Comparative MicroRNA Transcriptomic Analysis Provides Insights Into High-Altitude Acclimation Across Different Species

Animals lived in high altitude have evolved some anatomical and physiological traits that equip them for the extreme high-altitude environment,including larger lungs and hearts,blunted hypoxic pulmonary vasoconstriction,a strong environmental sense and a high energy metabolism.The mechanism of high altitude adaptation has become a topic of great interests in recent years.Extensive and in-depth investigations of high-altitude adaptation have been carried out at the level of morphology,anatomy,hemodynamics,physiology and genomics,but few investigations focused on the role of miRNA in high-altitude adaptation,especially the parallel comparison in multiple species.In this study,we generated high quality small RNA sequencing data from the low-and high-altitude populations across four typical livestock animals for six tissues(heart,liver,spleen,lung,kidney and muscle),investigated the evolutional scenario in four domesticated animals and examined the similarity in molecular mechanism of high-altitude acclimation across species.The major results are listed as following:(1)We collected 141 samples from six tissues(heart,liver,spleen,lung,kidney and muscle)across 4 typical livestock animals,and constructed and sequenced small RNA libraries for each sample.Total of 1.68 Gb raw reads were obtained and 11.41 Mb high quality reads were retained after data filtering;Using miRDeep2 software,we identified various number of miRNAs in four species ranging from 323 to 596 for mature sequences and ranging from 218 to 394 for miRNA precursors.Otherwise,we identified 193 to 304 miRNA families in four species,with an average family size of 1.19.(2)Birth and death analysis revealed an overall increase in the number of families in both mammals and birds from 78 ancestral families to 143–266 families in each species,and that the net increase rate of miRNA gene families in artiodactyl especially ruminant animals is very high.(3)Through sequence alignment and liftover,we identified miRNA orthologues for all pairwise species,and revealed the most number of miRNA orthologues in ruminant(171 orthologues),the second most number of miRNA orthologues in artiodactyla(111 orthologues),and the least number of miRNA orthologues in vertebrate(44 orthologues).(4)Pearson’s correlation analysis were performed between high-and low-altitude populations for a particular tissue.The result revealed higher Pearson’s correlation of heart,liver,spleen and muscle in high-altitude population and higher Pearson’s correlation of kidney and lung in low-altitude population,indicating that the artificial selection and natural selection have different preference for different tissues.Otherwise,the muscle which underwent dramatic artificial selection and the lung which experienced strong natural selection in high altitude environment showed the most significant difference between high-and low-altitude populations.(5)Hierarchical clustering and principal component analysis revealed that all samples can be firstly divided into vertebrate and mammals,thereafter mammalian samples can be separated by tissue and thereafter by species,which is inconsistent with the result from mRNA transcriptome.(6)Differentially expressed(DE)analysis revealed various number of DE miRNAs in each pairwise comparison.The number of DE miRNAs decreased from yak/cattle,pig,sheep and chicken,meanwhile kidney,liver and spleen have more DE miRNAs.We identified two miRNAs(miR-122 and miR-215)that simultaneously differentially expressed in three species,and identified 15 miRNAs that simultaneously differentially expressed in two species which were associated with iron homeostasis,glucose metabolism and cell apoptosis et al.(7)We detected widespread high expression of miR-2285 o in yak.Bioinformatics analysis revealed that protein tyrosine phosphatase,receptor type M(PTPRM)gene is the host gene of miR-2285 o,and that two linked single nucleotide polymorphisms in pre-miR-2285 o affect the biogenesis of miR-2285o-3p.Functional analysis indicated that miR-2285o-3p attenuated hypoxia-induced apoptosis through targeting phosphatase and tension homolog(PTEN)and very low density lipoprotein receptor(VLDLR).This study profiled comprehensive miRNAtranscriptome in four typical farm animals,including chicken,cattle/yak,sheep and pig,and analysized the evolutionary scenario of miRNAs.Compared with mRNAs,miRNAs showed more complex expressional pattern.In addition,a large number of differentially expressed miRNAs have been detected in each species and are associated with hypoxia-related process,such as angiogenesis,cell apoptosis.Furthermore,we also revealed the genetic mechanism underlying the expressional difference of miR-2285 o between yak and cattle,and clarified the function and molecular mechanism of miR-2285 o in high-altitude acclimatization.Our work provide an important reference for deeply digging the molecular mechanism of high-altitude adaptability in agricultural animals,and provide an important theoretical basis for utilizing characteristic genetic resources in our country,and provide valuable information for the development of livestock husbandry in high altitude.