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. 2022 Mar 10;12(6):694.
doi: 10.3390/ani12060694.

Identification and Molecular Analysis of m6A-circRNAs from Cashmere Goat Reveal Their Integrated Regulatory Network and Putative Functions in Secondary Hair Follicle during Anagen Stage

Taiyu Hui  1 , Yubo Zhu  1 , Jincheng Shen  1 , Man Bai  1 , Yixing Fan  1 , Siyu Feng  1 , Zeying Wang  1 , Junyin Zhao  1 , Qi Zhang  1 , Xingwang Liu  1 , Tiantian Gong  1 , Wenlin Bai  1
Affiliations

Identification and Molecular Analysis of m6A-circRNAs from Cashmere Goat Reveal Their Integrated Regulatory Network and Putative Functions in Secondary Hair Follicle during Anagen Stage

Taiyu Hui et al. Animals (Basel). .

Abstract

N6-methyladenosine (m6A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m6A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m6A modified circRNAs (m6A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m6A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m6A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m6A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m6A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m6A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m6A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m6A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m6A-circRNAs (m6A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m6A-circRNAs in SHF development and cashmere growth in goats.

Keywords: M6A-circRNAs; cashmere goat; expression characterization; regulatory network; secondary hair follicle.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
A schematic diagram of screening putative m6A-circRNAs with the design of divergent primer pairs. (A) A total of 15 putative m6A-circRNAs were screened out from full-length transcriptome sequencing data where the potential m6A sites were predicted using the SRAMP program (http://www.cuilab.cn/sramp, accessed on 11 July 2021); (B) The design scheme of divergent primer pairs for detecting the expression of putative m6A-circRNAs in skin tissue or SHFs of cashmere goat.
Figure 2
Figure 2
Diagram of putative m6A sites within 15 circRNA sequences from skin tissue of cashmere goat. The orange thin line represents circRNA sequence, and the motifs of m6A sites are shown within each circRNA sequence with the corresponding nucleotide positions. The putative m6A sites were predicted using the SRAMP program (http://www.cuilab.cn/sramp, accessed on 11 July 2021) and the only m6A site motifs are shown in each circRNA sequence with very high confidence in SRAMP analysis.
Figure 3
Figure 3
Expression analysis of 15 putative m6A-circRNAs in skin tissue of cashmere goats at both anagen and telogen stages presented as a hierarchical clustering heatmap. The “Up-Re” indicates the expression mean of putative m6A-circRNAs being significantly up-regulated in skin tissue at anagen of cashmere goats in comparison to telogen (p < 0.05). The “Down-Re” indicates the expression mean of putative m6A-circRNAs being significantly down-regulated in skin tissue at anagen of cashmere goats in comparison to telogen (p < 0.05). The “No-Diff” indicates no significant difference in expression mean of putative m6A-circRNAs in skin tissue of cashmere goats between anagen and telogen stages (p > 0.05). The resulting p-value for each comparison is presented in Table S1.
Figure 4
Figure 4
The ceRNA regulatory network of each putative m6A-circRNA with higher expression in anagen skin tissue of cashmere goats in comparison to telogen. The pink circle, red “V”, green diamond shapes represent m6A-circRNAs, miRNAs and target genes, respectively. (A) circRNA-ZNF638 = m6A-circRNA-ZNF638; (B) circRNA-DNAJB6 = m6A-circRNA-DNAJB6; (C) circRNA-TULP4 = m6A-circRNA-TULP4; (D) circRNA-CAT = m6A-circRNA-CAT; (E) circRNA-CAAP1 = m6A-circRNA-CAAP1; (F) circRNA-STAM2 = m6A-STAM2.
Figure 5
Figure 5
Integrated ceRNA network of the six putative m6A-circRNAs with higher expression in skin tissue at anagen of cashmere goats in comparison to telogen. The pink circle, red “V”, and green diamond shapes represent m6A-circRNAs, miRNAs and target genes, respectively. circRNA-ZNF638 = m6A-circRNA-ZNF638, circRNA-DNAJB6 = m6A-circRNA-DNAJB6, circRNA-TULP4 = m6A-circRNA-TULP4, circRNA-CAT = m6A-circRNA-CAT, circRNA-CAAP1 = m6A-circRNA-CAAP1, circRNA-STAM2 = m6A-STAM2. This integrated ceRNA network was constructed and visualized using the Cyotoscape (Version 2.8) procedure [35].
Figure 6
Figure 6
Pathway enrichment for the ceRNA regulatory genes of the six putative m6A-circRNAs with higher expression in anagen skin tissue of cashmere goats in comparison to telogen. The pathway enrichment was conducted using the CluePedia (a built-in plugin in Cytoscape procedure (http://www.ici.upmc.fr/cluepedia/, 12 October 2021). the enriched pathways and their corresponding genes were visualized with the same color nodes.
Figure 7
Figure 7
Expression analyses of the six anagen up-regulated m6A-circRNAs along with their host genes in SHFs of cashmere goats at telogen, anagen, and catagen stages. (A): circRNA-ZNF638 and its host gene ZNF638, (B): circRNA-TULP4 and its host gene TULP4, (C): circRNA-DNAJB6 and its host gene DNAJB6, (D): circRNA-CAT and its host gene CAT, (E): circRNA-STAM2 and its host gene STAM2, and (F): circRNA-CAAP1 and its host gene CAAP1. The expression difference was compared for each m6A-circRNA and corresponding host gene at telogen, anagen, and catagen, where the telogen expression data of both m6A-circRNA and the corresponding host gene was normalized to 1. The star symbol “*” stands for a significant difference (p < 0.05), and the “NS” stands for no significant difference (p > 0.05). The obtained p-value for each comparison is presented in Table S2. circRNA-ZNF638 = m6A-circRNA-ZNF638, circRNA-TULP4 = m6A-circRNA-TULP4, circRNA-DNAJB6 = m6A-circRNA-DNAJB6, circRNA-CAT = m6A-circRNA-CAT, circRNA-STAM2 = m6A-STAM2, and circRNA-CAAP1 = m6A-circRNA-CAAP1.
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