Survival analysis and development of a prognostic nomogram for patients with hepatitis B virus-associated hepatocellular carcinoma.

Background and aims: Hepatitis B virus (HBV) is a common cause of hepatocellular carcinoma (HCC) in China, and this study aimed to identify high-risk factors for overall survival and develop a nomogram prediction model. Methods: In the present retrospective cohort study, patients with HBV-associated HCC diagnosed from January 2009 to December 2018 were enrolled. Their clinical characteristics and time-to-event information were retrieved from electronic medical records. The zero time was the date of HCC diagnosis, and the endpoint was death or liver transplantation. Multivariable COX proportional hazard regression was used to screen independent risk factors for overall survival; then a nomogram model was developed to predict the survival probability of HCC patients. Results: A total of 1723 patients were enrolled, with 82.7 % male and a median age of 54.0 years. During a median follow-up time of 41.3 months, 672 cases (39.0 %) died. Age ≥60 years (HR = 1.209), Male (HR = 1.293), ALB <35 g/L (HR = 1.491), AST ≥80 U/L (HR = 1.818); AFP 20-400 ng/mL (HR = 2.284), AFP ≥400 ng/mL (HR = 2.746); LSM 9-22 kPa (HR = 2.266), LSM ≥22 kPa (HR = 4.326); BCLC stage B/C (HR = 4.079) and BCLC stage D (HR = 16.830) were the independent high-risk factors associated with HCC survival. A prognostic nomogram with a consistency index of 0.842 (95 % CI: 0.827-0.858) was developed. The calibration curve for long-term survival rate fitted well. Conclusions: This study identified independent risk factors affecting the survival of patients with HBV-associated HCC and constructed a predictive nomogram model, which can individually predict the overall survival and has good clinical application value.

Grape Seed Proanthocyanidins Improve the Quality of Fresh and Cryopreserved Semen in Bulls.

Oxidative stress leads to a decrease in semen quality during semen cryopreservation and fresh semen production. Grape seed proanthocyanidins (GSPs) are endowed with well-recognized antioxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Therefore, the objective of this experiment was to explore the effects of GSPs on the quality of fresh and cryopreserved semen to provide a basis for GSPs as a new dietary additive and semen diluent additive for males' reproduction. Fresh semen from three healthy bulls aged 3 to 5 years old were gathered and mixed with semen diluents dissolved with 0 µg/mL, 30 µg/mL, 40 µg/mL, 50 µg/mL, and 60 µg/mL GSPs respectively. The motility, physiological structures (acrosome integrity, membrane integrity, mitochondrial activity), and antioxidant capacity of frozen-thawed sperm were measured after storage in liquid nitrogen for 7 days (d). Bulls were fed with 20 mg/kg body weight (BW) GSPs in their diet for 60 days; the weight of the bull is about 600 kg. Then, the reproductive performance and antioxidant indexes of bulls were measured before and after feeding. The results demonstrated that GSPs supplementation significantly increased sperm motility, physiological structures, GSH-Px, and CAT enzyme activities and significantly decreased MDA content in sperm during semen cryopreservation. The optimal concentration of GSPs was 40 µg/mL (p < 0.05). After 20 mg/kg (body weight) GSP supplementation, sperm motility was significantly heightened (p < 0.05), the sperm deformity rate was significantly reduced (p < 0.05), and antioxidant enzyme activities (such as SOD, CAT, and GSH-Px) were significantly enhanced (p < 0.05), and the production of MDA was significantly suppressed (p < 0.05) in serum compared with that before feeding. In conclusion, these results reveal that a certain concentration of GSPs has a good protective effect on sperm damage caused by semen cryopreservation and the reproductive performance reduction caused by stress in bulls, which may be attributed to the antioxidant function of GSPs. In summary, GSPs are a useful cryoprotective adjuvant and dietary additive for bull sperm quality.

Lnc-TRTMFS promotes milk fat synthesis via the miR-132x/RAI14/mTOR pathway in BMECs.

