Integrated analysis of scRNA-seq and bulk RNA-seq reveals that GPRC5A is an important prognostic gene in pancreatic cancer and is associated with B-cell Infiltration in pancreatic cancer.

Introduction: Pancreatic cancer (PC) is a malignancy with poor prognosis. This investigation aimed to determine the relevant genes that affect the prognosis of PC and investigate their relationship with immune infiltration. Methods: : First, we acquired PC single-cell chip data from the GEO database to scrutinize dissimilarities in immune cell infiltration and differential genes between cancerous and adjacent tissues. Subsequently, we combined clinical data from TCGA to identify genes relevant to PC prognosis. Employing Cox and Lasso regression analyses, we constructed a multifactorial Cox prognostic model, which we subsequently confirmed. The prognostic gene expression in PC was authenticated using RT-PCR. Moreover, we employed the TIMER online database to examine the relationship between the expression of prognostic genes and T and B cell infiltration. Additionally, the expression of GPRC5A and its correlation with B cells infiltration and patient prognosis were ascertained in tissue chips using multiple immune fluorescence staining. Results: The single-cell analysis unveiled dissimilarities in B-cell infiltration between cancerous and neighboring tissues. We developed a prognostic model utilizing three genes, indicating that patients with high-risk scores experienced a more unfavorable prognosis. Immune infiltration analysis revealed a significant correlation among YWHAZ, GPRC5A, and B cell immune infiltration. In tissue samples, GPRC5A exhibited substantial overexpression and a robust association with an adverse prognosis, demonstrating a positive correlation with B cell infiltration. Conclusion: GPRC5A is an independent risk factor in PC and correlated with B cell immune infiltration in PC. These outcomes indicated that GPRC5A is a viable target for treating PC.

Identification of dynamic changes in volatile compounds and metabolites during the smoking process of Zhenba bacon by GC-IMS combined metabolomics.

Zhenba bacon is a traditional cured bacon product with a rich history that originated from Zhenba County, Shaanxi Province. This study aimed to investigate the patterns of volatile compound formation and changes in metabolites during the smoking process in Zhenba bacon. Firstly, the sensory properties and physicochemical properties of Zhenba bacon were analyzed. Gas chromatography-ion mobility spectrometry (GC-IMS) and nontargeted metabolomics technology were used to analyze Zhenba bacon from different smoking stages. The results show a gradual increase in the sensory acceptance and volatile flavor compounds such as aldehydes, ketones, and esters with the prolongation of smoking of Zhenba bacon. LC-MS analysis identified 191 co-expressed differentially metabolites, with amino acid and lipid metabolism being the main metabolic pathways according to KEGG enrichment analysis. Temporal expression analysis of bacon metabolites at each stage revealed a decrease in harmful steroid hormones such as cortisone and an increase in amino acids and lipid metabolites, such as arginine, lysine, acid, and cholesterol, that contribute to the flavor of bacon. In summary, duration of smoking increased, the amount of flavor substances in Zhenba bacon gradually increased, and the safety and quality of bacon reached the optimal level after 32 days of smoking. This study provides valuable insights into the dynamic changes in volatile flavor compounds in Zhenba bacon and establishes a theoretical foundation for quality control during its production.

Regular use of paracetamol and risk of liver cancer: a prospective cohort study.

BACKGROUND: Paracetamol induces hepatotoxicity and subsequent liver injury, which may increase the risk of liver cancer, but epidemiological evidence remains unclear. We conducted this study to evaluate the association between paracetamol use and the risk of liver cancer. METHODS: This prospective study included 464,244 participants free of cancer diagnosis from the UK Biobank. Incident liver cancer was identified through linkage to cancer and death registries and the National Health Service Central Register using the International Classification of Diseases (ICD)-10 codes (C22). An overlap-weighted Cox proportional hazards model was utilized to calculate the hazard ratio (HR) and 95% confidence interval (CI) for the risk of liver cancer associated with paracetamol use. The number needed to harm (NNH) was calculated at 10 years of follow-up. RESULTS: During a median of 12.6 years of follow-up, 627 cases of liver cancer were identified. Paracetamol users had a 28% higher risk of liver cancer than nonusers (HR 1.28, 95% CI 1.06-1.54). This association was robust in several sensitivity analyses and subgroup analyses, and the quantitative bias analysis indicated that the result remains sturdy to unmeasured confounding factors (E-value 1.88, lower 95% CI 1.31). The NNH was 1106.4 at the 10 years of follow-up. CONCLUSION: The regular use of paracetamol was associated with a higher risk of liver cancer. Physicians should be cautious when prescribing paracetamol, and it is recommended to assess the potential risk of liver cancer to personalize the use of paracetamol.

