Genome-wide identification and comparison of differentially expressed profiles of miRNAs and lncRNAs with associated ceRNA networks in the gonads of Chinese soft-shelled turtle, Pelodiscus sinensis

- comparison of differentially expressed profiles of miRNAs and lncRNAs with.
- associated ceRNA networks in the gonads of Chinese soft-shelled turtle, Pelodiscus sinensis.
- The regulatory mechanism of the expression of protein-coding genes involved in reproduction still remains to be elucidated.
- However, the regulatory patterns of miRNA and lncRNA in the sex biased expression of protein coding genes remains to be elucidated.
- In this study, we performed an integrated analysis of miRNA, messenger RNA (mRNA), and lncRNA expression profiles to explore their regulatory patterns in the female ovary and male testis of Pelodiscus sinensis..
- Results: We identified 10,446 mature miRNAs, 20,414 mRNAs and 28,500 lncRNAs in the ovaries and testes, and 633 miRNAs, 11,319 mRNAs, and 10,495 lncRNAs showed differential expression.
- The predicted target genes of these differentially expressed (DE) miRNAs and lncRNAs included abundant genes related to reproductive regulation.
- The target genes for the differentially expressed miRNAs and lncRNAs included abundant genes involved in gonadal development, including Wt1, Creb3l2, Gata4, Wnt2, Nr5a1, Hsd17, Igf2r, H2afz, Lin52, Trim71, Zar1, and Jazf1..
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- A thorough understanding of ncRNA-based cellular regulatory networks will aid in the improvement of P.
- Sex-dependent differences are often exhibited in the growth and size of aquaculture animals displaying sexual dimorphism [2].
- Reproduction is an important yet com- plex biological process in animals, and a comprehensive understanding of the genetic mechanisms underlying re- productive traits, particularly from the genomics per- spective.
- Similar to other reptiles and mammals, the soft-shelled turtle has the ability to store sperm in the ovary [4].
- Many previous studies have focused on sex determination and differentiation in the turtle.
- However, to the best of our knowledge, no study has explored the genetic mecha- nisms underlying the reproductive development of the soft-shelled turtle..
- MiRNAs regulate gene expression at the post- transcriptional level by binding to either perfect or imper- fect complementary sequences in the 3′ untranslated re- gions (UTRs) of targets and triggering either degradation.
- of the targets or inhibit their translation [11].
- MiR- 9 could bind to the foxl3 3′ UTR in Monopterus albus, which may be involved in the process of oocyte degener- ation [18].
- However, regulation of the reproductive system is complicated.
- Investigations re- garding lncRNA–miRNA–mRNA networks provide a better understanding of the role of lncRNA–miRNA inter- actions in mRNA regulation.
- In the.
- present study, miRNAs and lncRNAs of the ovary and testis were investigated in P.
- Results of the present study may provide the basis for a better understanding of the roles of miRNAs and lncRNAs in the turtle ovary and testis, leading to exploitation of the mechanisms of reproduction in Chinese soft-shelled turtle..
- Overview of the sequencing data.
- We constructed cDNA libraries of miRNAs, mRNAs, and lncRNAs using the RNA from the ovaries and testes..
- 1 Distribution of miRNAs, mRNAs and lncRNAs in ovaries and testes of Chinese soft-shelled turtle.
- (d) The outmost layer of the circos plot is a chromosome map of the turtle genome.
- The green layer of the circos plot is sense lncRNAs.
- The red layer of the circos plot is intergenic lncRNAs.
- The blue layer of the circos plot is intronic lncRNAs.
- The grey layer of the circos plot is antisense lncRNAs.
- 87.72% of the reads were mapped to intergenic regions in the P..
- Identification of the differential expression of mRNAs, miRNAs, and lncRNAs.
- Predic- tion of the potential targets of lncRNAs in cis and trans was performed to investigate the function of lncRNAs (Additional file 5).