As an important index to evaluate the quality of milk, milk fat content directly determines the nutrition and flavor of milk. Recently, growing evidence has suggested that long noncoding RNAs (lncRNAs) play important roles in bovine lactation, but little is known about the roles of lncRNAs in milk fat synthesis, particularly the underlying molecular processes. Therefore, the purpose of this study was to explore the regulatory mechanism of lncRNAs in milk fat synthesis. Based on our previous lncRNA-seq data and bioinformatics analysis, we found that Lnc-TRTMFS (transcripts related to milk fat synthesis) was upregulated in the lactation period compared to the dry period. In this study, we found that knockdown of Lnc-TRTMFS significantly inhibited milk fat synthesis, resulting in a smaller amount of lipid droplets and lower cellular triacylglycerol levels, and significantly decreased the expression of genes related to adipogenesis. In contrast, overexpression of Lnc-TRTMFS significantly promoted milk fat synthesis in bovine mammary epithelial cells (BMECs). In addition, Bibiserv2 analysis showed that Lnc-TRTMFS could act as a molecular sponge for miR-132x, and retinoic acid induced protein 14 (RAI14) was a potential target of miR-132x, which was further confirmed by dual-luciferase reporter assays, quantitative reverse transcription PCR, and western blots. We also found that miR-132x significantly inhibited milk fat synthesis. Finally, rescue experiments showed that Lnc-TRTMFS could weaken the inhibitory effect of miR-132x on milk fat synthesis and rescue the expression of RAI14. Taken together, these results revealed that Lnc-TRTMFS regulated milk fat synthesis in BMECs via the miR-132x/RAI14/mTOR pathway.

Milk fat is an important index to evaluate the quality of milk. The content of milk fat directly determines the quality and flavor of milk. Studies have shown that milk components can change with the expression of specific genes and noncoding RNA that regulate it in different lactation periods. In this study, after the interference and overexpression of Lnc-TRTMFS on milk fat metabolism in bovine mammany epithelial cells, we found that Lnc-TRTMFS could positively regulate milk fat synthesis in bovine mammary epithelial cells. The ceRNA network of Lnc-TRTMFS-miR-132x-RAI14 was constructed by software prediction and double fluorescein report test, and the salvage effect of Lnc-TRTMFS on milk fat synthesis was confirmed by salvage test. Most importantly, we found that Lnc-TRTMFS and miR-132x can regulate milk fat by regulating the mTOR pathway by regulating RAI14.

RNA-Seq and lipidomics reveal different adipogenic processes between bovine perirenal and intramuscular adipocytes.

Adipogenesis involves complex interactions between transcription and metabolic signalling. Exploration of the developmental characteristics of intramuscular adipocyte will provide targets for enhancing beef cattle marbling without increasing obesity. Few reports have compared bovine perirenal and intramuscular adipocyte transcriptomes using the combined analysis of transcriptomes and lipid metabolism to explore differences in adipogenic characteristics. We identified perirenal preadipocytes (PRA) and intramuscular preadipocytes (IMA) in Qinchuan cattle. We found that IMA were highly prolific in the early stages of adipogenesis, while PRA shows a stronger adipogenic ability in the terminal differentiation. Bovine perirenal and intramuscular adipocytes were detected through the combined analysis of the transcriptome and metabolome. More triglyceride was found to be upregulated in perirenal adipocytes; however, more types and amounts of unsaturated fatty acids were detected in intramuscular adipocytes, including eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3; DHA). Furthermore, differentially expressed genes in perirenal and intramuscular adipocytes were positively correlated with the eicosanoid, phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), and sphingomyelin contents. Associated differential metabolic pathways included the glycerolipid and glycerophospholipid metabolisms. Our research findings provide a basis for the screening of key metabolic pathways or genes and metabolites involved in intramuscular fat production in cattle.

Bta-miR-34b controls milk fat biosynthesis via the Akt/mTOR signaling pathway by targeting RAI14 in bovine mammary epithelial cells.