Establishment and characterization of a novel hilar cholangiocarcinoma cell line, CBC3T-1.

Cholangiocarcinoma (CCA) is a group of malignant heterogeneous cancer arising from the biliary tree. The tumor is characterized by insidious onset, high degree of malignancy, poor prognosis, and high recurrence rate. Immortalized cancer cell lines are the best and easiest models for in vitro cancer research. Here, we established a naturally immortalized highly tumorigenic hilar cholangiocarcinoma (hCCA) cell line, CBC3T-1. The CBC3T-1 cell line was cultured for over 60 passages. Thorough analysis showed that CBC3T-1 cells share characteristics similar to original tumor cells from patients with cholangiocarcinoma and display a stable phenotype, including features of epithelial origin, stem cell-like properties, as well as a high invasive and migratory capability and tumorigenicity in mice. Furthermore, this cell line showed the best sensitivity to paclitaxel, followed by gemcitabine. RNA sequencing and whole­exome sequencing showed that cancer-associated pathways and somatic mutations played a dominant role in the development of CCA. We established and characterized a new hCCA cell line, CBC3T-1, which contributes to a better understanding of bile duct cancer, and can be used to study tumorigenesis and progression and the role of anticancer drugs.

First report on establishment and characterization of the extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2.

BACKGROUND: Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice. These cells consist of both epithelial and mesenchymal cells. Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma. However, cholangiocarcinoma sarcoma cell lines are not available in cell banks. AIM: To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line, namely CBC2T-2. METHODS: We conducted a short tandem repeat (STR) test to confirm the identity of the CBC2T-2 cell line. Furthermore, we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies. The tumorigenic potential of CBC2T-2 cells was tested in vivo using non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The cells were injected subcutaneously and tumor formation was observed. In addition, immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts. The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma. Lastly, whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line. RESULTS: The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue. The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology. The cells exhibited a high proliferation ratio with a doubling time of 47.11 h. This cell line has migratory, invasive, and clonogenic abilities. The chromosomes in the CBC2T-2 cells were polyploidy, with numbers ranging from 69 to 79. The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice. CBC2T-2 cells and xenografts were positive for both the epithelial marker, CK19, and the mesenchymal marker, vimentin. These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics. The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma, and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option. CONCLUSION: We established the first human cholangiocarcinoma sarcoma cell line, CBC2T-2, with stable biogenetic traits. This cell line, as a research model, has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.

Proton pump inhibitors may enhance the risk of digestive diseases by regulating intestinal microbiota.

Proton pump inhibitors (PPIs) are the most used acid-inhibitory drugs, with a wide range of applications in the treatment of various digestive diseases. However, recently, there has been a growing number of digestive complications linked to PPIs, and several studies have indicated that the intestinal flora play an important role in these complications. Therefore, developing a greater understanding of the role of the gut microbiota in PPI-related digestive diseases is essential. Here, we summarize the current research on the correlation between PPI-related digestive disorders and intestinal flora and establish the altered strains and possible pathogenic mechanisms of the different diseases. We aimed to provide a theoretical basis and reference for the future treatment and prevention of PPI-related digestive complications based on the regulation of the intestinal microbiota.

Lanatoside C decelerates proliferation and induces apoptosis through inhibition of STAT3 and ROS-mediated mitochondrial membrane potential transformation in cholangiocarcinoma.

