRV1, DHCR24
GO:0031069 毛囊形态发生 Hair follicle morphogenesis 5 1.89E-02 KRT25, KRT27, FOXE1, KRT71, TMEM79
GO:0007173 表皮生长因子受体信号通路
Epidermal growth factor receptor signaling pathway		 6 1.62E-02 FAM83B, EREG, HBEGF, TGFA, AREG, SOX9
GO:0001942 毛囊发育
Hair follicle development		 6 4.11E-02 DSG4, ZDHHC21, FOXN1, SOX9, DNASE1L2, LGR5
skyblue2 GO:0090090 经典Wnt信号通路负调控
Negative regulation of canonical Wnt signaling pathway		 9 1.49E-02 WNT4, NOTCH1, WNT5B, SFRP4, FZD1, FOXO1, ISL1, FRZB, PTPRO
GO:0007155 细胞粘附
Cell adhesion		 16 4.20E-05 CTNNAL1, HAPLN1, TNC, COL15A1, ITGA3, POSTN, STAB2, GRHL2, TNFAIP6, ITGA9, LYVE1, TGFBI, VCAN, CNTN3, NCAN, SPP1

Fig. 4

KEGG enriched bubble diagram of target module"

Table 4

Part KEGG enrichment results of the target modules"

模块
Module		 ID KEGG条目
KEGG Term		 基因数
No. genes		 P值
P value		 基因
Genes
honeydew1 oas04512 ECM-受体相互作用
ECM-receptor interaction		 6 2.30E-02 LAMA1, ITGA11, COL2A1, THBS2, COL5A2, COL5A1
oas04510 粘着力
Focal adhesion		 11 4.72E-03 LAMA1, TLN2, PGF, ITGA11, PIK3R5, COL2A1, HGF, PIK3R3, THBS2, COL5A2, COL5A1
oas04340 Hedgehog信号通路
Hedgehog signaling pathway		 5 1.11E-03 PTCH1, PTCH2, HHIP, GLI2, GLI1
paleturquoise oas04530 紧密连接
Tight junction		 14 1.63E-02 CLDN8, PPP2R1B, PARD6B, CLDN4, MYL10, CLDN10, CLDN14, LLGL2, EPB41L3, IGSF5, CLDN1, MYH14, TJP3, MYH7B
oas04010 MAPK信号通路
MAPK signaling pathway		 24 4.05E-03 IL1R2, FGF5, MAPKAPK3, TGFB3, NR4A1, FGF22, FGF21, FGF12, FLNC, CACNA2D3, DUSP4, MAP3K6, FOS, MAP3K5, DUSP2, HSPA2, DUSP1, MAPK13, RPS6KA2, HSPB1, GADD45B, PLA2G4E, CD14, NGF
skyblue2 oas04512 ECM-受体相互作用
ECM-receptor interaction		 18 6.93E-10 COL4A4, COL4A3, TNC, ITGA3, COL5A3, ITGA9, LAMB3, COL6A6, LAMC3, LAMA5, COL6A5, ITGB6, RELN, SV2A, THBS1, COL24A1, SPP1, FN1
oas04310 Wnt信号通路
Wnt signaling pathway		 9 4.50E-02 WNT2, WNT10A, WNT4, WNT5B, DKK1, SFRP4, MMP7, FZD1, WIF1

Table 5

Top 5 mRNA and lncRNA sorted by degree"

honeydew1 paleturquoise skyblue2
基因名Gene name 度Degree 基因名Gene name 度Degree 基因名Gene name 度Degree
mRNA XLOC_170356 876 TEC 1462 XLOC_001041 915
ANGPT1 861 GRHL1 1455 CDH11 909
XLOC_074918 854 GSDMA 1451 POSTN 880
XLOC_024093 833 CLDN4 1448 ENSOARG00000012813 842
XLOC_106735 831 GABRP 1444 TNFAIP6 830
lncRNA ENSOART00000029117 391 TCONS_00489976 1079 TCONS_00041508 481
TCONS_00236664 104 TCONS_00346829 1074 TCONS_00008348 450
TCONS_00041597 61 TCONS_00039510 958 TCONS_00352973 362
TCONS_00453233 39 TCONS_00570568 876 TCONS_00497279 323
TCONS_00351448 34 ENSOART00000027499 813 TCONS_00127951 225

Fig. 5

The co-expression network of differentially expressed genes and lncRNAs in honeydew1 module The size of the dot represents the level of connectivity"

Fig. 6

The co-expression network of differentially expressed genes and lncRNAs in paleturquoise module The size of the dot represents the level of connectivity"

Fig. 7

The co-expression network of differentially expressed genes and lncRNAs in skyblue2 module The size of the dot represents the level of connectivity"

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