miRNAs-MUC13调控体系中与仔猪腹泻有关的基因与通路

doi:10.3969/j.issn.1004-1524.20230088

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (12): 2785-2793.DOI: 10.3969/j.issn.1004-1524.20230088

miRNAs-MUC13调控体系中与仔猪腹泻有关的基因与通路

路伏增(), 华卫东, 褚晓红, 戴丽荷, 陈晓宇, 章利丰, 王志刚, 徐如海()

浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2023-01-29 出版日期:2023-12-25 发布日期:2023-12-27
作者简介:路伏增(1979—),男,河南林州人,博士,副研究员,研究方向为猪育种。E-mail:1261445120@qq.com
通讯作者: *徐如海,E-mail:ruhaixu@163.com
基金资助:
浙江省农业新品种选育重大科技专项(2021C02068-4);国家重点研发计划子任务(2021YFD2000801);浙江省2021—2023年畜牧产业技术项目(2022R22S60D02);浙江省基础公益研究计划(LGN22C170009)

Genes and pathways related to piglet diarrhea in miRNAs-MUC13 regulation system

LU Fuzeng(), HUA Weidong, CHU Xiaohong, DAI Lihe, CHEN Xiaoyu, ZHANG Lifeng, WANG Zhigang, XU Ruhai()

Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-01-29 Online:2023-12-25 Published:2023-12-27

摘要/Abstract

摘要：

为研究microRNAs-Mucin13(miRNAs-MUC13)调控体系中与仔猪腹泻相关的分子调控机制,对miRNAs-MUC13调控体系中与仔猪腹泻有关的基因与通路进行了发掘与验证。用NCBI的blastn工具比对了小鼠MUC13基因和猪基因库中的核酸序列,发现两者的相似性在74%~87%,同源性较高。以小鼠为模型,选择miRDB、mirTarBase、TargetScan数据库预测调控MUC13基因的miRNA序列,通过StarBase数据库预测这些miRNA序列的靶基因,得到36个靶基因交集。用Cytoscape软件围绕MUC13和这36个靶基因构建仔猪腹泻基因互作网络,该网络包含19个筛选到的靶基因,基于MCOMD算法分析获得4个分子复合物集团。通过DAVID平台对这4个分子复合物进行基因和通路富集分析,结果发现,靶基因SRF和STAG2及其参与的有关通路与仔猪腹泻相关。以大肠埃希菌200和仔猪肠上皮细胞系(IPEC-1)作为试验材料,通过体外细胞试验设计仔猪腹泻模型进行验证,实时荧光定量PCR(qRT-PCR)检测结果显示,试验组MUC13、SRF、IL-6、MAPK、STAG2、NF-κB、TLR7等基因的表达量均显著(P<0.05)高于对照组,由此推测,SRF与STAG2基因在miRNAs-MUC13调控体系中分别通过MAPK、IL6、Toll-like受体信号通路参与仔猪腹泻调控。围绕miRNAs-MUC13调控体系发掘了4个分子复合物集团和2个核心基因(SRF和STAG2)并进行了试验验证,这为靶向治疗仔猪腹泻提供了理论参考和科学依据。

关键词: Mucin 13, microRNA, 仔猪腹泻, 基因网络

Abstract:

In order to discover the molecular regulatory mechanism related to piglet diarrhea, the genes and pathways related to piglet diarrhea in the microRNAs-Mucin13 (miRNAs-MUC13) regulatory system were explored and verified. NCBI blastn tool was used to compare the nucleic acid sequences of mouse MUC13 gene and pig gene bank, and found that the similarity between them was 74%-87%, with high homology. Taking the mouse as a model, the miRDB, mirTarBase and TargetScan databases were selected to predict the miRNA sequence that regulated the MUC13 gene. The target genes of these miRNA sequences were predicted through the StarBase database, and 36 target gene intersections were obtained. The interaction network of piglet diarrhea genes was built around MUC13 and these 36 target genes by using Cytoscape software. The network contains 19 screened target genes, and 4 molecular complex groups were obtained based on MCOMD algorithm analysis. The gene and pathway enrichment analysis of these 4 molecular complexes were carried out through DAVID platform. The results showed that the target genes SRF and STAG2 and their involved pathways were related to piglet diarrhea. Using Escherichia coli strain 200 and piglet intestinal epithelial cell line (IPEC-1) as the test materials, the piglet diarrhea model was designed and verified by the in vitro cell test. The real-time fluorescence quantitative PCR (qRT-PCR) results showed that the relative expressions of MUC13, SRF, IL-6, MAPK, STAG2, NF-κB, TLR7 in the test group were significantly higher than those of the control group (P<0.05), so it was speculated that SRF and STAG2 genes were involved in the regulation of piglet diarrhea through MAPK, IL 6 and Toll-like receptor signal pathway in the miRNAs-MUC13 regulation system. This study explored 4 molecular complex groups and 2 core genes (SRF and STAG2) around the miRNAs-MUC13 regulatory system, and carried out experimental verification, which provided theoretical reference and scientific basis for targeted treatment of piglet diarrhea.