- 2 Identifying differentially expressed miRNAs, mRNAs and lncRNAs by transcriptome sequencing in ovaries and testes of Chinese soft-shelled turtle.
- To annotate the molecular functions of the differen- tially expressed miRNAs, both RNA hybrid and Mi- Randa software were used to improve the prediction of miRNA targets, resulting in 8088 target genes in- cluding 2814 differentially expressed genes that were potentially regulated by 633 DEmiRNAs.
- GO categor- ies of miRNAs and lncRNAs were assigned to all tar- get genes based on the following three ontologies:.
- Functions of target genes in the cellular component category mainly fo- cused on cell part, cell, and membrane.
- The identified metabolic networks were related to neuroactive ligand-receptor interaction and regulation of the actin cytoskeleton.
- Validation of differentially expressed miRNAs and lncRNAs.
- To validate the sequencing data of miRNAs and lncRNAs, ten DEmiRNAs and ten DElncRNAs were randomly selected to test their relative expression in ovaries and testes.
- The expression of eight miRNAs and seven lncRNAs in ovaries and testes was consistent with the results of RNA sequen- cing.
- For instance, Dazl mRNA and MSTRG.71049.8 shared a common binding site of the miRNA novel-miR-1222..
- We also identified Wt1, CREB3l2, Gata4, Wnt2, Nr5a1, Hsd17, Igf2r, H2afz, Lin52, Trim71, Zar1, and Jazf1 in the ceRNA network.
- The turtle genome showed a large proportion of non- coding regions, indicating that this part of the genome.
- However, their exact functions in the soft-shelled turtle remain poorly understood.
- Despite limited studies that have identified lncRNAs in the turtle [3], the miR- NAs and lncRNAs in the database are still insufficient..
- In the present study, to understand the molecular mech- anism involved in the reproduction of P.
- sinensis, we analysed the genome-wide expression of miRNAs, lncRNAs, and mRNAs in the mature ovaries and testes during the reproductive season.
- After filtering, we ob- tained 10,796 miRNAs and 58,923 lncRNAs that were not reported previously in the miRbase and lncRNA da- tabases.
- The lengths of the miRNAs ranged from 18 to 25 nt.
- MiRNAs and lncRNAs have been shown to have stage-specific expres- sion in animals [16, 33].
- The database included many target genes for miRNAs and lncRNAs that might regulate.
- In the network, red circles = DEmiRNAs, blue triangles = DElncRNAs, and mazarine = DEmRNAs.
- A total of 8 miRNAs and 7 lncRNAs were verified using qRT-PCR.
- Mature miRNAs and lncRNAs are crucial for the regu- lation of gene expression in different ways [34, 35].
- The most abundant differentially expressed genes were involved in single organism process, followed by cellular process and biological regulation, indicating that abundant DEmiRNAs might be involved in the repro- ductive process and reproduction.
- In the single organism process, the targets of DEmiRNAs and DElncRNAs included Cyp19a1, Ar, Esrrb, Catsper2, and Pgr, etc., which were proven to be important for gonadal development, and the results indicated that DEmiRNAs and DElncRNAs might be involved in reproductive regulation..
- The DEmiRNAs identified in the soft-shelled turtle be- long to 439 families after mapping on the genome, in- cluding let-7, miR-10, miR-130, miR-133, miR-138, miR- 145, miR-143, miR-202, miR-224, and miR-378.
- In the majority of cases, the miRNAs and their targets were correlated with animal reproduction [36–39].
- In the present study, miR-10 and miR-202 ex- pression was significantly higher in the ovaries than the.
- LncRNAs are recognised as important functional regu- latory factors in the regulation of eukaryotic gene ex- pression in a variety of biological processes.
- LncRNA H19 could regulate the IGF-1 signalling path- way, which resulted in regulation of the proliferation and apoptosis of male germline stem cells in bovines [46].
- Furthermore, the H19 imprinting control region could acquire parent-of-origin-dependent methylation after fertilisation independent of the chromosomal inte- gration site or the prerequisite methylation acquisition in male germ cells [47].
