| Subterranean rodents could maintain their normal activities in hypoxia environments underground.Eospalax cansus（E.cansus）,as one kind of subterranean rodents found in China can survive for more than 10 h under 4%O2 without observable injury,while Sprague-Dawley rats can survive for more than 6 h under the same conditions in labs,indicating that E.cansus have good hypoxia adaptability.To explore the potential mechanism of hypoxia responses in E.cansus,we performed RNA-seq analysis of multiple tissues（Heart,Brain,Liver,Muscle）in E.cansus exposed to different oxygen levels（6.5%6h,10.5%44h,21%one week）.At the same time,we sequenced two additional tissues,lung and pancreas of E.cansus.By assembling the whole RNA-seq data and using the annotated gene set of the Spalax galili genome as reference,we obtained a total of 18037 genes and 73835 transcripts.The main distribution range of gene length is 201～4600 nt,and the average length is 3097 nt.Most genes contain 2～6transcripts,with an average of 4.09 transcripts.By aligning the gene sequences of other published species against E.cansus sequences,it was found that the gene sequence of S.galili had the best homology with most gene sequences（>84%）of E.cansus（highest blast score）.By annotating gene sequences to multiple databases,such as Nr,GO,Swiss-prot,Pfam,and KEGG,a total of 17911 gene sequences were annotated by at least one database.A total of 17760 CDS sequences were predicted（threshold length>100 aa）,of which 70.8%（12578）CDS were completed with start codon and stop codon.By analyzing the gene expression level,it was found that a total of 17663 genes had observable expression level（the expression level exceeds 1 FPKM in at least on sample）.The largest number of genes expressed in brain tissue was observed in six tissues,and a total of 10209 genes were generally expressed in six tissues,mainly involving basic biological functions.A total of 3002 genes were identified as tissue-specific expression genes.Brain had the most tissue-specific expression genes among six tissues,and the least tissue-specific genes were found in heart.Liver had the highest proportion of high expression genes across all tissue-specific genes of six tissues.The enrichment functions of tissue-specific genes were consistent with the functions of corresponding tissues.In addition,we identified 2872 genes that can preferentially express different transcripts in different tissues,which may contribute to the realization of tissue-specific functions.We predicted a total of 4594 differentially expressed genes（DEGs）in comparisons of different oxygen treatment groups in multiple tissues.The largest number of DEGs were found in heart（2027）and the least number of DEGs were found in brain tissue（1019）.The most DEGs（3469）were found in single tissue.GO enrichment results showed that DEGs in brain were mainly about immunity,stimulus response,metabolism,etc.DEG found in liver were mainly related to metabolic processes in the liver,such as sterol metabolism,lipid metabolism,cell ketone metabolism,etc.GO enrichments of DEGs in heart were related to response and regulation,such as response to wounding,response to stimulus,defense response,regulation of cell proliferation,etc.DEGs in muscle were related to development,regulation and response,such as,regulation of cell regulation,tissue development,regulation of response to stress,cellular response to hormone stimulus,etc.We found multiple DEGs containing the GO term“response to hypoxia”,which may play key roles in the response to hypoxia in relevant tissues.KEGG enrichment of DEGs in brain were JAK-STAT signal pathway,TNF signal pathway,hippo signal pathway,etc.The pathway enrichment in heart included complement pathway,HIF-1 signaling pathway,calcium signaling pathway,etc.The enriched pathways in muscle DEGs were PI3K-Art signaling pathways,extracellular matrix receptor interaction,protein digestion and absorption,etc.The pathways enriched with liver DEGs were PPAR,AMPK,cholesterol metabolism,bile secretion,etc.By analyzing expression patterns of important genes related to energy metabolism under hypoxic conditions,we speculated that E.cansus increases fatty acid oxidation and gluconeogenesis under hypoxia,while protein synthesis and fatty acid synthesis may decrease in liver.In addition,the up regulation of anti-apoptosis or anti-oxidant gene may contribute to the hypoxic adaption for E.cansus.We performed q-PCR validation on six important DEGs in liver tissue.Studies showed that long noncoding RNAs（lnc RNAs）play important roles in gene expression regulation in different levels.Some lnc RNAs were found as hypoxia induced lnc RNAs in cancer.By analyzing the transcriptome data of E.cansus,we predicted thousands of lnc RNAs in liver and heart.When comparing with m RNAs,these lnc RNAs usually have shorter length,lower expression level and lower GC content.Majors of lnc RNAs have expression peaks in hypoxia conditions.We found 1128 DE-lnc RNAs（differential expressed lnc RNAs）responding to hypoxia.To search the mi RNA regulation network for lnc RNAs,we predicted 471 and 92 DE-lnc RNAs acting as potential mi RNA target and target mimics,respectively.We also predicted the functions of DE-lnc RNAs based on the co-expression networks of lnc RNA-m RNA.The DE-lnc RNAs participated in the functions of biological regulation,signaling,developing,oxoacid metabolic process,lipid metabolic/biosynthetic process,and catalytic activity.As the first study of lnc RNAs in E.cansus,our results show that lnc RNAs are popular in transcriptome widely and can participate in multiple biological processes in hypoxia responses.Finally,in order to facilitate users to apply our pipelines for lnc RNA identification and functional prediction of lnc RNAs based on coexpression between lnc RNAs and m RNAs and ce RNA hypothesis（lnc RNA-mi RNA-m RNA regulational network）,we developed specific software for lnc RNAs identification and functional prediction,and released them to Git Hub for users to download freely. |
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