CN113913496A - Wool cyst development related circRNA expression profile as well as construction method and application thereof - Google Patents
Wool cyst development related circRNA expression profile as well as construction method and application thereof Download PDFInfo
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Abstract
The invention provides a circRNA expression profile related to wool cyst development and a construction method and application thereof. The construction method comprises the following steps: (1) RNA library construction and sequencing: constructing an RNA library for a sample of body-side skin of a sheep at a fetal stage and sequencing the RNA library; (2) identification of transcripts: predicting circRNA according to a circRNA identification standard; (3) differential expression analysis: performing differential expression analysis on the circRNA identified in the step (2); (4) GO enrichment and KEGG pathway analysis: GO enrichment and KEGG pathway analysis of circRNA related host genes differentially expressed in step (3); (5) construction of a cerRNA regulatory network: predicting the miRNA targeted by the circRNA, predicting the target gene of the targeted miRNA, and primarily constructing a circRNA-miRNA-mRNA regulation network. Provides important basis for exploring the molecular mechanism of the circRNA as the cerRNA in the generation and development of the hair follicle in the future.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a circRNA expression profile related to wool cyst development and a construction method and application thereof.
Background
There are two hair follicles in the skin of a cashmere goat, namely a primary hair follicle and a secondary hair follicle. The primary follicle produces coarse hair and the secondary follicle produces fine hair. The skin and hair follicle structural characteristics of cashmere goats are not only important for their biological characteristics, but also have a directly important impact on the yield and quality of the villi. The character of the hair follicle of the skin of the cashmere goat has direct and important influence on the yield and the quality of the fluff, the research in the field of the growth and development of the hair follicle of the cashmere goat is developed, and the final aim is to disclose the mechanism of the growth of the fluff and find out the important gene related to the growth of the fluff. The development of hair follicles may be related to several protein-encoding genes, and at present, most of the signaling molecules for regulating the morphogenesis of hair follicles are considered to belong to the Wnt pathway, Tumor Necrosis Factor (TNF), Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP), Sonic hedgehog (Shh), Transforming Growth Factor (TGF), NOTCH pathway, and the like. Some of the coding genes are stimulators of hair follicle development, and some are inhibitors, and are repeatedly used and mutually regulated.
circRNA is a relatively specific class of RNA that lacks free 5 'cap and 3' poly (A) structures and is nuclease-insensitive. The types of the circRNAs are classified into four types according to their sources, including four types, intron circRNA (intron circRNA, IcircRNA), exon circRNA (exon circRNA, EcircRNA), exon intron-binding circRNA (exon-intron circRNA, EIcircRNA), and intergenic circRNA (intergenic circRNA). The mechanism of action of circRNA mainly includes: regulating the expression of parent genes; interaction with RNA binding proteins; translating the protein; as a competitive endogenous RNA regulating gene expression.
Currently, it is a research hotspot that circRNA regulates the activity of life by competitively binding to miRNA to regulate the expression of genes. Research shows that the circLMO7 can enhance the expression quantity of miR-378a-3p target gene HDAC4 by competitively combining miR-378a-3p, promote muscle cell proliferation and inhibit the differentiation of bovine myoblast; the circARF3 adsorbs miR-103, relieves the targeted inhibition effect of miR-103 on TRAF3, and relieves the adiposis by promoting mitochondrion autophagy. The circRNA3669 is used as cerRNA to adsorb miR-26a and relieve the down-regulation effect of the miR-26a on RCN2 in the Endometrium Epithelial Cells (EEC) of the milk goat. circRNA has been studied in a variety of tissues and cells, however, research on the developmental regulation of hair follicle development in the embryonic stage of down producing goats is lacking.
Disclosure of Invention
In view of the above, the invention aims to provide a circRNA expression profile related to the occurrence and development of a wool follicle, and a construction method and application thereof, and provides an important basis for exploring a molecular mechanism of the circRNA as a ceRNA in the occurrence and development of hair follicles in the future.