- In the present study, we identified 28,500 lncRNAs including 10,495 DElncRNAs..
- Prediction of targets showed that a large number of DElncRNAs might regulate gonadal development, and further investigation should be undertaken to reveal their functions in the turtle..
- In the present study, we constructed lncRNA–miRNA–mRNA net- works for sex-specific expression based on high- throughput sequencing data in the turtle.
- The target genes of miRNAs and lncRNAs play important roles in the reproductive processes.
- the Cyp19a1 promoter, which is crucial for functional maintenance of the ovary [54].
- Further research on ceRNAs involved in reproductive regulation will be car- ried out in the turtle.
- Considering knowledge of the regulatory mechanism of gonadal development is scarce in non-mammal animals, our results help to enrich the understanding of the reproductive regulatory network in non-mammalian vertebrates..
- In the present study, we identified mRNAs, miRNAs, and lncRNAs using high-throughout sequencing data from the ovaries and testes of Chinese soft-shelled turtles and con- structed the associated ceRNA networks.
- We identified 11,319 DEmRNAs, 633 DEmiRNAs, and 10,495 DElncR- NAs in the ovary and testis.
- Furthermore, we constructed ceRNA networks, which included DEmRNAs, DEmiR- NAs, and DElncRNAs that regulated the reproduction of the turtle.
- All investigations in the present study were performed ac- cording to the Animal Experimental Guidelines of the Ethical Committee of the University of China.
- of Xinyang, China, which were aged 24 months and cultured in the same pond.
- To minimize suffering of the turtle, each turtle was euthanized with an overdose of 2 ml anaesthetic (2-phenoxyethanol;.
- Clustering of the index-coded samples was performed on a cBot Clus- ter Generation System using TruSeq PE Cluster Kit v4- cBot-HS (Illumina, USA) according to the manufac- turer’s instructions.
- Quality control of the reads was performed using FastQC (http://www.bioinformatics.babraham.ac.uk/pro- jects/fastqc.
- The Q20, Q30 and GC contents of the filtered reads were calculated and used for further analysis..
- The char- acteristic hairpin structure of the miRNA precursors was used to predict novel miRNA.
- miRDeep2 [61] and Mfold software were used for predicting the structure of the unannotated miRNAs and their precursors..
- To annotate the functions of the lncRNAs, we pre- dicted the target protein-coding genes of lncRNAs in cis and trans.
- Differentially expressed genes (DEGs) analysis of the miRNAs, lncRNAs, and mRNAs was performed using the DEGseq package .
- To predict the functions of the miRNAs and lncRNAs, the target genes and differentially expressed genes were annotated against the NCBI non-redundant protein database (Nr), the Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG), and clus- ters of orthologous groups of proteins.
- Quantitative real-time PCR analysis of miRNA and lncRNA In the present study, 8 miRNAs and 7 lncRNAs were verified by using quantitative real-time PCR (qRT-PCR)..
- The results of the qRT-PCR data are presented as the mean ± standard error of the mean value.
- We thank all of the people who contributed to the present study..
- These funding bodies had no role in the design of the study, extraction, analysis, and interpretation of data, or in writing the manuscript..
- The datasets generated and/or analysed during the current study are available in the NCBI Sequence Read Archive (SRA) repository (accession number is PRJNA623141).
- The genome information of the turtle was obtained from NCBI (accession number is PRJNA221645).
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- A 5 ′ UTR-overlapping LncRNA activates the male- determining gene doublesex1 in the crustacean Daphnia magna.
- Long non-coding RNAs potentially function synergistically in the cellular reprogramming of SCNT embryos.
- Crosstalk among lncRNAs, microRNAs and mRNAs in the muscle 'degradome' of rainbow trout.
- Transcription of CYP19A1 is directly regulated by SF-1 in the theca cells of ovary follicles in chicken

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