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Research article
Dequan Xu
Huazhong Agriculture University
Corresponding Author
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Background: MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis.
Results: In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-165 mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 ( PLCβ1) gene was verified to be a target of ssc-mir-423-5p .
Conclusions: This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.
Keywords
Meishan boar, Duroc boar, Testis, Sexual development, miRNA, Integrating analysis
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.pdf
Additional file 1: Table S1 The number of genes detected in the six samples.
- Additionalfile2.pdf
Additional file 2: Table S2 The number of genes involved in KEGG pathway and GO term.
- Additionalfile3.pdf
Additional file 3: Figure S1 Expression analyses of 4 DE miRNAs and 4 DEGs using qRT-PCR and RNA-Seq. Analyses of relative expression levels of miRNAs and genes in Duroc (black bars) and Meishan (gray bars) boars in the left; RPKM of miRNAs and genes in Duroc (blue bars) and Meishan (purple bars) boars a in the right. The X-axis indicates age of samples. The left Y-axis and right Y-axis show the relative expression level and RPKM of miRNAs and genes, respectively. a ssc-mir-181b, b ssc-mir-423-5p, c ssc-mir-196b-5p, d ssc-mir-4334-3p, e CYLD, f CDYL, g PLCβ1, h SOX30. **P< 0.01, *P< 0.05. DE miRNAs, differentially expressed miRNAs; DEGs, differentially expressed genes; qRT-PCR, quantitative real-time PCR; RNA-Seq, RNA sequencing; RPKM, Reads Per Kilobase per Million mapped reads; CYLD, Cylindromatosis; CDYL, chromodomain Y like; PLCβ1, phospholipase C beta 1; SOX30, SRY-box containing gene 30.
- Additionalfile4.pdf
Additional file 4: Table S3 Synthesis of ssc-mir-423-5p sequences. NC, negative control.
- Additionalfile5.pdf
Additional file 5: Figure S2 The expression levels of ssc-mir-423-5p were analyzed by qRT-PCR. a ssc-mir-423-5p expression level after transfecting ssc-mir-423-5p mimic, mimic NC. b ssc-mir-423-5p expression level after transfecting ssc-mir-423-5p inhibitor or inhibitor NC into ST cells. NC, negative control; ST, swine testis
- Additionalfile6.pdf
Additional file 6: Figure S3 Full image of Western Blotting. a PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p mimic and mimic NC. b PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p inhibitor and inhibitor NC.
- Additionalfile7.pdf
Additional file 7: Table S4 Primers of genes and miRNAs used for quantitative real-time PCR.
- Additionalfile8.pdf
Additional file 8: Table S5 Primers for amplification of porcine PLCβ1 3´UTR.
- Additionalfile9.xlsx
Additional file 9: Table S6 The identified miRNA information
- Additionalfile10.docx
Additional file 10: Figure S4 Western blotting analysis was used to detect PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p mimic /mimic NC or ssc-mir-423-5p inhibitor /inhibitor NC.
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Learn more about In Review
Research article
Dequan Xu
Huazhong Agriculture University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Genomics.
No community comments so far
Submission checks complete
On 27 Sep, 2020
Editorial decision: Accept
On 23 Sep, 2020
No comments provided
Editorial decision: Minor revision
On 18 Sep, 2020
Editor assigned
On 14 Sep, 2020
Submission checks complete
On 13 Sep, 2020
Editor invited
On 13 Sep, 2020
Version 4
Posted 10 Sep, 2020
No comments provided
Editorial decision: Minor revision
On 09 Sep, 2020
Editor assigned
On 07 Sep, 2020
Submission checks complete
On 06 Sep, 2020
Editor invited
On 06 Sep, 2020
No comments provided
Submission checks complete
On 03 Sep, 2020
Editorial decision: Minor revision
On 03 Sep, 2020
No comments provided
Editorial decision: Minor revision
On 12 Aug, 2020
Reviewer #2 agreed
On 25 Apr, 2020
Reviewer #1 agreed
On 23 Apr, 2020
Review #1 received
Received 23 Apr, 2020
Editor assigned
On 20 Apr, 2020
Reviewers invited
Invitations sent on 20 Apr, 2020
Submission checks complete
On 19 Apr, 2020
Editor invited
On 19 Apr, 2020
No comments provided
Editorial decision: Major revision
On 20 Mar, 2020
Review #2 received
Received 11 Mar, 2020
Reviewer #2 agreed
On 24 Feb, 2020
Review #1 received
Received 01 Feb, 2020
Reviewer #1 agreed
On 21 Nov, 2019
Reviewers invited
Invitations sent on 16 Nov, 2019
Submission checks complete
On 23 Sep, 2019
Editor assigned
On 05 Sep, 2019
First submitted
On 04 Sep, 2019
Editor invited
On 04 Sep, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Genomics.
Background: MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis.
Results: In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-165 mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 ( PLCβ1) gene was verified to be a target of ssc-mir-423-5p .
Conclusions: This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1.pdf
Additional file 1: Table S1 The number of genes detected in the six samples.
- Additionalfile2.pdf
Additional file 2: Table S2 The number of genes involved in KEGG pathway and GO term.
- Additionalfile3.pdf
Additional file 3: Figure S1 Expression analyses of 4 DE miRNAs and 4 DEGs using qRT-PCR and RNA-Seq. Analyses of relative expression levels of miRNAs and genes in Duroc (black bars) and Meishan (gray bars) boars in the left; RPKM of miRNAs and genes in Duroc (blue bars) and Meishan (purple bars) boars a in the right. The X-axis indicates age of samples. The left Y-axis and right Y-axis show the relative expression level and RPKM of miRNAs and genes, respectively. a ssc-mir-181b, b ssc-mir-423-5p, c ssc-mir-196b-5p, d ssc-mir-4334-3p, e CYLD, f CDYL, g PLCβ1, h SOX30. **P< 0.01, *P< 0.05. DE miRNAs, differentially expressed miRNAs; DEGs, differentially expressed genes; qRT-PCR, quantitative real-time PCR; RNA-Seq, RNA sequencing; RPKM, Reads Per Kilobase per Million mapped reads; CYLD, Cylindromatosis; CDYL, chromodomain Y like; PLCβ1, phospholipase C beta 1; SOX30, SRY-box containing gene 30.
- Additionalfile4.pdf
Additional file 4: Table S3 Synthesis of ssc-mir-423-5p sequences. NC, negative control.
- Additionalfile5.pdf
Additional file 5: Figure S2 The expression levels of ssc-mir-423-5p were analyzed by qRT-PCR. a ssc-mir-423-5p expression level after transfecting ssc-mir-423-5p mimic, mimic NC. b ssc-mir-423-5p expression level after transfecting ssc-mir-423-5p inhibitor or inhibitor NC into ST cells. NC, negative control; ST, swine testis
- Additionalfile6.pdf
Additional file 6: Figure S3 Full image of Western Blotting. a PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p mimic and mimic NC. b PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p inhibitor and inhibitor NC.
- Additionalfile7.pdf
Additional file 7: Table S4 Primers of genes and miRNAs used for quantitative real-time PCR.
- Additionalfile8.pdf
Additional file 8: Table S5 Primers for amplification of porcine PLCβ1 3´UTR.
- Additionalfile9.xlsx
Additional file 9: Table S6 The identified miRNA information
- Additionalfile10.docx
Additional file 10: Figure S4 Western blotting analysis was used to detect PLCβ1 protein expression levels at 72 h after ST cells were transfected with ssc-mir-423-5p mimic /mimic NC or ssc-mir-423-5p inhibitor /inhibitor NC.
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