Key words: Mucin 13, microRNA, piglet diarrhea, gene network

中图分类号:

S828

路伏增, 华卫东, 褚晓红, 戴丽荷, 陈晓宇, 章利丰, 王志刚, 徐如海. miRNAs-MUC13调控体系中与仔猪腹泻有关的基因与通路[J]. 浙江农业学报, 2023, 35(12): 2785-2793.

LU Fuzeng, HUA Weidong, CHU Xiaohong, DAI Lihe, CHEN Xiaoyu, ZHANG Lifeng, WANG Zhigang, XU Ruhai. Genes and pathways related to piglet diarrhea in miRNAs-MUC13 regulation system[J]. Acta Agriculturae Zhejiangensis, 2023, 35(12): 2785-2793.

图/表 7

参考文献 30

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基因名称 Gene name	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	基因编号 Gene number	熔解温度 Melting temperature/℃	产物大小 Product size/bp
MUC13	GTGATGCAGATGCCAGAGGT	AAAGAAAGCTTGTGCGGCAG	NM_001105293.1	60	135
SRF	GCACTGATTCAGACCTGCCT	CGCAGGTTTCGGTGTATCCT	XM_001929265.3	60	162
IL-6	CCTCCAGGAACCCAGCTATG	GTGGCATCACCTTTGGCATC	NM_214399.1	60	139
MAPK	ACAGGGTTCCTGACGGAGTA	ATGTGGTTCAGCTGGTCGAG	NM_001198922.1	60	179
STAG2	ACAACATTCATGTGACGGGC	AGACAATTCCGAAGTCGGGC	XM_003360436.1	60	133
NF-κB	ATGGAGTACCCTGAGGCTATAA	GTCCGCAATGGAGGAGAAG	EU399817.1	60	138
TLR7	CCAGCTGAAGACTGTCCCTG	GAGCTGAGGTCCAGATGTCG	NM_001097434.1	60	141
GAPDH	CGATGGTGAAGGTCGGAGTG	TGCCGTGGGTGGAATCATAC	NM_001206359.1	60	159

基因名称 Gene name	上游引物 Forward primer (5'→3')	下游引物 Reverse primer (5'→3')	基因编号 Gene number	熔解温度 Melting temperature/℃	产物大小 Product size/bp
MUC13	GTGATGCAGATGCCAGAGGT	AAAGAAAGCTTGTGCGGCAG	NM_001105293.1	60	135
SRF	GCACTGATTCAGACCTGCCT	CGCAGGTTTCGGTGTATCCT	XM_001929265.3	60	162
IL-6	CCTCCAGGAACCCAGCTATG	GTGGCATCACCTTTGGCATC	NM_214399.1	60	139
MAPK	ACAGGGTTCCTGACGGAGTA	ATGTGGTTCAGCTGGTCGAG	NM_001198922.1	60	179
STAG2	ACAACATTCATGTGACGGGC	AGACAATTCCGAAGTCGGGC	XM_003360436.1	60	133
NF-κB	ATGGAGTACCCTGAGGCTATAA	GTCCGCAATGGAGGAGAAG	EU399817.1	60	138
TLR7	CCAGCTGAAGACTGTCCCTG	GAGCTGAGGTCCAGATGTCG	NM_001097434.1	60	141
GAPDH	CGATGGTGAAGGTCGGAGTG	TGCCGTGGGTGGAATCATAC	NM_001206359.1	60	159

