藏黄牛与宣汉黄牛心脏miRNA表达谱比较

doi:10.3864/j.issn.0578-1752.2020.08.016

Abstract

Abstract:

【Objective】As a kind of non-coding RNA, miRNA is widely involved in various life activities of the organism. This study was aimed to explore the differential expression profiles of miRNA in the heart tissues between Tibetan cattle and Xuanhan cattle, so as to provide the basic data for further study on molecular mechanism of hypoxia adaptation in Tibetan cattle. 【Method】Each three healthy Tibetan and Xuanhan cattle were randomly selected for heart tissue sampling. RNA was extracted from tissues using the Trizol method. An 18 to 30nt fragment was selected by agarose gel electrophoresis, and 3' connector and 5' liner was ligated and then the fragment was enlarged. After gel electrophoresis, three Tibetan cattle and Xuanhan cattle libraries were established, respectively. High-throughput sequencing was performed by using the Illumina HiSeq4000 sequencing platform. The sequence was then filtered and the differentially expressed miRNA of Tibetan cattle and Xuanhan cattle were screened by comparing GenBank and Rfam databases. Functional annotation and signal pathway enrichment analysis of differentially expressed miRNA in Tibetan cattle and Xuanhan cattle. Finally, in order to verify the accuracy of the sequencing data, 8 miRNAs were randomly selected and the expression level of miRNA was detected by RT-qPCR. 【Result】The results showed that Tibetan cattle and Xuanhan cattle had high-quality reads of 17 463 446 and 13 662 812, respectively, while the clean reads were 16 552 296 and 12 055 304, respectively. The highest enrichment of high-quality nucleic acid sequences in Tibetan cattle and Xuanhan cattle were 21 nt, which were 37.5% and 32.1%, respectively. A total of 219 differential expressed miRNAs (48 up-regulated and 171 down-regulated) were obtained. There were 22 terms in the GO function annotation that significantly enriched in the molecular function of differentially expressed miRNAs target genes, such as GO: 0005488 (binding), GO: 0005515 (protein binding) and GO: 0043167 (ion binding). GO: 0005623 (cell), GO: 0044464 (cell component) and GO: 0005622 (cell) were among the 20 terms, which were significantly enriched in the cellular components. While there were 13 terms, which were significantly enriched in biological processes, such as GO: 0035556 (intracellular signal transduction), GO: 0032774 (RNA biosynthesis process) and GO: 0006351 (transcription, DNA templated). Analysis of KEGG signaling pathways revealed that miRNA target genes were significantly enriched to 232 signaling pathways, including the insulin signaling pathway (139 target genes), the mTOR signaling pathway (38 target genes) and the HIF-1 signaling pathway (92 target genes). Among them, 12 miRNA target genes worked together on these three signaling pathways. These results suggested that the differentially expressed miRNAs might participate in the regulation of hypoxia adaptation in Tibetan cattle through these three signaling pathways. Eight miRNAs were randomly selected for RT-qPCR, and the expression profiles were consistent with the sequencing data, indicating that the high-throughput sequencing data was reliable. 【Conclusion】Taken together, the expression profiles of miRNAs in the heart tissues of Tibetan and Xuanhan cattle were obtained in the present study, which laid a foundation for further research on the hypoxia adaptation mechanism of Tibetan cattle.

Key words: Tibetan cattle, Xuanhan cattle, miRNA, heart, high-throughput sequencing, hypoxia adaptability

CHEN LuLu,WANG Hui,WANG JiKun,WANG JiaBo,CHAI ZhiXin,CHEN ZhiHua,ZHONG JinCheng. Comparative Analysis of miRNA Expression Profiles in the Hearts of Tibetan Cattle and Xuanhan Cattle[J].Scientia Agricultura Sinica, 2020, 53(8): 1677-1687.