BACKGROUND: The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes, including milk fat synthesis. miR-34b is a member of the miR-34 miRNA cluster, which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods. Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis; therefore, it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells (BMECs). RESULTS: In this study, elevated miR-34b levels reduced milk fat synthesis, upregulated 1,999 genes, and downregulated 2,009 genes in BMECs. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway by reducing phosphorylation levels. Notably, the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b. Furthermore, we demonstrated that retinoic acid-induced protein 14 (RAI14) is a target of miR-34b via TargetScan and immunofluorescence assays. RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor. Moreover, the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway. CONCLUSIONS: Overall, our results identified a miR-34b-RAI14-Akt/mTOR regulatory network, while also providing a theoretical basis for the molecular breeding of dairy cows.

Insights into adaption and growth evolution: a comparative genomics study on two distinct cattle breeds from Northern and Southern China.

Mongolian cattle (MG, Bos taurus) and Minnan cattle (MN, Bos indicus) are two different breeds of Chinese indigenous cattle, representing North type and South type, respectively. However, their value and potential have not yet been discovered at the genomic level. In this study, 26 individuals of MN and MG were sequenced for the first time at an average of 13.9- and 12.8-fold, respectively. Large numbers of different variations were identified. In addition, the analyses of phylogenetic and population structure showed that these two cattle breeds are distinct from each other, and results of linkage disequilibrium analysis revealed that these two cattle breeds have undergone various degrees of intense natural or artificial selection. Subsequently, 496 and 306 potential selected genes (PSRs) were obtained in MN and MG, containing 1,096 and 529 potential selected genes (PSGs), respectively. These PSGs, together with the analyzed copy number variation (CNV)-related genes, showed potential relations with their phenotypic characteristics, including environmental adaptability (e.g., DVL2, HSPA4, CDHR4), feed efficiency (e.g., R3HDM1, PLAG1, XKR4), and meat/milk production (e.g., PDHB, LEMD3, APOF). The results of this study help to gain new insights into the genetic characteristics of two distinct cattle breeds and will contribute to future cattle breeding.

Transcription Factors ZEB1 and CREB Promote the Transcription of Bovine ABHD5 Gene.

Alpha/beta hydrolase domain 5 (ABHD5) plays a significant role in intracellular lipid metabolism, which is regulated by a complex network of transcription factors. The transcriptional regulation of the ABHD5 gene in cattle and other livestock, however, has not been previously investigated. Investigations in humans and animal models indicate that the transcription factors zinc finger E-box binding homeobox 1 (ZEB1) and cAMP-response element binding protein (CREB) may play important roles in the transcriptional regulation of ABHD5 in cattle. Our comparison of the sequence similarities in the transcription factor binding sites in Bos taurus, Bos indicus, Bos mutus, and Homo sapiens revealed high homology. Based on the data collected by the Cistrome Data Browser and its visualization window, we found that ZEB1 and CREB have significant ChIP-seq enrichments in the 5'-untranslated region (5' UTR) of the human ABHD5 gene. In bovine adipocytes, we detected ZEB1 and CREB binding sites in the ABHD5 gene. Mutations in the ZEB1 and CREB binding sites significantly reduced the promoter activity (p < 0.05 and p < 0.01, respectively). Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays demonstrated the binding of the transcription factors in vivo and in vitro, respectively. And overexpression or silencing the expression of the ZEB1 and CREB, respectively, resulted in significant changes to the ABHD5 promoter activity. Collectively, these results indicate that ZEB1 and CREB are important transcription factors that regulate ABHD5 gene expression in bovine adipocytes. They further our understanding of the transcriptional regulation and biological functions of the bovine ABHD5 gene.

Bovine Stearoyl-CoA Desaturase 1 Promotes Adipogenesis by Activating the PPARγ Receptor.

Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that mainly catalyzes the saturated fatty acids (SFAs) into the monounsaturated fatty acids (MUFAs). The expression level of SCD1 is positively correlated with the marbling score. However, the functional mechanism of SCD1 in adipogenesis is still unclear. In this study, we identified SCD1 as highly expressed in subcutaneous and visceral fat, peaking at 2 days after differentiation in bovine stromal vascular fraction (SVF) cells. When the SCD1 was overexpressed in bovine SVF cells, lipid droplets accumulation was increased from 142.46 ± 21.77 to 254.89 ± 11.75 µg/mg (P < 0.01). Further, the expression levels of FABP4, FASN, and ACCα were increased (P < 0.01), while the expression of PPARγ or C/EBPα was not changed at mRNA or protein level (P > 0.05). Dual-luciferase reporter assay showed that the activity of the PPARγ receptor was enhanced by 3.69 times (P < 0.01). Moreover, the contents of palmitoleate (C16:1) and oleate (C18:1) were significantly increased (P < 0.05). Furthermore, 100 µM exogenous oleate increased the lipid accumulation by 22.28 times (P < 0.01). These results suggest that oleate is probably a strong ligand of the PPARγ receptor to enhance adipogenesis.

MiR-143 Regulates Milk Fat Synthesis by Targeting Smad3 in Bovine Mammary Epithelial Cells.

Milk fat is the main nutritional component of milk and is also an important indicator for evaluating milk quality. Substantial evidence has implicated miRNAs in the synthesis of milk fat. miR-143 is one of the miRNAs closely related to lipid metabolism. Herein, miR-143 upregulation remarkably promoted the production of lipid droplets and increased the level of intracellular triglyceride (TAG). Meanwhile, miR-143 suppression overtly repressed TAG synthesis and lipid droplet accumulation in bovine mammary epithelial cells (BMECs). At the same time, miR-143 significantly upregulated the genes associated with lipid synthesis, including PPARγ, SCD1, CEBPß, and SREBP1. To examine the regulatory mechanism of miR-143 in milk fat synthesis, Smad3 was predicted as a new potential miR-143 target gene by TargetScan. Further studies found that miR-143 expression was inversely related to the levels of Smad3 mRNA and protein. Furthermore, luciferase reporter assays confirmed Smad3 to be a miR-143 direct target. Moreover, Smad3 gene silencing significantly increased intracellular TAG level in BMECs. These findings revealed that miR-143 promotes the TAG synthesis in BMECs via increasing the lipid synthesis related gens expression by targeting Smad3. The results of this study can be exploited in devising novel approaches for improving the nutritional value of milk in dairy cows.

Effect of ELOVL6 on the lipid metabolism of bovine adipocytes.

This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism.

Postpartum Uterine Involution and Embryonic Development Pattern in Chinese Holstein Dairy Cows.

Understanding the postpartum uterine involution pattern and embryonic development could facilitate bovine reproduction management, improve reproductive efficiency, and diagnosis of the reproductive disorder, which would contribute to the success of the dairy business. This study aimed to investigate postpartum uterine involution and embryonic developmental patterns or postconceptional marks of embryonic fetal development in Chinese Holstein dairy cows using B-mode ultrasonography. The results revealed a significant decline in the involution period with an increase of parity and age. The uterine involution period was shorter in multiparous cows when compared with cows with lower parities. Consistently, cows over 4 years old recovered faster than younger cows (2 or 3 years). Besides, the elder cows (over 4 years) had a relatively larger size of resumed cervix uteri and horns. Postpartum uterine involution pattern analysis revealed that the reproductive tract recovered very fast during the first 16 days postpartum for all the parity. Results of postconceptional marks of embryo development revealed a slow increase in diameter of the gravid uterine horn and crown-rump length (CRL) before day 60. In contrast, this increase was dramatic and rapid after the 60th day. We also established two models to estimate gestational age based on gravid uterine horn diameter or CRL. A formula was established to determine the gravid uterine horn size during postconceptional on day 30th-day 90th (r = 0.8714, P < 0.01). In addition, a significant positive correlation between CRL and gestational age (r = 0.98151, P < 0.01) was built. In conclusion, these results illustrated that parity and calving age had significant effects on uterine involution in Chinese Holstein cows. Crown-rump length and gravid uterine horn diameter are both efficient for evaluating the embryo growth. These current findings broaden the understanding of basic reproductive pattern in Chinese Holstein cows and could benefit bovine reproductive management primarily in postpartum and early pregnant cows to reduce the calving interval and avoid periparturient metabolic diseases.

