Transcriptome Profile Analysis of Mechanisms of Black and White Plumage Determination in Black-Bone Chicken.

BACKGROUND/AIMS: Melanin is a major and ubiquitous component of plumage colouration, and patterns of melanin pigmentation in birds are extremely varied. However, the molecular mechanism of pigmentation in avian plumage is still largely unknown. METHODS: To elucidate the molecular mechanisms involved in the formation of black and white plumage, this study takes advantage of high-throughput sequencing technology to compare differences in the transcriptome between black and white chicken feather bulbs. In total, we constructed six cDNA libraries from black (Group B) and white (Group W) feather bulbs in the dorsal plumage of Muchuan black-boned chickens. RESULTS: A comparison between Groups B and W revealed 61 differentially expressed genes, with 47 displaying higher, and 14 displaying lower, levels of expression in white feather bulbs. Our results revealed a set of candidate genes and two potential metabolic pathways involved in black-bone chicken plumage melanogenesis. These include four homeobox genes (HOXB9, HOXC8, HOXA9, and HOXC 9), two glutathione (GSH) metabolism-related genes (CHAC1 and GPX3), and the transforming growth factor beta (TGF-ß) signalling pathway. Two known genes, TYR and MITF, were also shown to play a role in melanin formation. CONCLUSION: our data provide a valuable resource for discovering genes important in plumage melanin formation and will help further elucidate the molecular mechanisms for black and white plumage.

A Functional Mutation in KIAA1462 Promoter Decreases Glucocorticoid Receptor Affinity and Affects Egg-Laying Performance in Yangzhou Geese.

The identification of genetic markers is valuable for improving the egg-laying performance in goose production. The single-nucleotide polymorphism (SNP) rs1714766362 in an intron of the goose KIAA1462 gene was found to be relevant to laying performance in our previous study. However, its function remains unclear. In this study, the full-length coding sequence of KIAA1462 gene was firstly characterized in Yangzhou geese. Q-PCR (Quantitative Real Time Polymerase Chain Reaction) results showed that KIAA1462 was highly expressed in the liver, ovary, and mature F1 follicles. For SNP rs1714766362, geese with the AA genotype showed better laying performance than the TT ones and exhibited a higher KIAA1462 expression level in the ovary. Gain- and loss-of function experiments in granulosa cells revealed that KIAA1462 affected the expression of the apoptosis marker gene caspase-3. Considering that rs1714766362 locates in an intron area, we compared the KIAA1462 promoter regions of AA and TT individuals and identified the SNP c.-413C>G (Genbank ss2137504176), which was completely linked to SNP rs1714766362. According to the transcription factor prediction results, the glucocorticoid receptor (GR) would bind to the SNP site containing the C but not the G allele. In this study, we proved this hypothesis by an electrophoretic mobility shift assay (EMSA). In summary, we identified a novel mutation in the promoter of KIAA1462 gene which can modulate GR binding affinity and affect the laying performance of geese.

C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene.

Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes.

Clinical effect of laparoscopic cholecystectomy in the treatment of chronic cholecystitis with gallstones.

Chronic cholecystitis is a common disease that causes inflammation in the gallbladder and is usually associated with gallstones. Laparoscopic cholecystectomy has been widely used as a minimally invasive surgical technique to treat this condition. However, the clinical effect of laparoscopic cholecystectomy in the treatment of chronic cholecystitis with gallstones needs further investigation. This study aimed to investigate the clinical effect of laparoscopic cholecystectomy in treating chronic cholecystitis with gallstones. 90 patients with chronic cholecystitis and gallstones were randomly divided into control and research groups. The control group underwent traditional open cholecystectomy, while the research group received laparoscopic cholecystectomy. Perioperative indexes, oxidative stress indexes, serum inflammatory factors, liver function indexes and the incidence of complications were observed and compared. Results showed that laparoscopic cholecystectomy significantly reduced the operation time, blood loss, anal exhaust time, abdominal pain duration, and hospital stay compared to traditional open cholecystectomy (P < 0.05). Moreover, laparoscopic cholecystectomy significantly reduced the levels of oxidative stress indexes (GSH-Px), inflammatory factors (IL-6, TNF-α, and CRP), and liver function indexes (TBIL, AST, and ALT) compared to traditional open cholecystectomy. Moreover, the complication rate of the research group was significantly lower than that of the control group (P < 0.05). In conclusion, laparoscopic cholecystectomy for chronic cholecystitis with gallstones is a safe and effective procedure that reduces the perioperative stress response and promotes the rapid recovery of the postoperative body. The findings of this study provide a basis for the clinical promotion of laparoscopic cholecystectomy as the preferred surgical treatment for chronic cholecystitis with gallstones.

