陈露露, 王会, 王吉坤, 王嘉博, 柴志欣, 陈智华^,, 钟金城^,西南民族大学青藏高原研究院/青藏高原动物遗传资源保护与利用四川省、教育部重点实验室,成都 610041

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

CHEN LuLu, WANG Hui, WANG JiKun, WANG JiaBo, CHAI ZhiXin, CHEN ZhiHua^,, ZHONG JinCheng^,Institute of Tibetan Plateau Research, Southwest Minzu University/Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Sichuan Province and Ministry of Education, Chengdu 610041

通讯作者: 钟金城,E-mail: zhongjincheng518@126.com 陈智华,E-mail: czh@swun.cn

责任编辑: 林鉴非
收稿日期:2019-04-30接受日期:2020-02-19网络出版日期:2020-04-16

基金资助:

国家肉牛牦牛产业技术体系项目.CARS-37 青藏高原生态畜牧业协同创新中心开放基金资助.QZGYXT02

Received:2019-04-30Accepted:2020-02-19Online:2020-04-16
作者简介 About authors
陈露露,E-mail: 1556370692@qq.com。









摘要
【目的】miRNA作为一类非编码RNA,广泛参与机体多种生命活动,研究旨在挖掘miRNA在藏黄牛和宣汉黄牛心脏组织中的差异miRNA,为进一步研究藏黄牛低氧适应的分子机制提供基础数据。【方法】随机选取健康的藏黄牛和宣汉黄牛各3头,迅速采集其心脏组织,使用Trizol法提取RNA,琼脂糖凝胶电泳切胶选取18—30nt的片段,连接3'端和5'端,反转录后进行扩增,凝胶电泳切胶纯化后建立藏黄牛和黄牛各3个文库,利用Illumina HiSeq4000测序平台进行高通量测序;将测序得到的序列进行过滤,比对GenBank和Rfam数据库,筛选出藏黄牛和宣汉黄牛的差异miRNA,进行功能注释及信号通路富集分析;随机选择8个miRNAs,利用实时荧光定量PCR检测其在心脏组织的表达量,以验证测序数据的准确性。【结果】藏黄牛和宣汉黄牛心脏组织的高质量读值的序列分别为17 463 446条和13 662 812条,干净读值为16 552 296条和12 055 304条,且在藏黄牛和宣汉黄牛中高质量核酸序列长度富集最多的均是21nt,分别为37.5%和32.1%;且共筛选出219个差异miRNAs,其中48个显著上调,171个显著下调;GO功能注释得到差异miRNA靶基因分子功能中显著富集的条目有22条,如,GO:0005488（结合）、GO:0005515（蛋白质结合）和GO:0043167（离子结合）;细胞组分中显著富集的条目有20条,如,GO:0005623（细胞）、GO:0044464（细胞组分）和GO:0005622（细胞内）;生物过程中显著富集的条目有13条,如,GO:0035556（细胞内信号转导）、GO:0032774（RNA生物合成过程）和GO:0006351（转录,DNA模板化）,KEGG信号通路分析得到差异表达miRNAs靶基因显著富集到胰岛素信号通路（139个靶基因）、mTOR信号通路（38个靶基因）和HIF-1 信号通路（92个靶基因）等232个信号通路中,其中有12个靶基因共同作用于这3个信号通路,说明miRNAs可能通过这3个信号通路,共同参与藏黄牛低氧适应性的调控;随机选取8个miRNAs进行荧光定量验证,其表达趋势与测序结果表达趋势基本一致,表明高通量测序数据可信度较高。【结论】得到了miRNA在藏黄牛与宣汉黄牛心脏组织中的表达谱,为揭示藏黄牛低氧适应性的调控机制研究奠定了基础。
关键词： 藏黄牛;宣汉黄牛;miRNA;心脏;高通量测序;低氧适应性

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.
Keywords：Tibetan cattle;Xuanhan cattle;miRNA;heart;high-throughput sequencing;hypoxia adaptability


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本文引用格式
陈露露, 王会, 王吉坤, 王嘉博, 柴志欣, 陈智华, 钟金城. 藏黄牛与宣汉黄牛心脏miRNA表达谱比较[J]. 中国农业科学, 2020, 53(8): 1677-1687 doi:10.3864/j.issn.0578-1752.2020.08.016
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 Acricultura Sinica, 2020, 53(8): 1677-1687 doi:10.3864/j.issn.0578-1752.2020.08.016