Bta-miR-34b regulates milk fat biosynthesis by targeting mRNA decapping enzyme 1A (DCP1A) in cultured bovine mammary epithelial cells1.

Milk fat is a main nutritional component of milk, and it has become one of the important traits of dairy cow breeding. Recently, there is increasing evidence that microRNAs (miRNA) play significant roles in the process of milk fat synthesis in the mammary gland. Primary bovine mammary epithelial cells (BMEC) were harvested from midlactation cows and cultured in DMEM/F-12 medium with 10% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL bovine insulin, 1 µg/mL hydrocortisone, and 2 µg/mL bovine prolactin. We found that miR-34b mimic transfection in BMEC reduced the content of intracellular triacylglycerol (TAG) and lipid droplet accumulation via triacylglycerol assay and Oil Red O staining; meanwhile, overexpression of miR-34b inhibited mRNA expression of lipid metabolism-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), and CCAAT enhancer binding protein alpha (C/EBPα). Whereas miR-34b inhibitor resulted in completely opposite results. Furthermore, q-PCR and western blot analysis revealed the mRNA and protein expression levels of DCP1A were downregulated in miR-34b mimic transfection group and upregulated in miR-34b inhibitor group. Moreover, luciferase reporter assays verified that DCP1A was the direct target of miR-34b and DCP1A gene silencing in BMEC-inhibited TAG accumulation and suppressed lipid droplet formation. In conclusion, these findings revealed a novel miR-34b-DCP1A axis that has a significant role in regulating milk fat synthesis and suggested that miR-34b may be used to improve the beneficial ingredients in milk.

Melatonin suppresses milk fat synthesis by inhibiting the mTOR signaling pathway via the MT1 receptor in bovine mammary epithelial cells.

Milk fat content is an important criterion for assessing milk quality and is one of the main target traits of dairy cattle breeding. Recent studies have shown the importance of melatonin in regulating lipid metabolism, but the potential effects of melatonin on milk fat synthesis in bovine mammary epithelial cells (BMECs) remain unclear. Here, we showed that melatonin supplementation at 10 µmol/L significantly downregulated the mRNA expression of lipid metabolism-related genes and resulted in lower lipid droplet formation and triglyceride accumulation. Moreover, melatonin significantly upregulated melatonin receptor subtype melatonin receptor 1a (MT1) gene expression, and the negative effects of melatonin on milk fat synthesis were reversed by treatment with the nonselective MT1/melatonin receptor subtype melatonin receptor 1b (MT2) antagonist. However, a selective MT2 antagonist did not modify the negative effects of melatonin on milk fat synthesis. In addition, KEGG analysis revealed that melatonin inhibition of milk fat synthesis may occur via the mTOR signaling pathway. Further analysis revealed that melatonin significantly suppressed the activation of the mTOR pathway by restricting the phosphorylation of mTOR, 4E-BP1, and p70S6K, and the inhibition of melatonin on milk fat synthesis was reversed by mTOR activator MHY1485 in BMECs. Furthermore, in vivo experiments in Holstein dairy cows showed that exogenous melatonin significantly decreased milk fat concentration. Our data from in vitro and in vivo studies revealed that melatonin suppresses milk fat synthesis by inhibiting the mTOR signaling pathway via the MT1 receptor in BMECs. These findings lay a foundation to identify a new potential means for melatonin to modulate the fat content of raw milk in Holstein dairy cows.

MEF2A Regulates the MEG3-DIO3 miRNA Mega Cluster-Targeted PP2A Signaling in Bovine Skeletal Myoblast Differentiation.