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References 42

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miRNAs miRNAs	引物序列（5'-3'） Primer sequence（5'-3'）
bta-miR-99a-5p	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAAGATC F: CTGGAGAACCCGTAGATCCGAT
bta-miR-100	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACAAGTT F: CTGGAGAACCCGTAGATCCGAA
miR-99-x	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCACAAGA F: CTGGAG AACCCGTAGATCCGAT
bta-miR-1468	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGCAAA F: CTGGAGGTCGTATCCAGTGCAG
bta-miR-1	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATACATCT F: CTGGAGTCGGATCCGTCTGAGC
bta-miR-148a	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACAAAGTT F: CTGGAGTCAGTGCACTACAGAA
miR-101-y	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGTCAGTT F: CTGGAGTACAGTACTGTGATAA
bta-miR-499	RT primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAACATCA F: CTGGAGTTAAGACTTGCAGTGA
miRNA统一反向引物 Unified reverse primer	GTGCAGGGTCCGAGGT

读段类型 Type of reads	藏黄牛1 数量Count	藏黄牛2 数量Count	藏黄牛3 数量Count	平均值 Average value	宣汉黄牛1 数量Count	宣汉黄牛2 数量Count	宣汉黄牛3 数量Count	平均值 Average value
总读值 Total reads	17274872 (100%)	14655372 (100%)	21634340 (100%)	17854861	14186785 (100%)	13820881 (100%)	13651263 (100%)	13886310
高质量读值 Reads with high quality	16864234 (97.6229%)	14331976 (97.7933%)	21194128 (97.9652%)	17463446	13970084 (98.4725%)	13591498 (98.3403%)	13426853 (98.3561%)	13662812
3' 端缺失 Reads with null 3'adapter	41744 (0.2475%)	31922 (0.2227%)	31922 (0.4363%)	35196	44975 (0.3219%)	29374 (0.2161%)	33735 (0.2513%)	36028
插入缺失读值 Reads with insert	101492 (0.6018%)	74047 (0.5167%)	51722 (0.2440%)	75754	108440 (0.7762%)	72639 (0.5344%)	93029 (0.6929%)	91369
5'端污染的读值 5'adapter contaminants	7946 (0.0471%)	15266 (0.1065%)	7948 (0.0375%)	10387	18678 (0.1337%)	20305 (0.1494%)	39930 (0.2974%)	26304
小于18nt的读值 Reads shorter than 18nt	437113 (2.5920%)	598024 (4.1727%)	480882 (2.2689%)	505340	811209 (5.8068%)	998246 (7.3446%)	1414065 (10.5316%)	1074507
含有poly(A)的序列 Reads with polyA	139 (0.0008%)	133 (0.0009%)	316 (0.0015%)	196	37 (0.0003%)	35 (0.0003%)	59 (0.0004%)	44
低频数读值(< 2) Reads with Low frequency	228502 (1.3550%)	270838 (1.8897%)	292937 (1.3822%)	264092	369657 (2.6461%)	300701 (2.2124%)	467408 (3.4811%)	379255
干净读值 Clean reads	16047298 (95.1558%)	13341746 (93.0908%)	20267845 (95.6295%)	16552296	12617088 (90.3150%)	12170198 (89.5427%)	11378627 (84.7453%)	12055304

基因名 Gene name	log₂(fc)	P值 P value	序列 Sequence
miR-2285-y	-2.185471095	0.010019532	TCCAAGTGAACTTTTTGGCT
miR-2284-y	-2.234579424	0.009299134	TCCAAGTGAACTTTTTGGCT
bta-miR-10a	-3.418663256	1.30E-06	TACCCTGTAGATCCGAATTTGTG
bta-miR-208b	-2.116094716	0.00109356	ATAAGACGAACAAAAGGTTTGT
bta-miR-146b	-3.180709957	9.28E-07	TGAGAACTGAATTCCATAGGCTGT
bta-miR-141	-8.183090025	6.55E-27	TAACACTGTCTGGTAAAGATGG
bta-miR-200a	-6.442558686	1.66E-10	TAACACTGTCTGGTAACGATGTT
bta-miR-2478	-2.342776751	0.000175165	GTATCCCACTTCTGACACCA
bta-miR-19b	-2.540817607	5.05E-05	TGTGCAAATCCATGCAAAACTGA
bta-miR-200c	-7.794012294	7.53E-22	TAATACTGCCGGGTAATGATGGA