0 引言

【研究意义】由于海拔升高导致分压降低,当升高的代谢需求超过氧气供应时,心血管和呼吸系统的容量受到抑制,所以缺氧是高海拔环境下的主要环境胁迫类型,会导致代谢需求与氧气对流运输之间的不平衡,最终可引起全身性低氧血症;氧气供应不足影响动物的生理生化功能,从而抑制其生长^[1,2]。宣汉黄牛广泛分布于我国四川宣汉、通江等平原地区,是我国优质的地方品种,但由于长期生活在平原,对于高原环境没有良好的适应机制。藏黄牛是以产乳为主, 乳、肉、役兼用的小型地方原始品种,由于近亲繁殖,造成了其个体小、成熟晚、生产性能低等缺陷,但其能适应低氧和低温的高海拔恶劣环境。分布在高海拔的藏黄牛与平原地区的宣汉黄牛在适应低氧环境机制上形成鲜明对比,本研究拟对藏黄牛和宣汉黄牛进行转录组比较分析,探索藏黄牛低氧适应的分子机制。【前人研究进展】微小核糖核酸（microRNA,miRNA）是由 18—22个核苷酸碱基组成的非编码内源性小分子 RNA,由DNA转录产生,广泛存在于动物、植物、微生物等生物体内^[3]。研究显示,在缺氧条件下, miRNA可以与低氧转录因子相互作用,参与生物体内的细胞代谢、细胞增殖、细胞分化、免疫应答、细胞凋亡和癌症的发生发展等一系列生物学过程^{[4,5,6,7,8,9,10]}。miRNA通过指导RNA诱导沉默复合物（RISC）对mRNA进行切割或者阻断,影响 mRNA 的表达,从而调控其蛋白表达水平^[11,12]。一个miRNA可以调控多个靶基因,同时也存在多个miRNA调节同一个靶基因,研究表明,人体约1/3的基因受到miRNA的调控^[13,14]。同时,低氧能调节miRNA活性并控制转录过程中关键蛋白的活性^[15]。在低氧条件下,miR-143和miR-101可以直接定位在组织细胞中的酵解酶己糖激酶,从而保护细胞免受缺氧性损伤^[16,17]。ZHANG等^[18]发现在鱼类缺氧时,酵解酶和己糖激酶显著上调,其调节的miRNA显着下调,表明这些酶调节的miRNA在应对缺氧时有重要作用。【本研究切入点】近年来有大量关于高原动物适应低氧的相关研究,但从miRNA角度研究藏黄牛适应低氧的研究鲜有报道。【拟解决的关键问题】本研究首次对藏黄牛和宣汉黄牛进行miRNA转录组比较分析,筛选低氧适应性相关miRNA及其靶信号通路,为进一步研究藏黄牛低氧适应机制提供了基础数据。

1 材料与方法

1.1 材料

2018年4月,于西藏自治区昌都市类乌齐县和成都市郫县各选取3头健康的藏黄牛和宣汉黄牛,采集心脏组织,DEPC冲洗,锡箔纸包装迅速置于液氮保存,带回青藏高原动物遗传资源保护与利用四川省、教育部重点实验室进行试验。

1.2 总RNA提取

Trizol法提取总RNA,使用Nanodrop ND-1000分光光度计和Bioanalyzer 2100生物分析仪（Agilent公司,美国）对RNA浓度及RNA完整性进行鉴定。

1.3 小RNA文库构建及Solexa测序

构建6个测序文库（藏黄牛3个,宣汉黄牛3个）：从样品中提取总RNA后,琼脂糖凝胶电泳切胶选取18—30nt的片段,连接3' 端和5' 端,对已连接两端的small RNA进行反转录和PCR扩增,琼脂糖凝胶电泳回收纯化约140 bp的条带后构建藏黄牛和宣汉黄牛各3个文库,构建好的文库使用 Agilent 2100以及qPCR进行质控,最后利用测序平台Illumina HiSeq4000进行测序。

1.4 数据处理统计

将测序得到的原始序列过滤掉低质量、有5'端和没有3'端、不含插入片段和插入片段小于18nt、含有polyA的序列,得到的有效序列进行总数、种类和长度分布统计,并做后续分析。

1.5 基因对比分析

1.5.1 比对GenBank和Rfam数据库 运用blastn软件,尽可能去除样本中的rRNA、scRNA、snoRNA、snRNA和tRNA。

1.5.2 基因组对比 通过Bowtie 1.1.2软件确定测序得到的sRNA序列在基因组上的位置,根据外显子和内含子在基因组的位置,找出来自mRNA降解片段的sRNA,通过 RepeatMasker version open-4.0.6软件将sRNA与重复序列进行比对,得到repeat associate sRNA。