Introduction: The incidence of cholangiocarcinoma (CCA) has increased worldwide in recent years. Given the poor prognosis associated with the current management approach of CCA, new therapeutic agents are warranted to improve the prognosis of this patient population. Methods: In this study, we extracted five cardiac glycosides (CGs) from natural plants: digoxin, lanatoside A, lanatoside C, lanatoside B, and gitoxin. Follow-up experiments were performed to assess the effect of these five extracts on cholangiocarcinoma cells and compounds with the best efficacy were selected. Lanatoside C (Lan C) was selected as the most potent natural extract for subsequent experiments. We explored the potential mechanism underlying the anticancer activity of Lan C on cholangiocarcinoma cells by flow cytometry, western blot, immunofluorescence, transcriptomics sequencing, network pharmacology and in vivo experiments. Results: We found that Lan C time-dependently inhibited the growth and induced apoptosis of HuCCT-1 and TFK-1 cholangiocarcinoma cells. Besides Lan C increased the reactive oxygen species (ROS) content in cholangiocarcinoma cells, decreased the mitochondrial membrane potential (MMP) and resulted in apoptosis. Besides, Lan C downregulated the protein expression of STAT3, leading to decreased expression of Bcl-2 and Bcl-xl, increased expression of Bax, activation of caspase-3, and initiation of apoptosis. N-acetyl-L-cysteine (NAC) pretreatment reversed the effect of Lan C. In vivo, we found that Lan C inhibited the growth of cholangiocarcinoma xenografts without toxic effects on normal cells. Tumor immunohistochemistry showed that nude mice transplanted with human cholangiocarcinoma cells treated with Lan C exhibited decreased STAT3 expression and increased caspase-9 and caspase-3 expression in tumors, consistent with the in vitro results. Conclusion: In summary, our results substantiates that cardiac glycosides have strong anti-CCA effects. Interestingly the biological activity of Lan C provides a new anticancer candidate for the treatment of cholangiocarcinoma.

Targeted knockout of Mx in the DF-1 chicken fibroblast cell line impairs immune response against Newcastle disease virus.

Newcastle disease virus (NDV) is an RNA virus taking poultry as the host, and the Newcastle disease (ND) caused by NDV is one of the diseases with serious damage to the health of poultry. Mx encoding by myxovirus resistance gene, induced by type I interferon (IFN), has a wide range of antiviral and GTPase activities in human, mice, and other species via inhibition virus replication. However, the antiviral ability of chicken Mx is still a controversial issue. To explore the effect of chicken Mx post-NDV infection, Mx-knockout DF-1 cells were constructed via CRISPR/Cas9 gene editing system. The number of copies of NDV was detected by RT-qPCR, and the mRNA expression levels of IRF-7, IFN-α, IFN-ß, TNF-α, p21, p27, and Bak in DF-1 cells were analyzed after NDV infection. Compared with control cells, virus titers were much higher in Mx-knockout DF-1 cells post-NDV infection. The deficiency of Mx aggravated the cell pathological features post-NDV infection, and promoted the expression levels of IRF-7, IFN-α, IFN-ß, and pro-inflammatory cytokine TNF-α in host cells. In addition, cells with Mx deficiency could alleviate the harm from virus by enhancing the expression of p21, p27, and Bak, which related to cell proliferation apoptosis. In conclusion, Mx played an important role in antivirus invasion. In the absence of Mx, cells could alleviate the harm from virus infection via retarding cell proliferation and enhancing cell apoptosis.

miR-486 Responds to Apoptosis and Autophagy by Repressing SRSF3 Expression in Ovarian Granulosa Cells of Dairy Goats.

The accumulation of ovarian granulosa cell (GC) apoptosis underlies follicular atresia. By comparing the previous sequencing results, miR-486 was found to be differentially expressed at higher levels in the monotocous goat than in the polytocous goat. Unfortunately, the miRNA-mediated mechanisms by which the GC fate is regulated are unknown in Guanzhong dairy goats. Therefore, we investigated miR-486 expression in small and large follicles, as well as its impact on normal GC survival, apoptosis and autophagy in vitro. Here, we identified and characterized miR-486 interaction with Ser/Arg-rich splicing factor 3 (SRSF3) using luciferase reporter analysis, detecting its role in GC survival, apoptosis and autophagy regulation through qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, mitochondrial membrane potential and monodansylcadaverine, etc. Our findings revealed prominent effects of miR-486 in the regulation of GC survival, apoptosis and autophagy by targeting SRSF3, which might explain the high differential expression of miR-486 in the ovaries of monotocous dairy goats. In summary, this study aimed to reveal the underlying molecular mechanism of miR-486 regulation on GC function and its effect on ovarian follicle atresia in dairy goats, as well as the functional interpretation of the downstream target gene SRSF3.

[Study on Platelet Adhesion and Aggregation Induced by Gradient Shear Stress Using Microfluidic Chip Technology].

