| Mammary gland is a special secretory organ for lactation in mammals.Mammary epithelial cell is a kind of typical cell in lactation system and is an important model to study physiological function of mammary gland in vitro.Lactation trait is one of important traits of milk goat,therefore,studies on molecular mechanism of milk protein and milk fat can provide theoretical basis for molecular breeding of milk goat and accelerate the breeding process.Mi RNA is a kind of multi-target non-coding RNA,which binds to target genes through seed sites to regulate target genes at post-transcriptional level,and then participates in various biological processes.The differential expression of miRNA in different lactation periods suggests that miRNA may play crucial roles in regulating lactation traits.Previous study by our research group showed that the expression of novel-miR-3880 was higher during peak lactation period in mammary gland of milk goats.At peak lactation period,metabolic rate in mammary gland is significantly accelerated and substrate uptake as well as protein and lipid synthesis were increased,so it was hypothesized that novel-miR-3880 might play a regulatory role in mammary gland development and milk synthesis.Ci RNA is a class of covalently closed circular non-coding RNAs produced by alternative splicing.Ci RNAs have multiple miRNA binding sites and can act as molecular sponge of miRNA to compete with the bind of miRNA target genes,thereby weakening the regulatory role of miRNA on target genes.However,little has been studied on novel-miR-3880 and ci RNAs in molecular mechanism of milk goat lactation.Our research group previously established a ci RNA database of dairy goat through high-throughput sequencing,and we performed bioinformatic analysis to predict ci RNAs that might absorb novel-miR-3880,to lay a foundation for the study of the mechanism of novel-miR-3880 in mammary gland.In this study,the effect of novel-miR-3880 on mammary gland structure during lactation was observed by transmission electron microscopy.High-throughput sequencing was used to screen differentially expressed genes(DEGs)of novel-miR-3880 in goat mammary epithelial cell(GMEC).The dual luciferase reporter assay,Real-time quantitative PCR(RT-q PCR)technology,and Western Blot technology was applied to further validate and screen the novel-miR-3880 target genes.Besides,the relationship between miRNA target genes and ci RNA was exploredthrough si RNA and overexpression vector in GMEC.The regulatory mechanism of ci RNA-miRNA-m RNA in lactation traits was studied by ELISA,Ed U,immunohistochemistry(IHC).The main results of this study are as follows: 1.Screening of novel-miR-3880 DEGs and the regulation on mammary gland development in goatIn this study,67 downstream DEGs of novel-miR-3880 were obtained through transcriptome sequencing.RT-q PCR was applied to verify the accuracy of the transcriptome sequencing results.GO enrichment and KEGG enrichment analysis for DEGs found that novel-miR-3880 DEGs are involved in important signaling pathways for mammary gland development,such as Prolactin signaling pathway,RIG-I-like receptor signaling pathway,TNF signaling pathway And NF-?B signaling pathway etc.Under transmission electron microscopy,mice mammary glands in control group had entered a degenerative phase,while the novel-miR-3880-treated mice had full acinus and abundant ducts,and average weight of their offsprings was significantly higher than that of control group.Therefore,we concluded that novel-miR-3880 promotes mammary gland development and secretes milk that can promote growth of pups.Compared with control group,novel-miR-3880 increased triglyceride content in mammary gland,while ensuring that blood triglyceride did not change.In GMEC,novel-miR-3880 promoted triglyceride synthesis and lipid droplet formation.Besides,novel-miR-3880 increased ?-casein secretion and decreased ?s1-casein,?s2-casein and ?-casein secretion.2.Regulation of novel-miR-3880 to RIG-I-like receptor signaling pathwayAfter novel-miR-3880 was transfected into GMEC,the expression levels of m RNA and protein of key genes ISG15 and RIG-I in the RIG-I-like receptor signaling pathway were detected,and found novel-miR-3880 reduced the expression of ISG15 and RIG-I in m RNA and protein levels.Sequence alignment revealed that the binding site of novel-miR-3880 was contained in the sequence of ISG15.However,the luciferase reprter assay results showed that novel-miR-3880 could not regulate the expression of ISG15 by binding to ISG15.RT-q PCR result revealed that si RIG-I promoted the expression of ISG15,while si ISG15 promoted the expression of RIG-I.In addition,si ISG15 promoted but si RIG-I inhibited interferon-related genes in the novel-miR-3880 