1.5.3 miRNA鉴定 利用Bowtie（version 1.1.2）软件与miRBase数据库中所有物种的miRNA 序列进行比对分析,鉴定出已知保守的miRNA 及其含量,利用MIREAP_v0.2 软件预测miRNA。

1.6 差异表达分析

利用edgeR 软件,使用edgeR默认参数,对已存在miRNA、已知miRNA和预测的新miRNA进行差异表达分析。差异表达miRNA的筛选标准为表达量变化2倍以上并且P<0.05。

1.7 靶基因预测及靶基因富集分析

使用RNAhybrid（v2.1.2）+svm_light（v6.01）,Miranda（v3.3a）,TargetScan（Version: 7.0）3种方法进行靶基因预测,然后取3种方法得到的靶基因预测结果的交集作为miRNA靶基因预测的结果。利用R语言中clusterProfilter软件包和org.Hs.eg.db数据库,根据靶基因注释信息进行GO富集和KEGG通路分析。

1.8 RT-qPCR验证

根据反转录试剂盒（TaKaRa公司）说明书,对总RNA进行反转录合成cDNA,标记后于-20℃保存。

随机选取8个miRNAs,用U6^[19]（F: GCTTCGG CAGCACATATACTAAAAT;R: CGCTTCACGAATT TGCGTGTCAT）作为内参基因,每个样品设置 3个重复。根据这8个miRNAs的成熟序列设计反转录及荧光定量PCR引物,引物序列见表1。利用Prime Script^TM RT反转录试剂盒和TB Green Premix Ex Taq^TMⅡ定量试剂盒,对目标miRNA在藏黄牛和宣汉黄牛心脏组织中的表达水平进行验证。

Table 1
表1
表1荧光定量引物
Table 1Fluorescent quantitative primers

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

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反应体系10 µL：5 µL SYBR premix Dimer Eraser（2×）,上下引物各0.4 µL,无菌去离子水3.2 µL,cDNA1.0 µL。

定量程序：95℃预变性30 s,95℃变性5 s,60℃退火30 s,72℃延伸30 s,39个循环。

采用2^-ΔΔCT法计算目的基因表达量,结果以“平均值±标准误差（Mean±SEM）”表示。

2 结果

2.1 数据处理统计

本次试验构建的6个文库,共获得95 223 513条原始读值,经过一系列过滤后得到藏黄牛和宣汉黄牛的高质量读值的序列分别为17 463 446条和13 662 812条,干净读值的序列分别为16 552 296条和12 055 304条（表2）。

Table 2
表2
表2数据过滤及去端头情况统计
Table 2Data filtering and de-joining statistics

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

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通过高通量测序分析得到藏黄牛和宣汉黄牛高质量核酸序列长度均主要分布在21nt,分别为37.5%和32.1%,具体见图1。

图1

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图1藏黄牛和宣汉黄牛miRNA的长度分布及频率百分比

Fig. 1Length distribution and frequency percentage of miRNAs in Tibetan cattle and Xuanhan cattle

2.2 miRNA 表达分析

差异表达分析共筛选出219个差异表达miRNAs,其中48个显著上调,171个显著下调（图2)。表3为差异表达中表达量较高的10个miRNAs。

图2

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图2藏黄牛和宣汉黄牛miRNA 差异表达统计图

Fig. 2Differential expression of miRNAs in Tibetan cattle and Xuanhan cattle



Table 3
表3
表3差异表达中表达量较高的10个 miRNAs
Table 310 miRNAs with high expression levels in differential expression

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

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2.3 miRNA靶基因预测及靶基因富集分析

2.3.1 miRNA 靶基因预测 由表4可知,藏黄牛靶基因预测数量为58 111个,宣汉黄牛为58 541个,对应的靶基因位点数量为2 584 253个和3 017 725个。

Table 4
表4
表4藏黄牛和宣汉黄牛miRNA靶基因位点预测统计
Table 4Predictive statistics of miRNA target genes loci in Tibetan cattle and Xuanhan cattle