OBJECTIVE: To study the effect of gradient shear stress on platelet aggregation by microfluidic chip Technology. METHODS: Microfluidic chip was used to simulate 80% fixed stenotic microchannel, and the hydrodynamic behavior of the stenotic microchannel model was analyzed by the finite element analysis module of sollidwork software. Microfluidic chip was used to analyze the adhesion and aggregation behavior of platelets in patients with different diseases, and flow cytometry was used to detect expression of the platelet activation marker CD62p. Aspirin, Tirofiban and protocatechuic acid were used to treat the blood, and the adhesion and aggregation of platelets were observed by fluorescence microscope. RESULTS: The gradient fluid shear rate produced by the stenosis model of microfluidic chip could induce platelet aggregation, and the degree of platelet adhesion and aggregation increased with the increase of shear rate within a certain range of shear rate. The effect of platelet aggregation in patients with arterial thrombotic diseases were significantly higher than normal group (P<0.05), and the effect of platelet aggregation in patients with myelodysplastic disease was lower than normal group (P<0.05). CONCLUSION: The microfluidic chip analysis technology can accurately analyze and evaluate the platelet adhesion and aggregation effects of various thrombotic diseases unde the environment of the shear rate, and is helpful for auxiliary diagnosis of clinical thrombotic diseases.

Transcriptomic analysis of thigh muscle of Lueyang black-bone chicken in free-range and caged feeding.

Lueyang black-bone chicken is free-range in hilly areas and has unique genetic characteristics and excellent muscle quality. However, the molecular mechanisms of breeding mode influence growth and meat quality in Lueyang black-bone chicken are still unclear. Here we analyzed the meat quality and transcriptome data of thigh muscle by comparing free-range and caged modes at the age of 60 and 120 days in Lueyang black-boned chicken. The results demonstrated that the free-range mode could improve the pH value, tenderness, and reducing the hardness of the thigh muscle. Intramuscular fat (IMF) content of the thigh muscle was markedly higher in the caged chickens compared with free-range animals at the age of 60 days. Functional pathway analysis illustrated that tight junction signaling was associated with the formation of slow-twitch fibers in free-range chickens at age of 120 days. All research data proved that the free-range mode could improve muscle quality by promoting the formation of slow-twitch fibers and IMF in thigh muscle in Lueyang black-bone chicken. Based on the animal benefit and healthy, the free-range feeding should be considered during the breeding process of broiler chicken. The results provide good knowledge of the functional molecular mechanisms associated with muscle quality in Lueyang black-bone chicken.

[Exploration and practice of the teaching system for "Human and Animal Physiology" in post pandemic era].

After the outbreak of COVID-19, the widespread application of online teaching has brought challenges and opportunities for higher education. Developing an effective teaching system is the focus of curriculum teaching reform in the post pandemic era. According to the characteristics of Human and Animal Physiology, the course teachers has developed a new teaching system by updating the teaching concept, reconstructing the contents of the course, changing the teaching modes, strengthening the integration of moral and intellectual education, and improving the assessment approaches. This teaching system is aimed at meeting the need of personalized learning for students and adapting to a new teaching environment. This article introduces the exploration and practice of the curriculum reform.

The N6-Methyladenosine Modification and Its Role in mRNA Metabolism and Gastrointestinal Tract Disease.

The N6-methyladenosine (m6A) modification is the most abundant internal modification of messenger RNA (mRNA) in higher eukaryotes. Under the actions of methyltransferase, demethylase and methyl-binding protein, m6A resulting from RNA methylation becomes dynamic and reversible, similar to that from DNA methylation, and this effect allows the generated mRNA to participate in metabolism processes, such as splicing, transport, translation, and degradation. The most common tumors are those found in the gastrointestinal tract, and research on these tumors has flourished since the discovery of m6A. Overall, further analysis of the mechanism of m6A and its role in tumors may contribute to new ideas for the treatment of tumors. m6A also plays an important role in non-tumor diseases of the gastrointestinal tract. This manuscript reviews the current knowledge of m6A-related proteins, mRNA metabolism and their application in gastrointestinal tract disease.

Application of a Simple Microfluidic Chip Analysis Technology to Evaluate the Inhibitory Role of Protocatechuic Acid on Shear-Induced Platelet Aggregation.

