Identification And Differential Expression Analysis Of MiRNA In Kazakh Horse Testes Of Different Developmental Stages

With the recent rise of horse industry in Xinjiang,the Kazakh horse,as an important and excellent indigenous breed,should be preserved.In meiosis,numerous genes control mammalian germ cells to develop and differentiate and these genes' expression is regulated post-transcriptionally too.MicroRNAs(miRNAs)are a class of short RNAs which regulate post-transcripionally gene expression in eukaryotes.Recent studies found some crucial steps in spermatogenesis are regulated by miRNAs.With the development of deep sequencing technology,many studies have determined the differential expression profiles of testis development in some species.But,miRNA expression profiles in horse testis haven't yet been studied.In order to characterize testicular development,miRNAs profiles and differential miRNA expression in horse testis and to characterize miR-34 family expression patterns in multiple organs and tissues in Kazakh horses,the study collected and measured foal,yearling and stallion testes(26 horses in total),determined(by dentation)and recorded horses' ages.Paraffin section technique was used to make testicular histological sections stained with HE or carmine and,for each animal,30 round and near round tubules were randomly selected to measure tubule diameters and to identify and enumerate separate kinds of germ cells(spermatogonia,spermatocyte,round spermatid and elongated spermatid).Based on histological analysis,foal,yearling and stallion(?5 years)were selected for deep sequencing.Total RNA was abstracted from foal,yearling and sallion(?5 years)testis tissues and were converted to cDNA which were sequenced,using Solexa sequencing technology,to construct three separate libraries(foal,yearling and stallion);clean reads were aligned against equine genome,NCBI Genebank and Rfam databases to identify equine miRNAs in combination with bioinformaics analysis.The three miRNA libraries were compared to analyze differentially expressed miRNAs.Target genes of differentially expressed miRNAs were predicted using GO and KEGG pathway analysis.The expression patterns of miR-34 family were studied in equine tesits,heart,liver,spleen,lung,kidney,skeleton muscle and fat tissues.The results were drawn as follows:1.During testis development of Kazakh horse,testis increase in length and width.The testis of 3 year old horse is smaller than that of full grown horse(p<0.05).The mean seminiferous tubule diameter and numbers of germ cell are less than these of full grown horse.The foal testis has testicular cords rather than seminiferous tubules;yearling testis has developing seminiferous tubules and numerous leydig cells;3 year old horse testis has full developed tubules with less round spermatids and elongated spermatids;5-12 year old horse testis has full grown testis and more round spermatids and elongated spermatids.2.The raw reads in testis libraries were 9250474(foal),9820184(yearling)and 9813836(stallion),respectively.The short RNAs in foal and yearling libraries had normal distribution,with a 21-23 nt peak,representing miRNAs;the stallion library short RNAs showed a bimodal distribution with a major peak at 22-23 nt and a weak peak at 25-27 nt,which representing miRNAs and piRNAs respectively.Foal,yearling and stallion libraries have 375,391 and 388 known and reserved equine miRNAs and 47,111 and 78 novel miRNAs respectively.Based on differential analysis of miRNA expression in horse testes,miRNAs were divided into nine groups.The constant-up group had 48 miRNAs,including four verified and spermatogenesis close-related miRNAs: miR-34 b,miR-34 c,miR-449 a and 449 b.GO and KEGG pathway analysis reveal the constant-up group is related to cell proliferation and cancer.3.miR-34 a has strong expression in foal liver and lung and stallion spleen;it has moderate expression in testis of foal,yearling and stallion,lung and kidney of stallion,and foal kidney and heart;it has weak expression skeletal muscle and fat.miR-34 b has high expression in foal lung,have moderate expression stallion spleen,kidney,testis and lung and foal kidney,and have low expression in heart,liver,skeletal muscle and fat.Similar to miR-34 b expression,miR-34 c has elevated expression in foal lung,and have moderate expression in stallion spleen,kidney,lung,testis and low expression in heart,fat and skeletal muscle.MiR-34 a has relatively stable expression during testis development.however,miR-34 b and miR-34 c have significantly increased expression in stallion testis(p<0.01).MiR-34 family have identical seed sequence(GGCAGUG).MiR-34 a has 86% and 82% sequence similarity with miR-34 b and miR-34 c.Results reveal miR-34 b and miR-34 c have similar expression patterns in equine multiple organs and tissues.Based on the above results,the conclusions were drawn as follows:1.During Kazakh horse development,5 month old horse(foal)testis is not developing with testicular cords;1.5 year old horse(yearling)has developing testis with under-developed seminiferous tubules;3 year old horse has well-developed testis with less spermatids and elongated spermatids;horse(? 5 years old)(stallion)had fully-developed testis with all kinds of germ cells in abundance,indicating active spermatogenesis.2.In the three libraries,we identified total 459 known and reserved miRNAs,including 375 miRNAs in foal library,391 miRNAs in yearling library and 388 miRNAs in stallion library.The 25-27 nt RNAs account for more than one third of clean reads,which represent piRNAs.piRNAs are closely related with meiosis,and we predict piRNA is a hot spot in animal reproduction science in near future.3.MiR-34 a has a ubiquitous expression in horse and miR-34 b and miR-34 a have tissue-specific expression.MiR-34 b has similar expression patterns with miR-34 a in these organs and tissues,which suggests miR-34 b and miR-34 c have function redundancy.