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

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2.3.2 miRNA 靶基因富集分析 对差异表达miRNA的靶基因进行GO功能注释结果表明：miRNA靶基因显著富集55条GO条目,其中生物过程22条,细胞组分20条,分子功能13条。由图3可知,在生物过程中,细胞过程和单一生物过程富集水平最高,且显著富集前三的是GO:0035556（细胞内信号转导）、GO:0032774（RNA生物合成过程）和GO:0006351（转录,DNA模板化）;在分子功能中,结合和催化活性富集水平最高,且富集显著前三的是GO: 0005488（结合）、GO:0005515（蛋白质结合）和GO:0043167（离子结合）;在细胞组分中,细胞和细胞部分富集水平最高,且富集显著前三的是GO: 0005623（细胞）、GO:0044464（细胞组分）和GO: 0005622（细胞内）。

图3

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图3藏黄牛和宣汉黄牛miRNA靶基因GO 富集分类柱状图（A:生物过程 B:分子功能 C：细胞组分）

Fig. 3Quantitative histogram of GO enrichment of miRNA target genes in Tibetan cattle and Xuanhan cattle（A :Biological process B: Molecular function C: Cellular component）



KEGG Pathway富集分析结果表明,差异表达miRNAs靶基因显著富集到MAPK信号通路、mTOR信号通路、轴突导向和胰岛素信号通路等信号通路,图4为其中20条信号通路;在低氧适应相关通路中发现胰岛素信号通路涉及差异表达miRNAs的靶基因135个,mTOR信号通路涉及差异表达miRNAs的靶基因38个,HIF-1信号通路涉及差异表达miRNAs的靶基因65个,且有12个靶基因（PIK3C、PIK3R、RP-S6e、EIF4E、MAPK1-3、AKT、RPS6KB、IGF1、mTOR、EIF4EBP1、HIF1A、PRKCG）共同参与胰岛素信号通路、HIF-1信号通路和mTOR信号通路（表5）。

图4

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图4藏黄牛和宣汉黄牛miRNA靶基因Pathway 通路分析

Fig. 4Pathway analysis of miRNA target genes in Tibetan cattle and Xuanhan cattle



Table 5
表5
表53个信号通路涉及的靶基因
Table 5Target genes involved in the three signaling pathways

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

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2.4 RT-qPCR验证

由图5可知,RT-qPCR结果表明,miRNA在宣汉黄牛和藏黄牛心脏组织中的表达趋势与高通量测序所得表达趋势基本一致,说明测序得到数据的可靠性高;且在RT-qPCR 验证中发现bta-miR-99a-5p在藏黄牛心脏组织中的表达量显著高于宣汉黄牛心脏组织中的表达量（P<0.05）,其他基因在藏黄牛和宣汉黄牛心脏组织中的表达则不具有显著性差异（P>0.05）。

图5

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图5随机挑选的8个基因在藏黄牛和宣汉黄牛心脏中的表达水平（A：测序结果;B：验证结果）

不同字母表示具有显著性差异(P<0.05)
Fig. 5Expression levels of 8 randomly selected genes in Tibetan cattle and Xuanhan cattle hearts (A: sequencing results; B: verification results)

Different letters indicate significant difference (P<0.05)

3 讨论

随着科学技术的飞速发展,高通量测序因为操作简单、效率高、通量大等优点在科学实验中得到了广泛应用;它能够快速地获得成千上万条基因序列,以此开发出了大量的微卫星分子标记资源^[20]。

心脏有左心房、左心室、右心房和右心室4个腔室,是脊椎动物最重要的器官之一,主要作用是推动血液流动,为组织和器官提供充足的血流量,是机体各项生命活动的重要动力来源。藏黄牛长期生活在高海拔地区,为了能够适应恶劣环境,对低氧、低温逐渐形成了适应性特征,如肺脏重为其体重的1.1%—1.7%,心脏的重量占其体重的0.5%—0.8%^[21]。

miRNA是参与转录后表达水平调节的重要因子,是表型调控的重要潜在位点,而缺氧会影响某些特定组miRNA的表达 ^[22,23],本研究通过高通量测序分析,去除质量值低于20的碱基数超过1个或含有N的片段,以及rRNA,scRNA,snoRNA,snRNA,tRNA,得到高质量片段,结果分析表明藏黄牛和宣汉黄牛miRNA的长度均集中分布在21 nt,符合典型的Dicer酶切割成熟的miRNA的长度分布^[24]。在RT-qPCR验证试验中,bta-miR-499的表达趋势与测序数据不一样,可能是由于样本量较少和试验方法不同而造成的,但其他miRNA的表达趋势和测序结果中miRNA的表达趋势基本相同,表明本研究所得数据有较高的可信度。研究显示,缺氧诱导因子（HIF）可作为氧稳态的主要调节剂,能够刺激红细胞生成;脯氨酰羟化酶蛋白(EGLN1)错义突变编码的脯氨酰羟化酶2（PHD2）是HIF依赖的负调节剂,在适应低氧的情况下可防止红细胞增多症;细胞过度磷酸化缺陷型的过度表达能够干扰MAPK依赖性,从而调节HIF的活性^[25,26]。本试验的差异表达分析及生物信息分析结果表明,bta-miR-499y的靶基因EGLN1和HIF1,miR-101-y的靶基因EGLN1、bta- miR-101的靶基因EGLN1,bta-miR-2284p的靶基因MAPK均属于低氧适应性基因。推测这些miRNAs可能通过这些低氧适应性相关基因从而参与低氧适应性的调控。

