BMP15 Modulates the H19/miR-26b/SMAD1 Axis Influences Yak Granulosa Cell Proliferation, Autophagy, and Apoptosis.

Bone morphogenetic protein 15 (BMP15) regulates the growth and development of follicles. In particular, the long non-coding RNA H19 plays an important role in mammalian reproduction. However, the function and regulatory mechanism of the interaction of BMP15 with H19 in yak granulosa cell (GC) proliferation, autophagy, and apoptosis are poorly understood. In our study, quantitative reverse-transcription-polymerase chain reaction analysis showed that H19 were highly expressed in yak healthy follicles. H19 was induced by BMP15 protein in yak GCs. In addition, we confirmed that overexpression of H19 promoted yak GC proliferation and autophagy and inhibited apoptosis. Bioinformatic analysis and luciferase reporter assays demonstrated that H19 directly binds to miR-26b, and SMAD1 was identified as a target of miR-26b. miR-26b overexpression inhibited GC proliferation and autophagy and promoted apoptosis through decreased SMAD1 expression, which was attenuated by H19 overexpression. RNA immunoprecipitation-quantitative polymerase chain reaction and dual-luciferase assays showed that miR-26b was sponged by H19 to preserve SMAD1 expression. Furthermore, SMAD1 mRNA expression was induced and miR-26b expression was reduced after yak GCs were treated with BMP15 protein. In conclusion, our results demonstrated that the H19/miR-26b/SMAD1 axis responds to BMP15 to regulate yack GC proliferation, autophagy, and apoptosis.

The Resistance Mechanism of Mycoplasma bovis From Yaks in Tibet to Fluoroquinolones and Aminoglycosides.

Mycoplasma bovis (M. bovis) is one of the important pathogens for yaks. Aminoglycosides and fluoroquinolones are frequently used medications for the treatment of M. bovis. Drug-resistant strains were inevitable with the abuse of antibiotics. The resistance of M. bovis to aminoglycosides was related to the base mutations in drug target genes. Amino acid mutations at the quinolone resistance-determining region (QRDR) in gyrA, gyrB, parC, and parE conferred resistance to fluoroquinolones. In order to investigate the resistance mechanism of M. bovis from yaks in Tibet to aminoglycosides and fluoroquinolones, six frequently used antibiotics and ten clinical M. bovis strains were administered for a drug sensitivity test for in vitro-induced highly resistant strains, a drug stable-resistance test, cross-resistance test, and analysis of target gene mutations. The results showed that the clinical strains of M. bovis from yaks in Tibet had varying degrees of resistance to fluoroquinolones and aminoglycosides. The mechanism of resistance to fluoroquinolones and aminoglycosides was identified preliminarily for M. bovis from yaks: the single-site base mutation mediated the resistance of M. bovis from yaks and both base mutations led to highly resistant strains (aminoglycosides: rrs3 and rrs4; fluoroquinolones: gyrA and parC). The active efflux system results of M. bovis showed that there was no active efflux system based on fluoroquinolones and aminoglycosides expressed in M. bovis from yaks. The research could provide a reference for clinical treatment of M. bovis.

Epidemiological Survey of Mycoplasma bovis in Yaks on the Qinghai Tibetan Plateau, China.

Mycoplasma bovis (M. bovis) is one of the most important pneumonia pathogens in yaks. It may result in more economic losses due to the cold and anoxia condition at Qinghai Tibetan plateau. However, to date, limited information on M. bovis infection in yaks is available in China. For this purpose, the seroprevalence of M. bovis was investigated in yaks living in the mentioned area through commercial ELISA kits. A total of 959 yaks were incorporated into this study. The prevalence of the disease in yaks was 48.70%. The serological results revealed a relatively high prevalence of M. bovis infection in yaks. The present study may greatly contribute to the prevention of this disease. More importance should be given to the potential threat caused by M. bovis in the special plateau.

Isolation, identification and biological characteristics of Mycoplasma bovis in yaks.