样品名 Sample name	miRNA数量 miRNA number	靶基因数量 Target gene number	靶基因位点数量 Target site number
总数 Total	2033	59271	5486903
藏黄牛Tibetan cattle	962	58111	2584253
宣汉黄牛Xuanhan cattle	1103	58541	3017725

富集通路 Enrichment pathway	靶基因 Target gene
胰岛素信号通路 Insulin signaling pathway	FASN, PYG, GYS, HK, PHKG, PIK3C, G6PC, PCK, ACACB, BAD, CALM, PIK3R, HRAS, RP-S6e, GSK3B, SOS, EIF4E, FBP, PKA, GRB2, BRAF, RAF1, MAP2K1, MAP2K2, MAPK13, MKNK, ELK1, CRK, JNK, AKT,INSR, RPS6KB, SOCS1, SOCS2, SOCS3, SOCS4, CBL, PRKAR, PTPRF, PTPN1, PRKCI, SORBS1, PPP1C, PDPK1, RAPGEF1, SHC1, IRS2, LIPE, PPP1R3, PHKA-B, SLC2A4, FLOT, APS, RHOQ, EXOC7, TRIP10, SREBP1, PRKAA, PRKAB, PRKAG, FOXO1, PPARGC1A, MTOR, RAPTOR, EIF4EBP1, TSC1, TSC2, RHEB, IKBKB, KRAS, NRAS, ARAF, ACACA, PKLR, GCK, PDE3B, SHIP2, IRS1, IRS3, IRS4, SHC2, SHC3, SHC4, PPP1R3F, PRKCZ, HRAS, GSK3B, CXCR4, PPP3C, RASA1, MAPK1_3, RAC1, PAK1, PAK2, RHOA, ROCK1, GNAI, MET, EPHA1, EPHA2, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, EFNA, EFNB, FYN, ITGB1, PTK2, PAK3, PAK4, PAK6, PAK7, LIMK1, LIMK2, CFL, PPP3R, SEMA4, SEMA7, L1CAM, PLXNC, ABL1, NRP1, ROBO1, ROBO2, DCC, PLXNA, PLXNB
mTOR信号通路 mTOR signaling pathway	PIK3C, PTEN, PIK3R, PRKCA, RP-S6e, TNF, EIF4B, EIF4E, BRAF, MAPK1_3, RPS6KA, AKT, RPS6KB, VEGFA, IGF1, PDPK1, PRKAA, MTOR, RAPTOR, EIF4EBP1, TSC1, TSC2, RHEB, IKBKB, STK11, MLST8, RICTOR, HIF1A, ULK1_2_3, DDIT4, STRADA, CAB39, IRS1, AKT1S1, RRAGA_B, RRAGC_D, PRKCB, PRKCG
HIF-1信号通路 HIF-1 signaling pathway	GAPDH, PDHA, PDHB, HK, PIK3C, PFKFB3, PLCG1, ENO, BCL2, NFKB1, PIK3R, PRKCA, RP-S6e, TNFRSF3, EIF4E, RBX1, CUL2, VHL, SERPINE1, EGF, EGFR, MAP2K1, MAPK1_3, MKNK, AKT, EP300, CAMK2, INSR, IFNG, RPS6KB, STAT3, RELA, IL6R, ERBB2, IGF1R, FLT1, TEK, IFNGR1, IFNGR2, IL6, EPO, VEGFA, IGF1, ANGPT1, ANGPT2, ANGPT4, PLCG2, TFRC, CDKN1B, CDKN1A, MTOR, EIF4EBP1, SLC2A1, NOX, HIF1A, EGLN, TLR4, PDK1, NOS2, NOS3, TF, EDN1, TIMP1, PRKCB, PRKCG

Comparative Analysis of miRNA Expression Profiles in the Hearts of Tibetan Cattle and Xuanhan Cattle

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