The invention provides a method for constructing circRNA related to wool cyst development on one hand, which comprises the following steps:
(1) RNA library construction and sequencing: constructing an RNA library for a sample of body-side skin of a sheep at a fetal stage and sequencing the RNA library;
(2) identification of transcripts: predicting circRNA according to a circRNA identification standard;
(3) differential expression analysis: performing differential expression analysis on the circRNA identified in the step (2);
(4) GO enrichment and KEGG pathway analysis: GO enrichment and KEGG pathway analysis of circRNA related host genes differentially expressed in step (3);
(5) construction of a cerRNA regulatory network: predicting the miRNA targeted by the circRNA, predicting the target gene of the targeted miRNA, and primarily constructing a circRNA-miRNA-mRNA regulation network.
Further, on the basis of the technical scheme provided by the invention, in the step (1), the sample comprises four phases of the sheep fetal stage: 45 days, 55 days, 65 days and 75 days.
Further, on the basis of the technical scheme provided by the present invention, in the step (2), the circRNA identification standard comprises: the mismatch is less than or equal to 2, the Back-helicedjuntions reads are more than or equal to 1, and the distance between two helices sites on the genome is less than 100 kb.
Further, on the basis of the technical scheme provided by the invention, in the step (3), the SRPBM value of the circRNAs is calculated to perform differential expression analysis;
preferably, differential circRNA screening is performed using fold difference and pvalue;
more preferably, genes satisfying both log2FoldChange absolute value equal to 1 and pvalue equal to 0.05 are designated as yes, and those not are designated as no.
Further, in the step (4), GO enrichment and KEGG pathway analysis of the circRNA related host genes which are differentially expressed. The results show the GO term or KEGG path with P value <0.05 or top 20.
Further, on the basis of the technical scheme provided by the invention, in the step (5), miRNA library data and mRNA library data are integrated, and the circRNA and mRNA and miRNA binding sites are analyzed by using miRanda and Targetscan to construct a cerRNA network of the circRNA-miRNA.
Furthermore, in the step (5), two types of software, namely Targetscan (http:// www.targetscan.org/mammm _31/) and MiRanda (http:// www.microrna.org/microrna/home. do), are used for predicting the CircRNA-targeted miRNA, predicting the target gene of the targeted miRNA, preliminarily constructing a CircRNA-miRNA-mRNA regulation and control network, and finally visualizing the mRNA by using Cytoscape.
Further, on the basis of the technical scheme provided by the invention, the sheep is an inner Mongolian cashmere goat.
In the research, high-throughput sequencing is used to obtain the expression profiles of the circRNAs in the skin hair follicles of the inner Mongolian cashmere goats at different embryonic stages (45 days, 55 days, 65 days and 75 days), and 21784 circRNAs are identified. Meanwhile, the 6 ratios formed in four stages are respectively as follows: d75vsd45, circRNA up-regulated 59, down-regulated 33; d75vsd55, circRNA up-regulated 61, down-regulated 102; d75vsd65, circRNA up-regulated 32, down-regulated 33; d65vsd55, circRNA up-regulation 67, down-regulation 169; d65vsd45, circRNA up-regulation 96, down-regulation 63; d55vsd45, circRNA up-regulated 76, down-regulated 42. 6 circRNAs with different expressions are randomly selected, and RT-qPCR is used for verifying the reliability of a sequencing result. Then GO and KEGG pathway analysis is carried out on the parent gene corresponding to the circRNA, and the result shows that GO-enriched biological processes related to hair follicle growth and development mainly comprise hair follicle stimulating, hair follicle mapping, cell degrading and the like, and signal pathways related to hair follicle development comprise Notch signaling pathway and NF-kappa B signaling pathway. The circRNA differentially expressed by d75vsd45 is combined with miRNA and mRNA databases to construct a coexpression network of the circRNA-miRNA-mRNA, and an interaction relationship of 102 pairs of circRNA-miRNA and 126 pairs of miRNA-mRNA is formed. The dual-luciferase is further used for verifying the binding relationship of circRNA3236-chi-miR-27b-3p and circRNA3236-chi-miR-16b-3p, and the result shows that the circRNA3236 has a targeted binding relationship with chi-miR-27b-3p, circRNA3236 and chi-miR-16b-3 p.
