MiR-21-3p Centric Regulatory Network in Dairy Cow Mammary Epithelial Cell Proliferation.

MicroRNA-mediated gene regulation is important for the development of the mammary gland and the lactating process. A previous study has shown that the expression of microRNA-21 (miR-21) is different in the dry and early lactation period of the dairy cow mammary gland, but the molecular mechanisms underlying the lactation cycle are not fully understood. Here, the function of miR-21-3p on bovine mammary gland epithelial cells (BMECs) was detected by MTT assay and flow cytometry analysis, which showed that miR-21-3p significantly promoted the cell viability and proliferation. Then, the regulating mechanism of miR-21-3p on cell viability and proliferation was elucidated. Dual luciferase assay, RT-qPCR, and Western blot results revealed that IGFBP5 was a target gene of miR-21-3p. It was known that lncRNA could act as a competing endogenous RNA to sequester miRNAs and reduce the regulatory effect of miRNA-targeted genes. Based on our previous lncRNA-seq data and bioinformatics analysis, lncRNA NONBTAT017009.2 was potentially associated with miR-21-3p, and its expression was specifically inhibited with the transfection of miR-21-3p mimic into BMECs. Inversely, the overexpression of NONBTAT017009.2 significantly decreased the expression level of miR-21-3p in BMECs, while the expression of IGFBP5, the target gene of miR-21-3p, was significantly upregulated. In addition, the promoter region of miR-21 contained two STAT3 binding sites, and the dual luciferase reporter assays revealed that the overexpression of STAT3 significantly reduced the promoter activity of miR-21, implying that the transcription factor STAT3 may act as an upstream regulator affecting the regulation process of miR-21-3p. The overexpression of STAT3 significantly inhibited the expression of miR-21-3p, while the mRNA expression of IGFBP5 was significantly increased compared with the control group. Besides, there are no STAT3 binding sites in the promoter region of IGFBP5 as we predicted by gene-regulation and JASPAR software. Therefore, it could infer that STAT3 might regulate the expression of IGFBP5 by miR-21-3p. Taken together, these results established a regulatory network of miR-21-3p to illustrate the regulating mechanism on promoting cow mammary epithelial cell proliferation.

Gene cloning and seamless site-directed mutagenesis using single-strand annealing (SSA).

The full length of interested genes can be usually cloned by assembling exons or RACE products through overlap PCR. However, the procedure requires multiple PCR steps, which are prone to random mutagenesis. Here, we present a novel SSA-based method for gene cloning and seamless site-directed mutagenesis. We firstly cloned the full-length coding sequence of Cashmere goat (Capra hircus) Hoxc13 gene by assembling exons amplified from genomic DNA. Secondly, we created a Hoxc13 loss-function mutant seamlessly and further illustrated that direct repeat length of 25 bp is enough to trigger the SSA repair in routine E. coli strains including DH5α, Trans1t1, JM109, and Top10. Moreover, we cloned another full-length mutant of Foxn1 gene from Cashmere goat cDNA using further shortened direct repeats of 19 bp. In summary, our study provided an alternative method to overcome the difficulties during overlap PCR in some particular cases for gene cloning.

Transcriptome sequencing to detect the potential role of long non-coding RNAs in bovine mammary gland during the dry and lactation period.

BACKGROUND: It is known that long non-coding RNAs (lncRNAs) play an important role in various biological processes, including cell proliferation, differentiation and apoptosis. However, their functions and profiles in lactation cycle of dairy cows are largely unknown. In this study, lncRNA-seq technique was employed to compare the expression profiles of lncRNAs and mRNAs from Chinese Holstein mammary gland in dry and lactation period. RESULT: Totally 3746 differentially expressed lncRNAs (DELs) and 2890 differentially expressed genes (DEGs) were identified from the dry and lactation mammary glands of Holstein cows. Functional enrichment analysis on target genes of lncRNAs indicated that these genes were involved in lactation-related signaling pathways, including cell cycle, JAK-STAT, cell adhesion, and PI3K-Akt signaling pathways. Additionally, the interaction between lncRNAs and their potential miRNAs was predicted and partly verified. The result indicated that the lactation-associated miR-221 might interact with lncRNAs TCONS_00040268, TCONS_00137654, TCONS_00071659 and TCONS_00000352, which revealed that these lncRNAs might be important regulators for lactation cycle. CONCLUSION: This study provides a resource for lncRNA research on lactation cycle of bovine mammary gland. Besides, the interaction between lncRNAs and the specific miRNA is revealed. It expands our knowledge about lncRNA and miRNA biology as well as contributes to clarify the regulation of lactation cycle of bovine mammary gland.

Integrated analysis of coding genes and non-coding RNAs during hair follicle cycle of cashmere goat (Capra hircus).

BACKGROUND: Cashmere growth is a seasonal and cyclic phenomenon under the control of photoperiod and multiple stimulatory and inhibitory signals. Beyond relevant coding genes, microRNA (miRNA) and long non coding RNA (lncRNA) play an indispensable role in hair follicle (HF) development and skin homeostasis. Furthermore, the influence of lncRNA upon miRNA function is also rapidly emerging. However, little is known about miRNAs, lncRNAs and their functions as well as their interactions on cashmere development and cycling. RESULT: Here, based on lncRNA and miRNA high-throughput sequencing and bioinformatics analysis, we have identified 1108 lncRNAs and 541 miRNAs in cashmere goat skin during anagen and telogen. Compared with telogen, 1388 coding genes, 41 lncRNAs and 15 miRNAs were upregulated, while 1104 coding genes, 157 lncRNAs and 8 miRNAs were downregulated in anagen (adjusted P-value ≤0.05 and relative fold-change ≥2). Subsequently, we investigated the impact of lncRNAs on their target genes in cis and trans, indicating that these lncRNAs are functionally conserved during HF development and cycling. Furthermore, miRNA-mRNA and miRNA-lncRNA interaction were identified through the bioinformatics algorithm miRanda, then the ceRNA networks, miR-221-5p-lnc_000679-WNT3, miR-34a-lnc_000181-GATA3 and miR-214-3p-lnc_000344-SMAD3, were constructed under defined rules, to illustrate their roles in cashmere goat HF biology. CONCLUSION: The present study provides a resource for lncRNA, miRNA and mRNA studies in cashmere cycling and development. We also demonstrate potential ceRNA regulatory networks in cashmere goat HF cycling for the first time. It expands our knowledge about lncRNA and miRNA biology as well as contributes to the annotation of the goat genome.