CircRNA expression profiles in human visceral preadipocytes and adipocytes.

Circular RNAs (circRNAs) regulate several physiological and pathological processes, but their role in visceral lipid deposition has not been explored. In the present study, human preadipocytes from visceral fat tissue (HPA­v) were induced to form adipocytes, and the circRNA expression profiles in HPA­v and adipocytes were detected using circRNA microarrays. The microarray data revealed that 2,215 and 1,865 circRNAs were significantly up­ and downregulated, respectively, in adipocytes compared with HPA­v. Moreover, the parental genes of differentially expressed circRNAs were associated with fatty acid metabolism based on Kyoto Encyclopedia of Genes and Genomes analysis. Three circRNAs (hsa_circ_0136134, hsa_circ_0017650, and hsa­circRNA9227­1) were selected for quantitative PCR (qPCR) validation, and the qPCR results were consistent with the microarray results. Furthermore, MiRanda software was used to predict the microRNAs (miRNAs) potentially targeting the top 10 up­ and downregulated circRNAs, and 14 miRNAs with more than two miRNA response elements targeting these circRNAs. This is the first study of the expression profiles of circRNAs in HPA­v and adipocytes and may suggest potential therapeutic targets for the visceral obesity.

Transcriptome profile analysis identifies candidate genes for the melanin pigmentation of breast muscle in Muchuan black-boned chicken.

Melanin-based coloration in the meat of black-boned chicken is a major economic issue in China. Variation in the pigmentation (hypopigmentation) of chicken muscle causes direct economic losses every year. To determine the molecular mechanisms involved in the melanogenesis of muscle tissue, this study used high-throughput sequencing to compare differences in the transcriptome between black (BM) and white (WM) chicken breast muscles. We constructed 6 cDNA libraries from BM and WM groups in Muchuan black-boned chickens. A comparison between the BM and WM groups revealed 264 differentially expressed genes, of which 152 were upregulated, whereas 112 were downregulated in black muscle. Gene ontology and a Kyoto Encyclopedia of Genes and Genomes pathway analysis identified several differentially enriched biological functions and processes of the 2 muscles. Seven promising candidate genes [PMEL, Ras-related protein RAB29, and 5 solute carrier superfamily genes: SLC6A9, SLC38A4, SLC22A5, SLC35F3, and SLC16A3] may play an important role in the melanogenesis of chicken muscle. Our data provide a valuable resource for identifying genes whose functions are critical for muscle melanogenesis, and will assist studies of the molecular mechanisms of melanogenesis regulation in chicken muscle.

Increased EXT1 gene copy number correlates with increased mRNA level predicts short disease-free survival in hepatocellular carcinoma without vascular invasion.