Understanding the molecular mechanisms of skeletal myoblast differentiation is essential for studying muscle developmental biology. In our previous study, we reported that knockdown of myocyte enhancer factor 2A (MEF2A) inhibited myoblast differentiation. Here in this study, we further identified that MEF2A controlled this process through regulating the maternally expressed 3 (MEG3)-iodothyronine deiodinase 3 (DIO3) miRNA mega cluster and protein phosphatase 2A (PP2A) signaling. MEF2A was sufficient to induce MEG3 expression in bovine skeletal myoblasts. A subset of miRNAs in the MEG3-DIO3 miRNA cluster was predicted to target PP2A subunit genes. Consistent with these observations, MEF2A regulated PP2A signaling through its subunit gene protein phosphatase 2 regulatory subunit B, gamma (PPP2R2C) during bovine myoblast differentiation. MiR-758 and miR-543 in the MEG3-DIO3 miRNA cluster were down-regulated in MEF2A-depleted myocytes. Expression of miR-758 and miR-543 promoted myoblast differentiation and repressed PPP2R2C expression. Luciferase activity assay showed that PPP2R2C was post-transcriptionally targeted by miR-758 and miR-543. Taken together, these results reveal that the MEG3-DIO3 miRNAs function at downstream of MEF2A to modulate PP2A signaling in bovine myoblast differentiation.

Fatty acid-binding protein 1 increases steer fat deposition by facilitating the synthesis and secretion of triacylglycerol in liver.

Castration is an important means of improving the beef quality via increasing fat deposition. However, little is known about the molecular mechanism underlying the fat deposition after castration. Here, the intramuscular fat (IMF) content of the steer group was shown to be much higher than the bull group. To understand transcriptional changes in the genes involved in fat deposition following castration, differential expression patterns of mRNAs in liver tissue were investigated in steers and bulls using RNA sequencing. In total, we obtained 58,282,367-54,918,002 uniquely mapped reads, which covered 90.13% of the currently annotated transcripts; 5,864 novel transcripts and optimized 9,088 known genes were determined. These results indicated that castration could change the expression patterns of mRNAs in liver tissue, and 282 differentially expressed genes (DEGs) were detected between steers and bulls. KEGG pathway analysis showed that the DEGs were mostly enriched in PPAR signaling pathway, steroid biosynthesis, steroid hormone biosynthesis, and biosynthesis of fatty acids. Furthermore, eight DEGs were corroborated via quantitative real-time PCR and we found that FABP1 gene knockdown in bovine hepatocytes prominently reduced intracellular triacylglycerol (TAG) synthesis and very low density lipoprotein (VLDL) secretion in culture medium. In summary, these results indicate that FABP1 may promote fat deposition by promoting the production and secretion of TAG and VLDL in steer liver.

Molecular characterization of ABHD5 gene promoter in intramuscular preadipocytes of Qinchuan cattle: Roles of Evi1 and C/EBPα.

The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/ß hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5'-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of -109/+307 was identified as proximal minimum core promoter region of ABHD5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

Cooperative and Independent Functions of the miR-23a~27a~24-2 Cluster in Bovine Adipocyte Adipogenesis.

The miR-23a~27a~24-2 cluster is an important regulator in cell metabolism. However, the cooperative and independent functions of this cluster in bovine adipocyte adipogenesis have not been elucidated. In this study, we found that expression of the miR-23a~27a~24-2 cluster was induced during adipogenesis and this cluster acted as a negative regulator of adipogenesis. miR-27a and miR-24-2 were shown to inhibit adipogenesis by directly targeting glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) and diacylglycerol O-acyltransferase 2 (DGAT2), both of which promoted adipogenesis. Meanwhile, miR-23a and miR-24-2 were shown to target decorin (DCN), glucose-6-phosphate dehydrogenase (G6PD), and lipoprotein lipase (LPL), all of which repressed adipogenesis in this study. Thus, the miR-23a~27a~24-2 cluster exhibits a non-canonical regulatory role in bovine adipocyte adipogenesis. To determine how the miR-23a~27a~24-2 cluster inhibits adipogenesis while targeting anti-adipogenic genes, we identified another target gene, fibroblast growth factor 11 (FGF11), a positive regulator of adipogenesis, that was commonly targeted by the entire miR-23a~27a~24-2 cluster. Our findings suggest that the miR-23a~27a~24-2 cluster fine-tunes the regulation of adipogenesis by targeting two types of genes with pro- or anti-adipogenic effects. This balanced regulatory role of miR-23a~27a~24-2 cluster finally repressed adipogenesis.