The second aspect of the invention provides a circRNA expression profile related to wool cyst development, which is obtained by adopting a construction method of circRNA related to wool cyst development.
The third aspect of the invention provides a construction method of the circRNA related to wool follicle genesis and development, or an application of the expression profile of the circRNA related to wool follicle genesis and development in the regulation of sheep skin hair follicle genesis and development.
The expression profile of the circRNA in the skin hair follicles of the embryonic stage of the down producing goat is established, and the parent gene of the circRNA is found to be possibly involved in the development of the down producing goat hair follicles. A coexpression network of circRNA-miRNA-mRNA for differentially expressing circRNA is constructed and preliminarily verified. The technical scheme adopted by the invention has the following beneficial effects:
(1) the invention establishes an expression profile of circRNA in skin hair follicles in the embryonic stage of the down producing goat, and finds that a parent gene of the circRNA may participate in the development of the down producing goat hair follicles.
(2) The invention constructs a coexpression network of circRNA-miRNA-mRNA for differentially expressing circRNA, and carries out preliminary verification. Provides important basis for exploring the molecular mechanism of circRNA as cerRNA in the generation and development of hair follicles
Drawings
FIG. 1 shows the results of the identification of circRNA in example 1, with the left side showing the number of circRNA and the right side showing the number of host genes for circRNA.
FIG. 2 is a graph showing the predicted results of circRNA in example 1. Wherein, A: characterization of circRNA in sheep follicles, distribution of the amount of circRNA per gene. The x-axis represents the number of circRNAs/host genes and the y-axis represents the number of circRNAs. B: the diagram shows the exon length of the exon-derived circular RNA, the x-axis indicates the number of exons contained in the circular RNA, and the y-axis indicates the exon length. C: the distribution of the identified circular RNAs on each chromosome is represented by the number of chromosomes on the x-axis and the number of circular RNAs sorted by chromosome. D: classification of circular RNAs in wool sacs of wool of. .
FIG. 3 is a graph showing the results of the expression of CircRNA in example 1. A: a block diagram showing the abundance of CircRNA expression in each sample. B: density plots of the density distribution of the expression of the CircRNA in each sample.
FIG. 4 shows the differential expression of circRNAs for different groups in example 1. Left shows up-regulated circRNA and right shows down-regulated circRNA.
FIG. 5 shows the expression amount and expression tendency of circRNA in example 1 at different periods. 0.8< | Rs | <1 indicates strong correlation; 0.6< | Rs | <0.8 indicates that there is strong correlation; 0.4< | Rs | <0.6 indicates moderate correlation; 0.2< | Rs | <0.4 indicates that the correlation is weak; 0< | Rs | <0.2 indicates no correlation; the closeness between | Rs | and 1 represents closeness and correlation between the two variables.
FIG. 6 shows the analysis of the regulatory network of the Flora goat hair follicle CircRNA-miRNA mRNA in example 1. Between d45vsd75, 6A represents an up-regulated circRNA network and 6B a down-regulated circRNA network.
FIG. 7 shows ircRNA3236 targets chi-miR-27b-3p and chi-miR-16b-3p in example 2. Wherein, 7A: predicted binding sites and mutation sites of chi-miR-27b-3p in CircRNA 3236. 7B: predicted binding sites and mutation sites of chi-miR-16a-3p in CircRNA 3236. 7C: the interaction between the CircRNA3236 and the chi-miR-27b-3p is detected by a dual luciferase reporter gene. 7D: and detecting the circRNA3236 and the chi-miR-16b-3p by using a dual-luciferase reporter gene.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The invention is described in detail below with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
All animal experiments in the following examples were performed according to the "guide for laboratory animals" of the department of science and technology (Beijing, China). All procedures were performed as recommended by the European Committee (1997) and approved by the university of inner Mongolia agricultural laboratory animal ethics Committee.