This study aimed to develop a simple microfluidic chip analysis technology to study the inhibitory effect of protocatechuic acid on shear-induced platelet aggregation. The microfluidic chip designed in this study simulates 80% fixed narrow microchannels. This microchannel narrow model uses the finite element analysis module of the three-dimensional modeling software solidwork to analyze fluid dynamic behavior. Blood treated with protocatechuic acid at 1, 2, 4, 8, or 16 µg/mL was passed through the microchannel stenosis model at a shear rate of 10,000 s-1. The platelet adhesion and aggregation behaviors were then measured using fluorescence microscopy and observed in real time. Simultaneously, the antiplatelet aggregation effect of protocatechuic acid was analyzed using thromboelastography and photoelectric turbidimetry. The designed stenosis model of the microfluidic chip can produce a gradient of fluid shear rate, and the gradient of fluid shear rate can induce platelet aggregation. Under this model, the degree of platelet adhesion and aggregation increased as the shear rate increased. In the experimental concentration range of 0-8 µmol/mL, protocatechuic acid exerted a concentration-dependent inhibition of platelet aggregation. In contrast, thromboelastography and photoelectric turbidimetry failed to demonstrate an inhibitory effect. The microfluidic chip analysis technology developed in this study can be used to study the effect of protocatechin in inhibiting platelet aggregation induced by shear rate in vitro. This technology is simple to operate and can be used as a new type of antiplatelet aggregation analysis technology for screening studies of novel potential antiplatelet aggregation drugs.

Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1.

BACKGROUND: MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes, including proliferation, development and apoptosis. Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters. The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development. RESULTS: cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing. In total, 142 differentially expressed unigenes (DEGs) were detected between two libraries, including 78 down-regulated and 64 up-regulated genes. GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development. STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes. In vitro, bioinformatics analysis and 3'-UTR assays confirmed that STC1 was a target of miR-101-3p. ELISA was performed to detect the estrogen (E2) and progesterone (P4) levels. CCK8, EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells. Results showed that miR-101-3p regulated STAR, CYP19A1, CYP11A1 and 3ß-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus promoted E2 and P4 secretions. MiR-101-3p also affected the key protein PI3K, PTEN, AKT and mTOR in PI3K-AKT pathway by STC1, thereby suppressing proliferation and promoting apoptosis of granulosa cells. In vivo, the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation (FISH). Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups. Small and stunted ovarian fragments, decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin (HE) staining, thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion. Inhibition of miR-101-3p manifested opposite results. CONCLUSIONS: Taken together, our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells, and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.

In Vitro and In Vivo Studies of Polyvinyl Pyrrolidone-Coated Sparfloxacin-Loaded Ring Contact Lens to Treat Conjunctivitis.

Currently, conjunctivitis is treated by frequent high dose administration of sparfloxacin eye drop solution. However, the eye drops are inconvenient because of low bioavailability, short ocular drug residence time, and need of frequent instillation, which lead to patient noncompliance affecting the routine life style of patients. Silicone contact lenses can be used to sustain the release of sparfloxacin. However, the presence of sparfloxacin alters the optical and physical properties of the contact lens. To overcome the issues, a novel polyvinyl pyrrolidone (PVP)-coated sparfloxacin-laden ring contact lens was designed to provide sustained ocular drug delivery without altering the optical and swelling properties of contact lens. The ring was implanted within the periphery of the lens. Sparfloxacin was loaded by soaking (Sp-S), direct loading (Sp-L), and ring casting method (Sp-R). PVP (comfort agent) was coated on the surface of contact lens by novel short surface curing technique. The in vitro sparfloxacin release data of Sp-S (up to 12-36 h) and Sp-L batches (up to 12-24 h) showed high burst release, whereas Sp-R batch showed sustained release up to 36-48 h without significant (p > 0.05) alteration of the optical and swelling properties. All the batches showed sustained release of PVP up to 48 h. The in vivo release studies in the rabbit tear fluid showed improvement in the sparfloxacin [>MIC for Staphylococcus aureus] and PVP retention time in comparison to eye drop solution. The in vivo efficacy study in the S aureus-induced conjunctivitis showed improved healing effect with the single PVP-coated Sp-R-300 contact lens in comparison to the frequent high-dose sparfloxacin eye drop therapy. The study demonstrated the successful application to codeliver sparfloxacin and PVP from the contact lens for the extended period to treat conjunctivitis.

Endometrial genome-wide DNA methylation patterns of Guanzhong dairy goats at days 5 and 15 of the gestation period.