基因名称 Gene name	基因编号 Gene number	基因名称 Gene name	基因编号 Gene number
Stag2	NM_001077712	Gnaq	NM_008139
Fam108b	NM_146096	Dag1	NM_010017
Nfe2l1	NM_001130451	Mtap2	NM_008632
Ier5	NM_010500	Nrxn3	NM_172544
Rnf38	NM_001038993	Tardbp	NM_001003899
Zzz3	NM_198416	Prkacb	NM_011100
Tspan7	NM_019634	Neurod1	NM_010894
Ankrd40	NM_027799	Zfp608	NM_175751
Adipor2	NM_197985	Nrxn1	NM_177284
Srf	NM_020493	Dnajb12	NM_019965
Fubp1	NM_057172	Cd24a	NM_009846
Arl8b	NM_026011	Neurod2	NM_010895
Gabrb2	NM_008070	Klf12	NM_010636
Tnrc6b	NM_177124	Calm1	NM_009790
Ivns1abp	NM_001039512	Vsnl1	NM_012038
Gabra2	NM_008066	Nufip2	NM_001024205
Rtn1	NM_001007596	Gal3st3	NM_001024717
Zfhx2	NM_001039198	Zmynd11	NM_144516

基因名称 Gene name	基因编号 Gene number	基因名称 Gene name	基因编号 Gene number
Stag2	NM_001077712	Gnaq	NM_008139
Fam108b	NM_146096	Dag1	NM_010017
Nfe2l1	NM_001130451	Mtap2	NM_008632
Ier5	NM_010500	Nrxn3	NM_172544
Rnf38	NM_001038993	Tardbp	NM_001003899
Zzz3	NM_198416	Prkacb	NM_011100
Tspan7	NM_019634	Neurod1	NM_010894
Ankrd40	NM_027799	Zfp608	NM_175751
Adipor2	NM_197985	Nrxn1	NM_177284
Srf	NM_020493	Dnajb12	NM_019965
Fubp1	NM_057172	Cd24a	NM_009846
Arl8b	NM_026011	Neurod2	NM_010895
Gabrb2	NM_008070	Klf12	NM_010636
Tnrc6b	NM_177124	Calm1	NM_009790
Ivns1abp	NM_001039512	Vsnl1	NM_012038
Gabra2	NM_008066	Nufip2	NM_001024205
Rtn1	NM_001007596	Gal3st3	NM_001024717
Zfhx2	NM_001039198	Zmynd11	NM_144516

分类 Category	所属通路 Pathway affiliated	基因数量 Gene number	所含基因名称 Name of gene contained	P值 P value	发现错误率 False discovery rate
分子复合物1 Molecular Complex 1	KEGG通路:MAPK信号通路 KEGG_PATHWAY: MAPK signaling pathway	11	AKT1, TRP53, FOS, MAPK1, ELK4, MAPK3, ELK1, MAPK8, PAK1, SRF, MYC	2.51×10^-8	2.55×10^-5
	BIOCART:MAL在Rho介导的SRF 激活中的作用 BIOCARTA: Role of MAL in Rho-mediated activation of SRF	6	MAPK1, MAPK3, RHOA, MAPK8, MAL, SRF	3.50×10^-6	0.004 047
	BIOCARTA:PDGF信号通路 BIOCARTA: PDGF signaling pathway	6	FOS, MAPK3, ELK1, MAPK8, SRF, PIK3R1	2.74×10^-5	0.031 626
	BIOCARTA:EGF信号通路 BIOCARTA:EGF signaling pathway	6	FOS, MAPK3, ELK1, MAPK8, SRF, PIK3R1	2.74×10^-5	0.031 626
	KEGG通路:细胞周期 KEGG_PATHWAY: Cell cycle	5	TRP53, CDKN1A, SMAD3, MYC, STAG2	0.001 506	1.518 954
	BIOCARTA:IL-6信号通路 BIOCARTA: IL-6 signaling pathway	4	FOS, MAPK3, ELK1, SRF	0.002 880	3.275 084
	KEGG通路:Toll样受体信号通路 KEGG_PATHWAY: Toll-like receptor signaling pathway	7	AKT1, FOS, MAPK1, MAPK3, MAPK8, PIK3R1, STAG2	3.63×10^-5	0.036 899
分子复合物2 Molecular Complex 2	KEGG通路:MAPK信号通路 KEGG_PATHWAY: MAPK signaling pathway	3	BDNF, NTRK2,PFN2	0.015 087	11.720 810
分子复合物3 Molecular Complex 3	KEGG通路:ABC转运器 KEGG_PATHWAY: ABC transporters	3	ABCB1B, ABCC2, ABCG2	6.01×10^-5	0.006 015
分子复合物4 Molecular Complex 4	KEGG通路:p53信号通路 KEGG_PATHWAY: p53 signaling pathway	3	BBC3, DDB2, GADD45A	0.001 039	0.735 622

miRNAs-MUC13调控体系中与仔猪腹泻有关的基因与通路

Genes and pathways related to piglet diarrhea in miRNAs-MUC13 regulation system

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