Mycoplasma bovis (M. bovis) is one of the important pathogens which may cause bovine respiratory disease syndrome (BRDS), and results in huge economic losses for yaks (Bos gaurus) breeding industry. However, there is limited information about M. bovis in yaks. In our study, 145 nasal mucus samples from yaks with pneumonia were collected to clarify. Bacteriological determination was carried out through biochemical identification and Polymerase Chain Reaction (PCR) detection. And ten strains of Mycoplasma bovis (M. bovis) were found from collected samples. Then, the growth curve of isolated strains was determined by the change of optical density (OD630), pH value and Color Change Cnit (CCU). K-B disk method was also used for antimicrobial susceptibility testing. Results of colony morphology and biochemical testing were consistent with the biological characters of M. bovis. The nucleotide sequences of uvrC specific gene and 16S rRNA gene among the 10 strains were highly homologous. The growth curve assay showed that the isolates cultured in PPLO medium were in lag phase for 24 h, entered stable period in 42 h, and entered decline phase after 78 h. The isolates were found resistant to macrolides, aminoglycosides and lincomycin at various degrees, but they were sensitive or moderately sensitive to doxycycline and kanamycin under antimicrobial susceptibility analysis. In conclusion, the results provided certain reference for the follow-up research and guiding for the treatment of M. bovis in yaks.

Down-Regulation of miR-378d Increased Rab10 Expression to Help Clearance of Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1ß, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.

microRNA-125b Regulates Apoptosis by Targeting Bone Morphogenetic Protein Receptor 1B in Yak Granulosa Cells.

The intronic microRNA, miR-125b, plays a vital role in promyelocytic and hematopoietic stem cells, and in the development and apoptosis of cancer cells. In this study, we showed that miR-125b regulates granulosa cell (GC) apoptosis in the yak ovary. Bioinformatic analyses and luciferase reporter assays demonstrated that bone morphogenetic protein receptor type 1B (BMPR1B) is an miR-125b target. miR-125b overexpression induced apoptosis in yak GC, and affected the mRNA and protein expression of BMPR1B and the ratio of Bcl2/Bax. Silencing of miR-125b decreased the rate of yak GC apoptosis and increased the ratio of Bcl2/Bax. In addition, the effects of an miR-125b inhibitor were overturned by cotransfection with siRNA-BMPR1B2 (siRNA-299) in yak GC. Together, these results demonstrated that miR-125b regulates GC apoptosis in the yak ovary by targeting BMPR1B.

TGF-ß1 resulting in differential microRNA expression in bovine granulosa cells.

To explore the expression profile of the cellular miRNAs in bovine ovarian granulosa cells responding to transforming growth factor-ß1 (TGF-ß1), the effect of TGF-ß1 on cell proliferation was firstly investigated by CCK-8 method and the results showed that there was a significant inhibitory effect on bovine granulosa cell proliferation treated with 5/10 ng/mL human recombinant TGF-ß1 for 24 h compared to the control (P < 0.05). Then, we performed high-throughput sequencing of two small RNA libraries prepared from cultured bovine granulosa cells stimulated with or without 10 ng/mL human recombinant TGF-ß1. A total of 13,257,248 and 138,726,391 clean reads per library were obtained from TGF-ß1 and control groups, respectively. There were 498 and 499 bovine-specific exist miRNAs (exist miRNAs), 627 and 570 conserved known miRNAs (known miRNAs), and 593 and 585 predicted novel miRNAs in TGF-ß1 and control groups, respectively. A total of 78 miRNAs with significant differential expression, including 39 up-regulated miRNAs and 39 down-regulated miRNAs were identified in the TGF-ß1 group compared with the control. Real-time quantitative PCR analyses of bta-miR-106a and bta-miR-1434-5p showed that their up-expressions were interrupted by SB431542, an inhibitor that blocks TGFß1/Smad signaling, which supported the sequencing data. GO analysis showed involvement of the predicted genes of the differentially expressed miRNAs in a broad spectrum of cell biological processes, cell components, and molecular functions. KEGG pathway analysis of the predicted miRNA targets further indicated that these differentially expressed miRNAs are involved in various signaling pathways, such as Wnt, MAPK, and TGF-ß signaling, which might be involved in follicular development. These results provide valuable information on the composition, expression, and function of miRNAs in bovine granulosa cells responding to TGF-ß1, and will aid in understanding the molecular mechanisms of TGF-ß1 in granulosa cells.