Quantitative parameters of dynamic contrast-enhanced magnetic resonance imaging to predict lymphovascular invasion and survival outcome in breast cancer.

BACKGROUND: Lymphovascular invasion (LVI) predicts a poor outcome of breast cancer (BC), but LVI can only be postoperatively diagnosed by histopathology. We aimed to determine whether quantitative parameters of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can preoperatively predict LVI and clinical outcome of BC patients. METHODS: A total of 189 consecutive BC patients who underwent multiparametric MRI scans were retrospectively evaluated. Quantitative (Ktrans, Ve, Kep) and semiquantitative DCE-MRI parameters (W- in, W- out, TTP), and clinicopathological features were compared between LVI-positive and LVI-negative groups. All variables were calculated by using univariate logistic regression analysis to determine the predictors for LVI. Multivariate logistic regression was used to build a combined-predicted model for LVI-positive status. Receiver operating characteristic (ROC) curves evaluated the diagnostic efficiency of the model and Kaplan-Meier curves showed the relationships with the clinical outcomes. Multivariate analyses with a Cox proportional hazard model were used to analyze the hazard ratio (HR) for recurrence-free survival (RFS) and overall survival (OS). RESULTS: LVI-positive patients had a higher Kep value than LVI-negative patients (0.92 ± 0.30 vs. 0.81 ± 0.23, P = 0.012). N2 stage [odds ratio (OR) = 3.75, P = 0.018], N3 stage (OR = 4.28, P = 0.044), and Kep value (OR = 5.52, P = 0.016) were associated with LVI positivity. The combined-predicted LVI model that incorporated the N stage and Kep yielded an accuracy of 0.735 and a specificity of 0.801. The median RFS was significantly different between the LVI-positive and LVI-negative groups (31.5 vs. 34.0 months, P = 0.010) and between the combined-predicted LVI-positive and LVI-negative groups (31.8 vs. 32.0 months, P = 0.007). The median OS was not significantly different between the LVI-positive and LVI-negative groups (41.5 vs. 44.0 months, P = 0.270) and between the combined-predicted LVI-positive and LVI-negative groups (42.8 vs. 43.5 months, P = 0.970). LVI status (HR = 2.40), N2 (HR = 3.35), and the combined-predicted LVI model (HR = 1.61) were independently associated with disease recurrence. CONCLUSION: The quantitative parameter of Kep could predict LVI. LVI status, N stage, and the combined-predicted LVI model were predictors of a poor RFS but not OS.

Molecular Profiling of DNA Methylation and Alternative Splicing of Genes in Skeletal Muscle of Obese Rabbits.

DNA methylation and the alternative splicing of precursor messenger RNAs (pre-mRNAs) are two important genetic modification mechanisms. However, both are currently uncharacterized in the muscle metabolism of rabbits. Thus, we constructed the Tianfu black rabbit obesity model (obese rabbits fed with a 10% high-fat diet and control rabbits from 35 days to 70 days) and collected the skeletal muscle samples from the two groups for Genome methylation sequencing and RNA sequencing. DNA methylation data showed that the promoter regions of 599 genes and gene body region of 2522 genes had significantly differential methylation rates between the two groups, of which 288 genes had differential methylation rates in promoter and gene body regions. Analysis of alternative splicing showed 555 genes involved in exon skipping (ES) patterns, and 15 genes existed in differential methylation regions. Network analysis showed that 20 hub genes were associated with ubiquitinated protein degradation, muscle development pathways, and skeletal muscle energy metabolism. Our findings suggest that the two types of genetic modification have potential regulatory effects on skeletal muscle development and provide a basis for further mechanistic studies in the rabbit.

Untargeted Metabolomics Analysis Revealed Lipometabolic Disorders in Perirenal Adipose Tissue of Rabbits Subject to a High-Fat Diet.