Identification of Potential Key Genes Associated with Adipogenesis through Integrated Analysis of Five Mouse Transcriptome Datasets.

Adipose tissue is the most important energy metabolism and secretion organ, and these functions are conferred during the adipogenesis process. However, the cause and the molecular events underlying adipogenesis are still unclear. In this study, we performed integrated bioinformatics analyses to identify vital genes involved in adipogenesis and reveal potential molecular mechanisms. Five mouse high-throughput expression profile datasets were downloaded from the Gene Expression Omnibus (GEO) database; these datasets contained 24 samples of 3T3-L1 cells during adipogenesis, including 12 undifferentiated samples and 12 differentiated samples. The five datasets were reanalyzed and integrated to select differentially expressed genes (DEGs) during adipogenesis via the robust rank aggregation (RRA) method. Functional annotation of these DEGs and mining of key genes were then performed. We also verified the expression levels of some potential key genes during adipogenesis. A total of 386 consistent DEGs were identified, with 230 upregulated genes and 156 downregulated genes. Gene Ontology (GO) analysis showed that the biological functions of the DEGs primarily included fat cell differentiation, lipid metabolic processes, and cell adhesion. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were mainly associated with metabolic pathways, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, regulation of lipolysis in adipocytes, the tumor necrosis factor (TNF) signaling pathway, and the FoxO signaling pathway. The 30 most closely related genes among the DEGs were identified from the protein⁻protein interaction (PPI) network and verified by real-time quantification during 3T3-L1 preadipocyte differentiation. In conclusion, we obtained a list of consistent DEGs during adipogenesis through integrated analysis, which may offer potential targets for the regulation of adipogenesis and treatment of adipose dysfunction.

Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast.

Myocyte enhancer factor 2A (MEF2A) is widely distributed in various tissues or organs and plays crucial roles in multiple biological processes. To examine the potential effects of MEF2A on skeletal muscle myoblast, the functional role of MFE2A in myoblast proliferation and differentiation was investigated. In this study, we found that the mRNA expression level of Mef2a was dramatically increased during the myogenesis of bovine skeletal muscle primary myoblast. Overexpression of MEF2A significantly promoted myoblast proliferation, while knockdown of MEF2A inhibited the proliferation and differentiation of myoblast. RT-PCR and western blot analysis revealed that this positive effect of MEF2A on the proliferation of myoblast was carried out by triggering cell cycle progression by activating CDK2 protein expression. Besides, MEF2A was found to be an important transcription factor that bound to the myozenin 2 (MyoZ2) proximal promoter and performed upstream of MyoZ2 during myoblast differentiation. This study provides the first experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that MEF2A regulates myoblast differentiation via regulating MyoZ2.

MiR-27a-5p Increases Steer Fat Deposition Partly by Targeting Calcium-sensing Receptor (CASR).

Castration increases fat deposition, improving beef quality in cattle. Here, the steer group exhibited a significantly higher intramuscular fat (IMF) content than the bull group. To determine the potential roles of microRNAs (miRNAs) in castration-induced fat deposition, differential expression patterns of miRNA in liver tissue were investigated in bulls and steers. A total of 7,827,294 clean reads were obtained from the bull liver library, and 8,312,483 were obtained from the steer liver library; 452 conserved bovine miRNAs and 20 novel miRNAs were identified. The results showed that the expression profiles of miRNA in liver tissue were changed by castration, and 12 miRNAs that were differentially expressed between bulls and steers were identified. Their target genes were majorly involved in the metabolic, PI3K-Akt, and MAPK signaling pathways. Furthermore, six differentially expressed miRNAs were validated by quantitative real-time PCR, and luciferase reporter assays verified that calcium-sensing receptor (CASR) was the direct target of miR-27a-5p. Meantime, we found that the expression level of CASR was significantly higher in steers than in bulls, and revealed that CASR gene silencing in bovine hepatocytes significantly inhibited triacylglycerol (TAG) accumulation and reduced secretion of very low density lipoprotein (VLDL). These results obtained in the liver indicate that miR-27a-5p may increase fat deposition partly by targeting CASR in steers.