Example 1
A method for constructing circRNA related to wool cyst development comprises the following steps:
(1) preparing test animal and sample
Selecting three-year ewes in inner Mongolia Jinlai animal husbandry for synchronous estrus treatment, and recording mating time. After three skin samples of each sheep at fetal stage of 45 days, 55 days, 65 days and 75 days were collected, the skin samples were immediately treated with DPEC water and placed in liquid nitrogen. Subsequently, the cells were stored in a-80 ℃ refrigerator for RNA-seq and RT-qPCR assays.
(2) RNA library construction and sequencing
Total RNA was isolated and purified using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's procedure. RNA quantity and purity were quantified for each sample using a NanoDrop ND-1000(NanoDrop, Willington, DE, USA). Integrity of RNA was assessed with agilent 2100. By usingConsumption of ribosomal RNA by approximately 5. mu.g of total RNATMThe rRNA removal kit (Illumina, San Diego, USA) and the remaining RNA fragments were reverse transcribed using the RNA sequence library preparation kit (Illumina) to form the final product cDNA. Finally, paired-end sequencing was performed on Illumina Hiseq 4000 (alligator, hangzhou, zhejiang, china) according to the supplier's recommended protocol. High-throughput sequencing was performed on inner mongolian down goats (albus type) at four stages of the fetal phase.
Firstly, 12 enucleated ribosomal RNA libraries at the fetal stage of the cashmere goat are constructed, namely d45-1, d45-2, d45-3, d55-1, d55-2, d55-3, d65-1, d65-2, d65-3, d75-1, d75-2 and d75-3 (wherein d represents days).
These pools were RNA-seq using the Illumina Hiseq 4000 platform, from which 1063299566 raw reads from 12 pools were obtained (Table 1). In these original Reads, 1023889360 valid Reads were obtained by removing Reads with a tape linker (adapter), Reads with a proportion of N (N indicates no definite base information) greater than 5%, and low-quality Reads (the number of bases with a quality value Q ≦ 10 is more than 20% of the total read). The Q20 (the proportion of the base with the quality value of more than or equal to 30 and the error rate of less than 0.001) of each library is 99.90%, the Q30 (the proportion of the base with the quality value of more than or equal to 20 and the error rate of less than 0.01) is more than 98.1% (Table 1), and the sequencing accuracy is high.
TABLE 1
Comparing 1023889360 Valid Reads to the reference genome, comparing to the reference genome the number of Reads to greater than 94% of Valid Reads, comparing to the unique position of the reference genome the number of Reads to greater than 77% of Valid Reads, and comparing to the multiple positions of the reference genome the number of Reads to greater than 17% of Valid Reads. Therefore, the utilization rate of the data is normal, and the obtained raw data meets the analysis requirements of subsequent circRNA in quantity and quality (Table 2).
TABLE 2
(3) Identification of transcripts
According to the structural characteristics and splicing sequence characteristics of the circRNA, two types of software, namely CIRC Explotter 2 and CIRI, are used for predicting the circRNA. And the results of both software were integrated according to the circRNA start and stop positions. The following is the circRNA identification standard 1.mismatch is less than or equal to 2; 2. Back-helicedjuntions reads is more than or equal to 1; two splice sites were less than 100kb apart on the genome. The circRNA was identified more precisely according to the above identification screening criteria.