Uterine receptivity for the embryo is established and maintained through a series of precise cellular and molecular events, such as DNA methylation. There have been no studies to elucidate entire genome DNA methylation changes associated with embryo receptivity development of the endometrium (RE). In the present study, there was development of a complete genome-wide DNA methylome maps of the RE using whole-genome bisulphite sequencing and bioinformatics analysis. As many as 163.06 Gb of sequencing data averaging 81.53 Gb per sample were obtained for genome bisulphite sequencing of endometrium samples. There were distinct genome-wide DNA methylation patterns in pre-receptive endometrium (PE; Day 5 of gestation) and RE (Day 15 of gestation). There were as many as 16,467 differentially methylated regions (DMRs); 21,391 DMRs were less methylated in RE samples compared with PE samples (P-values ≤ 0.05 and |log2 (fold change)| ≥ 2). Compared with PE samples, methylation ratios of IGF2BP2, ACOX2, PTGDS, VEGFB and PTGDR2 genes were markedly less in RE samples (P-value ≤ 0.05 and |log2 (fold change)| ≥ 2). Conversely, in RE samples there was a markedly greater methylation ratio of IGFBP3 and IGF1R genes. The results of KEGG analysis indicated that these genes were involved in the signalling pathways for insulin, mitogen-activated protein kinase, gonadotropin-releasing hormone, vascular endothelial growth factor and progesterone-mediated oocyte maturation, which participated in differential regulation of goat endometrial development during receptive and prereceptive phases. The results of previous and the present study indicate resulting proteins of IGF2BP2, PTGDS, VEGFB, PGR, IGFBP3 and IGF1R gene expression may have important functions in regulating endometrial receptivity for the embryo.

Genome-wide differences in DNA methylation changes in caprine ovaries between oestrous and dioestrous phases.

BACKGROUND: DNA methylation plays a vital role in reproduction. Entire genome DNA methylation changes during the oestrous phase (ES) and dioestrous phase (DS) in the ovaries of Guanzhong dairy goats were investigated using bisulphite sequencing to understand the molecular biological mechanisms of these goats' oestrous cycle. RESULTS: We discovered distinct genome-wide DNA methylation patterns in ES and DS ovaries. A total of 26,910 differentially methylated regions were upregulated and 21,453 differentially methylated regions were downregulated in the ES samples compared with the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Differentially methylated region analysis showed hypomethylation in the gene body regions and hypermethylation in the joining region between upstream regions and gene bodies. The methylation ratios of the STAR, FGF2, FGF12, BMP5 and SMAD6 genes in the ES samples were lower than those of the DS samples (P-values ≤0.05 and fold change of methylation ratios ≥2). Conversely, the methylation ratios of the EGFR, TGFBR2, IGF2BP1 and MMD2 genes increased in the ES samples compared with the DS samples. In addition, 223 differentially methylated genes were found in the GnRH signalling pathway (KO04912), ovarian steroidogenesis pathway (KO04913), oestrogen signalling pathway (KO04915), oxytocin signalling pathway (KO04921), insulin secretion pathway (KO04911) and MAPK signalling pathway (KO04010). CONCLUSIONS: This study is the first large-scale comparison of the high-resolution DNA methylation landscapes of oestrous and dioestrous ovaries from dairy goats. Previous studies and our investigations have shown that the NR5A2, STAR, FGF2 and BMP5 genes might have potential application value in regulating caprine oestrus.

Chi-miR-3031 regulates beta-casein via the PI3K/AKT-mTOR signaling pathway in goat mammary epithelial cells (GMECs).

BACKGROUND: MicroRNAs can regulate gene expression at the posttranscriptional level through translational repression or target degradation. Our previous investigations examined the differential expression levels of chi-miR-3031 in caprine mammary gland tissues in colostrum and common milk stages. RESULTS: The present study detected the role of chi-miR-3031 in the lactation mechanisms of GMECs. High-throughput sequencing was used to analyze transcriptomic landscapes of GMECs transfected with chi-miR-3031 mimics (MC) and a mimic negative control (NC). In the MC and NC groups, we acquired 39,793,503 and 36,531,517 uniquely mapped reads, respectively, accounting for 85.85 and 81.66% of total reads. In the MC group, 180 differentially expressed unigenes were downregulated, whereas 157 unigenes were upregulated. KEGG pathway analyses showed that the prolactin, TNF and ErbB signaling pathways, including TGFα, PIK3R3, IGF2, ELF5, IGFBP5 and LHß genes, played important roles in mammary development and milk secretion. Results from transcriptome sequencing, real-time PCR and western blotting showed that chi-miR-3031 suppressed the expression of IGFBP5 mRNA and protein. The expression levels of ß-casein significantly increased in the MC and siRNA-IGFBP5 groups. We observed that the down-regulation of IGFBP5 activated mTOR at the Ser2448 site in GMECs transfected with MC and siRNA-IGFBP5. Previous findings and our results showed that chi-miR-3031 activated the PI3K-AKT-mTOR pathway and increased ß-casein expression by down-regulating IGFBP5. CONCLUSIONS: These findings will afford valuable information for improving milk quality and contribute the development of potential methods for amending lactation performance.