Exostosin-1 (EXT1) has been demonstrated to participate in the progression of many cancers. However, it has not been previously described in patients with hepatocellular carcinoma (HCC) without vascular invasion. In this study, we got the accurate data of EXT1 mRNA Z-score from the CBio data portal of The Cancer Genome Atlas (TCGA), which was used to express the level of EXT1 gene expression. We analyzed the EXT1 gene expression between HCC and normal liver tissue and compared the clinical significance of tumor tissue's EXT1 gene expression of HCC patients without vascular invasion based on data from TCGA database. The association between EXT1 gene expression and disease-free survival (DFS) was further analyzed. EXT1 gene copy number was also analyzed in this study. Univariate and multivariate analyses showed that high EXT1 gene expression group was significantly poorer than that of the low EXT1 gene expression group (P = .004). In addition, EXT1 gene expression was positively associated with α-fetoprotein (AFP), which is a well-known marker for HCC. There was a significant positive correlation between EXT1 copy number and upregulated EXT1 gene (P < .0001). In conclusion, upregulation of EXT1 could be an important indicator to the short DFS of HCC patients without vascular invasion. EXT1 gene copy number amplification is one of the mechanisms underlying the upregulation of EXT1.

The c.-360 T>C mutation affects PGAM2 transcription activity and is linked with the water holding capacity of the longissimus lumborum muscle in pigs.

The phosphoglycerate mutase 2 (PGAM2) gene encodes a key enzyme in the glycolytic process. This study examined a functional mutation in the PGAM2 gene and evaluated its relationship with water holding capacity (WHC). RT-qPCR analysis showed the PGAM2 mRNA level was significantly higher in the low-WHC group than in the high-WHC group (P<0.05). The c.-360 T>C mutation was identified through sequencing and found to have opposite allele distributions in the two groups. The allele was further genotyped in 170 Duroc×Large White×Yorkshire crossbred pigs using allele-specific PCR. The CC genotype was associated with lower WHC and higher PGAM2 mRNA levels, whereas the TT genotype corresponded to a higher WHC and lower PGAM2 mRNA levels (P<0.05). A luciferase activity assay also showed that the CC-genotype promoter had higher activity than the TT-genotype promoter (P<0.05). In conclusion, we discovered the c.-360 T>C mutation in the PGAM2 gene, which is a promising marker for improving pork WHC.

Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

Identification of Laying-Related SNP Markers in Geese Using RAD Sequencing.

Laying performance is an important economical trait of goose production. As laying performance is of low heritability, it is of significance to develop a marker-assisted selection (MAS) strategy for this trait. Definition of sequence variation related to the target trait is a prerequisite of quantitating MAS, but little is presently known about the goose genome, which greatly hinders the identification of genetic markers for the laying traits of geese. Recently developed restriction site-associated DNA (RAD) sequencing is a possible approach for discerning large-scale single nucleotide polymorphism (SNP) and reducing the complexity of a genome without having reference genomic information available. In the present study, we developed a pooled RAD sequencing strategy for detecting geese laying-related SNP. Two DNA pools were constructed, each consisting of equal amounts of genomic DNA from 10 individuals with either high estimated breeding value (HEBV) or low estimated breeding value (LEBV). A total of 139,013 SNP were obtained from 42,291,356 sequences, of which 18,771,943 were for LEBV and 23,519,413 were for HEBV cohorts. Fifty-five SNP which had different allelic frequencies in the two DNA pools were further validated by individual-based AS-PCR genotyping in the LEBV and HEBV cohorts. Ten out of 55 SNP exhibited distinct allele distributions in these two cohorts. These 10 SNP were further genotyped in a goose population of 492 geese to verify the association with egg numbers. The result showed that 8 of 10 SNP were associated with egg numbers. Additionally, liner regression analysis revealed that SNP Record-111407, 106975 and 112359 were involved in a multiplegene network affecting laying performance. We used IPCR to extend the unknown regions flanking the candidate RAD tags. The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens. Five novel genes were cloned for geese which harbored the candidate laying-related SNP, including membrane associated guanylate kinase (MAGI-1), KIAA1462, Rho GTPase activating protein 21 (ARHGAP21), acyl-CoA synthetase family member 2 (ACSF2), astrotactin 2 (ASTN2). Collectively, our data suggests that 8 SNP and 5 genes might be promising candidate markers or targets for marker-assisted selection of egg numbers in geese.