The results showed that there were more than 2800 circrnas identified in each library, and more than 1700 parental genes identified in each library (figure 1). Most circRNAs were found to contain 2 to 4 exons (fig. 2A). Meanwhile, the exon length of the circRNAs includes that only one exon is longer than that of the circRNAs composed of multiple exons (fig. 2B). Chromosome distribution analysis shows that circRNAs are distributed on almost all chromosomes; among them, chromosomes 1, 10 and 11 have higher numbers of circRNAs than other chromosomes (fig. 2C). Finally, exon cirRNAs accounted for 92.01% of all cirRNAs in the down producing goat skin follicles (FIG. 2D)
(4) Differential expression analysis
To further explore the regulatory role of circRNAs in the early development of down producing goat hair follicles, six ratio pairs were formed at four stages and differential expression analysis was performed by calculating SRPBM values of circRNAs (fig. 3A and 3B). When differential expression analysis is performed on the identified circrnas, differential circRNA screening is performed by default using Fold difference (Fold Change) and pvalue: i.e. both a log2FoldChange (sprom) absolute value of 1 or more and a pvalue value of 0.05 or less.
The results show that: d75vsd45, circRNA up-regulated 59, down-regulated 33; d75vsd55, circRNA up-regulated 61, down-regulated 102; d75vsd65, circRNA up-regulated 32, down-regulated 33; d65vsd55, circRNA up-regulation 67, down-regulation 169; d65vsd45, circRNA up-regulation 96, down-regulation 63; d55vsd45, circRNA up-regulated 76, down-regulated 42 (fig. 4).
Further investigation of the differential expression pattern of circRNA revealed that d65vsd55 differentially expressed most circRNA and d75vs65 differentially expressed least circRNA (fig. 4).
(5) Results of circRNAs verified by qRT-PCR
To verify the accuracy of the sequencing results of the circRNAs, the relative expression of 6 differentially expressed circRNAs, circRNA2049, circRNA3411, circRNA2225, circRNA5681, circRNA1604, circRNA4351) was determined using qRT-PCR (fig. 5), which was consistent with the transcriptome sequencing data.
The specific method of qRT-PCR: total RNA was extracted from 12 cashmere goat fetal skin samples using Trizol reagent (Takara, Dalian, Liaoning, China) according to the manufacturer's instructions. Subsequently, RNA was reverse transcribed to cDNA using a Kit with PrimeScript RT Reagent Kit and gDNA Eraser (Takara, Dalian, Liaoning, China). Then, at
qRT-PCR was performed on a real-time fluorescent quantitation system (Roche, Basel, Switzerland) using TB GreenPremix Ex-Taq II (Takara, Dalian, Liaoning, China) under the conditions: 95 ℃ for 30s, and then 40 cycles at 95 ℃ for 10s, 30s at 60 ℃ and then 10s at 72 ℃. Jien using beta-actin as reference, use 2-△△CTThe method calculates the expression level.
(6) Host gene GO enrichment analysis and KEGG pathway analysis
Gene ontology analysis includes three fields describing the cellular and molecular roles of genes and gene products (biological processes (BP), Cellular Components (CC), and Molecular Functions (MF)). KEGG is a pathway database for systematic analysis of gene function, linking genomic and functional information. In order to discuss the regulation and control effect of the host genes of DE circRNAs in the development of down producing goat hair follicles, GO and KEGG pathway enrichment analysis is carried out on the host genes of the circRNAs differentially expressed in each comparison group. The GO enrichment result shows that gene enrichment exists in the biological processes related to hair follicle growth and development, such as hair follicle morphogensis, hair follicle growth, cell depletion and the like; the KEGG pathway analysis shows that there is gene enrichment in the signal pathways such as Notch signaling pathway, NF-kappa B signaling pathway, PI3K-AKt signaling pathway, etc. Therefore, the parent gene corresponding to the circRNA with differential expression may participate in the process of hair follicle growth and development, and further play a role in regulation.
(7) construction of a CerA regulatory network
The competitive endogenous rna (cerna) hypothesis is a new model of post-transcriptional gene regulation. According to this hypothesis, the expression of the indicated mirnas was reduced by the cernas. To construct the cerRNA network of circRNA-miRNA, miRNA library data and mRNA library data were integrated and the binding sites of circRNA and mRNA to miRNA were analyzed using miRanda and Targetscan.