在差异表达分析中,我们共挖掘了219个差异表达miRNAs（48个显著上调,171个显著下调）,说明miRNA主要通过负调控基因的表达从而参与到机体生长发育的过程中。但也有相关研究表明miRNA的作用机制也会因细胞所处的发育阶段或特殊的生理条件而存在差异 ^[27]。有研究发现,miRNA在牦牛和黄牛心脏组织中的差异表达基因富集到胰岛素信号通路、mTOR信号通路和HIF-1信号通路等与低氧适应性调控密切相关的信号通路中;藏鸡适应高原环境也是通过HIF-1信号通路、黑色素生成、DNA修复等通路实现^[28,29]。这与本研究得到的KEGG信号通路分析结果基本一致,说明miRNA靶向的差异表达基因具有较高的保守性。本试验差异表达miRNAs靶基因功能富集分析表明,富集最多的GO条目涉及细胞过程和代谢过程,推测差异表达miRNAs参与心肌细胞增殖及能量代谢等生理过程。

mTOR信号通路属于磷脂酰肌醇-3激酶,其通过MTOR C1（MTOR complex1, MTOR C1）和MTOR C2（MTOR complex2,MTOR C2）实现对细胞生长、增殖等生理功能的调控^[30]。MTOR C1对心脏有保护作用;而MTOR C2能够保护心脏结构和功能^[31,32]。前人研究表明激活PI3K-AKT-mTOR信号通路能促进细胞生长^[33],它诱导激活的miR-199a^[34]和miR-21^[35]能通过靶向GLUT1（胰岛素依赖性葡萄糖转运蛋白）调节葡萄糖代谢。HIF依赖性代谢适应有助于活细胞保持完整性,维持能量供应,并在低氧条件下维持其功能;缺氧情况下,能够启动缺氧诱导因子-1a（HIF-1a）的表达,并进一步诱导VEGF（血管内皮生长因子）和MAPK（丝裂原活化蛋白激酶）等靶基因的表达,促进血管生成及发育、呼吸系统发育和心肌细胞发育等细胞组织生理变化^{[36,37,38,39]}。有研究发现,miR-20a能通过靶向HIF-la来调节VEGF的表达^[40]。bta-miR-101主要富集到胰岛素信号通路和mTOR信号通路,推测bta-miR-101能够促进细胞生长;bta-miR-499和bta-miR-126-3p等miRNA同时作用于胰岛素信号通路、mTOR信号通路和HIF-1信号通路,由此推测,bta-miR-499和bta-miR-126-3p等miRNA可能通过HIF-1信号通路增加氧气的运输量,为机体提供更多氧气以适应高海拔环境。同时,也有研究发现,miR-101可以在缺氧情况下减少心肌细胞凋亡^[41];miR-499能够抑制心肌梗死后心肌纤维化并且改善心脏推动血液循环的功能^[42];miR-126-3p促进VEGF表达^[43]。本研究结果表明,有12个靶基因（PIK3C、PIK3R、RP-S6e、EIF4E、MAPK1-3、AKT、RPS6KB、IGF1、mTOR、EIF4EBP1、HIF1A、PRKCG）共同参与胰岛素信号通路、HIF-1信号通路和mTOR信号通路,推测差异表达miRNAs可能通过这些靶基因参与藏黄牛的低氧适应性机制的调控。

4 结论

本研究通过对藏黄牛和宣汉黄牛miRNA高通量测序分析,筛选出了219个差异表达miRNAs,其靶基因富集到胰岛素信号通路、mTOR信号通路和HIF-1信号通路等对心脏的结构保护、能量产生及储存具有重要作用的通路中,推测这些差异表达基因在藏黄牛适应低氧环境的过程中具有重要调控作用。本研究为进一步探讨高原动物低氧适应机制提供了基础数据。

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