A high-fat diet (HFD) is widely recognized as a significant modifiable risk for insulin resistance, inflammation, Type 2 diabetes, atherosclerosis and other metabolic diseases. However, the biological mechanism responsible for key metabolic disorders in the PAT of rabbits subject to HFD remains unclear. Here, untargeted metabolomics (LC-MS/MS) combined with liquid chromatography (LC) and high-resolution mass spectrometry (MS) were used to evaluate PAT metabolic changes. Histological observations showed that the adipocytes cells and density of PAT were significantly increased in HFD rabbits. Our study revealed 206 differential metabolites (21 up-regulated and 185 down-regulated); 47 differential metabolites (13 up-regulated and 34 down-regulated), comprising mainly phospholipids, fatty acids, steroid hormones and amino acids, were chosen as potential biomarkers to help explain metabolic disorders caused by HFD. These metabolites were mainly associated with the biosynthesis of unsaturated fatty acids, the arachidonic acid metabolic pathway, the ovarian steroidogenesis pathway, and the platelet activation pathway. Our study revealed that a HFD caused significant lipometabolic disorders. These metabolites may inhibit oxygen respiration by increasing the adipocytes cells and density, cause mitochondrial and endoplasmic reticulum dysfunction, produce inflammation, and finally lead to insulin resistance, thus increasing the risk of Type 2 diabetes, atherosclerosis, and other metabolic syndromes.

Untargeted Metabolomic Characteristics of Skeletal Muscle Dysfunction in Rabbits Induced by a High Fat Diet.

Type 2 diabetes and metabolic syndrome caused by a high fat diet (HFD) have become public health problems worldwide. These diseases are characterized by the oxidation of skeletal muscle mitochondria and disruption of insulin resistance, but the mechanisms are not well understood. Therefore, this study aims to reveal how high-fat diet causes skeletal muscle metabolic disorders. In total, 16 weaned rabbits were randomly divided into two groups, one group was fed a standard normal diet (SND) and the other group was fed a high fat diet (HFD) for 5 weeks. At the end of the five-week experiment, skeletal muscle tissue samples were taken from each rabbit. Untargeted metabolomic analysis was performed using ultra-performance liquid chromatography combined with mass spectrometry (UHPLC-MS/MS). The results showed that high fat diet significantly altered the expression levels of phospholipids, LCACs, histidine, carnosine, and tetrahydrocorticosterone in skeletal muscle. Principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) showed that, compared with the SND group, skeletal muscle metabolism in HFD group was significantly up-regulated. Among 43 skeletal muscle metabolites in the HFD group, phospholipids, LCACs, histidine, carnosine, and tetrahydrocorticosteroids were identified as biomarkers of skeletal muscle metabolic diseases, and may become potential physiological targets of related diseases in the future. Untargeted metabonomics analysis showed that high-fat diet altered the metabolism of phospholipids, carnitine, amino acids and steroids in skeletal muscle of rabbits. Notably, phospholipids, LCACs, histidine, carnopeptide, and tetrahydrocorticosteroids block the oxidative capacity of mitochondria and disrupt the oxidative capacity of glucose and the fatty acid-glucose cycle in rabbit skeletal muscle.

Intramuscular adipocyte and fatty acid differences between high-fat and control rabbit groups subject to a restricted diet.

Fatty acids of intramuscular fat (IMF) in rabbits can influence meat quality, but it is unclear which fatty acids benefit to human health. A rabbit model of weight gain and weight loss was constructed using two rabbit groups and two growth stages. Stage 1 included control group1 fed a commercial diet(CG1) and experimental group1 fed a high fat diet (EG1). Stage 2 include control group2(CG2) and experimental group2 (EG2) both fed a restricted commercial diet. We detected differences in blood biochemical indicators as well as changes in intramuscular adipose cells and intramuscular fatty acid content in control and experiment groups at two stages. High fat induction can make rabbits become obese, have higher concentrations of glucose (GLU), total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) and free fatty acid (FFA), and lower concentrations of insulin (INS). In addition, a high-fat diet promotes hypertrophy of precursor adipocytes in femoral muscles. Conversely, a restricted diet causes weight loss, decreases the concentration of TG, FFA, and INS in CG2 and EG2, and increases the deposition of unsaturated fatty acids in the femoral muscle. The content of monounsaturated trans oleic acid (C18:1n-9T) in EG2 was significantly higher than in CG2, whereas oleic acid (C18:1n-9C) was significantly lower in EG2 than in CG2. The polyunsaturated fatty acids Linolenate (C18:3 n-3) and cis-5,8,11,14,17-Eicosapentaenoate (C20:5 n-3) increased in CG2 and EG2. The content of Linoleate (C18:2 n-6) and γ-Linolenic acid (C18:3 n-6) significantly increased in CG2. The content of cis-11,14-Eicosatrienoic acid (C20:2) decreased significantly in CG2, but increased significantly in EG2.Thus, a high-fat diet can increase the formation of unhealthy fatty acids. Conversely, weight loss due to a restricted diet leads to an increase in unsaturated fatty acids in the femoral muscle, indicating that it reduces obesity symptoms and it may improve meat quality in rabbit.