The exon circrnas in d75vsd45 were selected to construct a ceRNA regulatory network, predicting 46 circRNA-miRNA and 49 miRNA-mRNA interactions in the up-down-up regulation mode (fig. 6A). As shown in FIG. 6A, upregulated circRNA9106 can act as a sponge for multiple miRNAs (chi-miR-1, chi-miR-18a-3p and chi-miR-93-3 p).
Notably, the three circRNA8058, circRNA663 and circRNA8624 contain the seed target of chi-miR-133a-5p, identified by finding miRNA targets. In addition, these miRNAs could down-regulate the expression of their target genes (chi-miR-133a-5p was predicted to bind to FLRT1, SOX5, and RBPJL genes).
Further a bottom-up co-expression network was constructed using miRanda and Targetscan, where 56 circRNA-miRNA and 77 miRNA-mRNA interactions were predicted (fig. 6B). In a downward-upward-downward co-expression network, three circRNAs of circRNA3382, circRNA1448 and circRNA1896 which are down-regulated contain a seed target point of chi-miR-26b-3p, and are predicted to be combined with a plurality of target genes such as MCTP1, MEF2C, GPC6 and FZD5, and the circRNA3236 is predicted to have two target genes (miR-16b-3p and miR-27b-3 p).
Example 2 Targeted binding of CircRNA3236 to chi-miR-27b-3p and chi-miR-16b-3p
circRNA3236 was down-regulated in d75vsd45, and Targetscan and miRanda software predicted that circRNA3236 targets chi-miR-27b-3p and chi-miR-16b-3p, with two binding sites, respectively. Thus, two mutational vectors were constructed to validate specific binding sites (fig. 7A and 7B). The results show that in this experiment, there is a binding effect between the two. After mu1 mutation, chi-miR-27b-3p failed to down-regulate the expression of luciferase in circRNA3236-mut1 (p >0.05), indicating successful mutation. Mu1 is the binding site of chi-miR-27b-3p to circRNA3236, and the failure of chi-miR-16b-3p to down-regulate the expression of luciferase in circRNA236-mut4 after mutation (p >0.05) indicates the success of mutation. Mu4 is the binding site for chi-miR-16b-3p and circRNA3236 (FIGS. 7C and 7D).
The specific steps of the dual-luciferase reporter gene detection are as follows: Chi-miR-27b-3p mimics and Chi-miR-27b-3p mimics are synthesized by Shanghai Han dynasty Biotechnology limited in China. miRNA mimics were transfected into HEK 293T cells. Plasmids were transfected into HEK 293T cells using LipoFiter transfection reagent according to the manufacturer's instructions. The psiCHECK2-circRNA3236-WT construct was generated by inserting a fragment of circRNA3236 containing the miRNA binding sequence into the psiCHECK-2 vector (Promega) at the 3' end of the Renilla luciferase gene. The miRNA binding sequence is mutated into a complementary sequence by overlap extension PCR, and a mutant psiCHECK2-circRNA3236-MUT structure is obtained. For circRNA3236 luciferase assay, HEK 293T cells were transfected with miRNA mimetics and either psiCHECK2-circRNA3236-WT or a mutant psiCHECK2-circRNA3236-Mut reporter plasmid. Luciferase activity was measured 48 hours post-transfection using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. By comparing the activity ratio of firefly/renilla luciferase, the relative activity of luciferase was calculated.
Example 3
In order to further explore the functional mechanism of the circRNA in the development process of the hair follicles of the down producing goats, the target genes of the circRNA targeted miRNA and the targeted miRNA differentially expressed by d75vsd45 are predicted, so that the ceRNA network is constructed. Wherein 16 circRNAs are targeted to 6 miRNAs in the up-regulation-down-regulation mode, 6 miRNAs are targeted to 23 target genes, 14 circRNAs are targeted to 16 miRNAs in the down-regulation-up-regulation mode, and 16 miRNAs are targeted to 26 target genes. Constructs circRNA9127-chi-miR-1-WNT3, circRNA2046-chi-miR-93-3p-FLRT1,
and the signal pathways of the circRNA2962-chi-miR-20b-SMAD6, the circRNA3236-chi-miR-27b-3p-SFRP1, the circRNA3236-chi-miR-16b-3p-SLITRK3, the circRNA1476-chi-miR-10b-3p-WNT2 and the like. Wherein the SFRP1, WNT3, SMAD6, and other genes are all important channels related to hair follicle development in previous researches, including WNT, TGF-beta, TNF and other signal channels. Therefore, it is further presumed that the circRNA as a ceRNA may exert a regulatory effect in the hair follicles of the skin at the fetal stage of the cashmere goat through related genes in the pathways of WNT, TGF-. beta.and TNF.