DEGs.Therefore,it is speculated that it might be relevant to the negative regulatory relationship between ISG15 and RIG-I.However,when ISG15 is overexpressed,the regulation of RIG-I is not significantly regulated,and under the condition of ISG15 high expression,the regulation of novel-miR-3880 to RIG-I is not significant.GMEC apoptosis results showed that novel-miR-3880,si ISG15 and si RIG-I inhibited GMEC apoptosis.It can be seen in the Western Blot results that novel-miR-3880 and si ISG15 can inhibit the expression of pro-apoptotic genes Caspase3,p53 and TLR4,and promote the expression of anti-apoptotic gene Bcl-2.When ISG15 is overexpressed,the expressions of Caspase3,p53 and TLR4 were elevated,and Bcl-2 expression was decreased,but si RIG-I inhibited the expression of Caspase3 and p53,promoted the expression of Bcl-2 and TLR4.Finally,si RIG-I still suppressed GMEC apoptosis.It is speculated that ISG15-increased-expression induced by RIG-I knockdown might be one of the reasons for TLR4 increase.3.Effects of ci RNA13761 on GMECCi RNA13761 is derived from exons 36,37 and 38 of DOCK1 and is widely expressed in goat tissues.RT-q PCR results indicate that there was a mutual inhibition relationship between ci RNA13761 and DOCK1.Through luciferase reporter assay and RT-q PCR detection,it was found that the expression of novel-miR-3880 in GMEC can be reduced by ci RNA13761 sponge.In GMEC viability and proliferation assays,ci RNA13761 was found to inhibit GMEC viability and proliferation.In addition,ci RNA13761 can participate in GMEC casein secretion,triglyceride synthesis and lipid droplet formation,reduce GMEC triglyceride synthesis and lipid droplet formation,promote ?s1-casein,?s2-casein and ?-casein secretion,and inhibit ?-casein secretion,while novel-miR-3880 overexpression can inhibit the regulation of ci RNA13761 on GMEC.4.Identification of novel-miR-3880 target genes and construction of ci RNA13761/novel-miR-3880/ELF2 regulatory networkELF2 belongs to the Ets transcription factor family and mediates key cellular processes such as development,differentiation,growth,and transformation.There are two sites containing the novel-miR-3880 seed sequence in ELF2,and the two dual luciferase vectors were constructed.It was found that novel-miR-3880 can bind to the binding site in ELF2-1 by luciferase repoter assay.ELF2-1 and ci RNA13761 can form a competitive combination with the seed site of novel-miR-3880,thereby regulating biological processes.RT-q PCR and Western Blot results showed the presence of ci RNA13761/novel-miR-3880/ELF2 regulatory network in GMEC.There exists a mutual inhibition between novel-miR-3880 and ci RNA13761,and novel-miR-3880 inhibited m RNA and protein expression of ELF2,while ci RNA13761 promoted the expression of ELF2 m RNA and protein;ELF2 inhibited the expression of novel-miR-3880,and promoted the expression of ci RNA13761 and DOCK1;novel-miR-3880 promoted the expression of DOCK1,ci RNA13761 inhibited the expression of DOCK1;si DOCK1 promoted novel-miR-3880 and ci RNA13761 expression,but had no significant regulation to ELF2 expression.The detection of casein and triglyceride content and oil red O results show that ELF2 inhibited GMEC triglyceride synthesis and lipid droplet formation,inhibited ?-casein secretion,and promoted ?s1-casein,?s2-casein and ?-casein secretion..5.Research on ci RNA13761/novel-miR-3880/ELF2 network to regulate GMECMTOR regulates protein lipid synthesis and cell growth and can form a functional complex with lactation initiator PI3K/AKT.Phosphorylation of m TOR is an important way for the activation of S6K1,which is a key gene for milk protein and milk fat synthesis.Western Blot results show that ci RNA13761/novel-miR-3880/ELF2 network and DOCK1 were involved in regulating the phosphorylation level of PI3K/AKT/m TOR/S6K1 pathway in GMEC,regulating the expression of Bcl-2/Bax.RT-q PCR detection revealed that PI3 K,AKT,m TOR,and S6K1 have feedback regulation on ci RNA13761/novel-miR-3880/ELF2 network,and inhibition of PI3 K,AKT,m TOR or S6K1 increased the expression of novel-miR-3880 and DOCK1,reduced ci RNA13761 and ELF2 expression.Cell proliferation results show that the PI3K/AKT/m TOR/S6K1 pathway played a key role in the regulation of GMEC proliferation,and ci RNA13761/novel-miR-3880/ELF2 can regulate the proliferation of GMEC cells through the PI3K/AKT/m TOR/S6K1 pathway.In summary,novel-miR-3880 regulates GMEC apoptosis through RIG-I pathway,participates in ci RNA13761/novel-miR-3880/ELF2 regulatory network,and regulate GMEC growth,milk fat and casein formation through PI3K/AKT/m TOR/S6K1 and Bcl-2/Bax pathways,finally achieve the goal of promoting mammary gland development. |
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