In conclusion, the invention constructs the expression profiles of the circRNAs in the skin hair follicles of the inner Mongolian cashmere goats at different embryonic stages (45 days, 55 days, 65 days and 75 days), and 21784 circRNAs are identified. The circrnas differentially expressed in groups were screened for 6 ratios. 6 circRNAs with different expressions are randomly selected to carry out RT-qPCR experiments, and sequencing results are reliable. The GO and KEGG analysis results of the parent genes indicate that the circRNA and the parent genes thereof possibly have certain relation, and the circRNA possibly plays a role in the biological process of the growth and development of the hair follicle of the cashmere goat through Notch signaling pathway and NF-kappa B signaling pathway. Meanwhile, a coexpression network of the circRNA-miRNA-mRNA is constructed, and the interaction relationship of 102 pairs of circRNA-miRNA and 126 pairs of miRNA-mRNA is formed. The dual-luciferase verification result shows that the circRNA3236-chi-miR-27b-3p and the circRNA3236-chi-miR-16b-3p have a targeted binding relationship, so that an important basis is provided for future research on a molecular mechanism of the circRNA serving as the cerRNA in the generation and development of hair follicles, and important information is provided for the research on the mechanism of the circRNA in human hair follicles.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (8)
1. A method for constructing circRNA related to wool cyst development is characterized by comprising the following steps:
(1) RNA library construction and sequencing: constructing an RNA library for a sample of body-side skin of a sheep at a fetal stage and sequencing the RNA library;
(2) identification of transcripts: predicting circRNA according to a circRNA identification standard;
(3) differential expression analysis: performing differential expression analysis on the circRNA identified in the step (2);
(4) GO enrichment and KEGG pathway analysis: GO enrichment and KEGG pathway analysis of circRNA related host genes differentially expressed in step (3);
(5) construction of a cerRNA regulatory network: predicting the miRNA targeted by the circRNA, predicting the target gene of the targeted miRNA, and primarily constructing a circRNA-miRNA-mRNA regulation network.
2. The constructing method according to claim 1, wherein in the step (1), the sample comprises four phases of the fetal stage of sheep: 45 days, 55 days, 65 days and 75 days.
3. The method for constructing according to claim 1, wherein in the step (2), the circRNA identification standard comprises: the mismatch is less than or equal to 2, the Back-specific junctions reads are more than or equal to 1, and the distance between two specific sites on the genome is less than 100 kb.
4. The constructing method according to claim 1, wherein in the step (3), the differential expression analysis is performed by calculating SRPBM values of the circRNAs;
preferably, differential circRNA screening is performed using fold difference and pvalue;
more preferably, genes satisfying both log2FoldChange absolute value equal to 1 and pvalue equal to 0.05 are designated as yes, and those not are designated as no.
5. The method of constructing according to claim 1, wherein in the step (5), miRNA library data and mRNA library data are integrated, and a cerRNA network of the circRNA-miRNA is constructed by analyzing the binding sites of the circRNA and mRNA to miRNA using MiRanda and Targetscan.
6. The method of any one of claims 1 to 5, wherein the sheep is an inner Mongolian cashmere goat.
7. A circRNA expression profile associated with wool cyst development obtained by the method of any one of claims 1 to 6.
8. The method for constructing as claimed in any one of claims 1 to 6, or the use of the expression profile of circRNA related to wool follicle development as claimed in claim 9 for regulating the development of sheep skin hair follicles.
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