Multi-Omics Analysis of Key microRNA-mRNA Metabolic Regulatory Networks in Skeletal Muscle of Obese Rabbits.

microRNAs (miRNAs), small non-coding RNA with a length of about 22 nucleotides, are involved in the energy metabolism of skeletal muscle cells. However, their molecular mechanism of metabolism in rabbit skeletal muscle is still unclear. In this study, 16 rabbits, 8 in the control group (CON-G) and 8 in the experimental group (HFD-G), were chosen to construct an obese model induced by a high-fat diet fed from 35 to 70 days of age. Subsequently, 54 differentially expressed miRNAs, 248 differentially expressed mRNAs, and 108 differentially expressed proteins related to the metabolism of skeletal muscle were detected and analyzed with three sequencing techniques (small RNA sequencing, transcriptome sequencing, and tandem mass tab (TMT) protein technology). It was found that 12 miRNAs and 12 core genes (e.g., CRYL1, VDAC3 and APIP) were significantly different in skeletal muscle from rabbits in the two groups. The network analysis showed that seven miRNA-mRNA pairs were involved in metabolism. Importantly, two miRNAs (miR-92a-3p and miR-30a/c/d-5p) regulated three transcription factors (MYBL2, STAT1 and IKZF1) that may be essential for lipid metabolism. These results enhance our understanding of molecular mechanisms associated with rabbit skeletal muscle metabolism and provide a basis for future studies in the metabolic diseases of human obesity.

Growth, behavioural, serum biochemical and morphological changes in female rabbits fed high-fat diet.

This study aimed to determine whether high-fat diet (HFD) could cause growth, behavioural, biochemical and morphological changes in young female rabbits. Thirty-six female rabbits were randomly divided into two groups fed with either a high-fat diet (HFD) or a standard normal diet (SND) for 5 weeks. Growth and behavioural changes were recorded during the 5-week feeding period. Tissue samples, including blood and adipose tissue, were obtained after slaughter. HFD rabbits weighed more by the end of the feeding period, had a higher percent body weight and adipose tissue weight change and had longer body and bust lengths than SND rabbits. HFD rabbits significantly reduced their feed intake and feeding frequency during the fourth and fifth weeks. HFD rabbits also showed lower frequency of drinking and resting and increased stereotypical behaviour. Besides, HFD rabbits showed significant physiological abnormalities. HFD rabbits had higher serum cholesterol (TC) and triglycerides (TG) levels than SND rabbits at the end of the feeding period, and higher free fatty acid (FFA) levels than rabbits in the SND group after the third week of feeding. Serum thyroxine (T4) increased significantly in week 2 and week 5 and triiodothyronine (T3) increased significantly in week four. However, there was no significant change in serum glucose (GLU) and insulin (INS) levels. Additionally, HFD reduced the area and diameter of perirenal and subcutaneous fat cells and increased their density. Our findings suggest that HFD rabbits had higher weight gains, accumulation of fat, and more behavioural changes than SND rabbits. Although high levels of fat in the diet had a low impact on hyperglycaemia, it could lead to hyperlipidemia and hyperthyroidism. Our results also suggest that sustained HFD may cause the proliferation of adipocytes in young female rabbits.

MiR-9-5p promotes rabbit preadipocyte differentiation by suppressing leptin gene expression.

BACKGROUND: MicroRNAs (miRNAs) are a class of small non-coding RNAs, which participate in the regulation of cell differentiation. Previous studies have demonstrated that miR-9-5p plays a key role in cancer cell development, but the mechanisms by which miR-9-5p regulates adipogenesis remain poorly understood. The present study intended to investigate its significance in producing rabbits with high-quality meat by observing the regulatory effect of miR-9-5p in preadipocytes and finding the related targets. METHODS: In this study, a dual-luciferase reporter assay was employed to validate the targeting relationship between miR-9-5p and leptin gene. We also utilized quantitative reverse transcription PCR (qRT-PCR), western blot, oil red-O staining assay, and determination of triglyceride content to analyze the regulation of miR-9-5p and leptin gene during adipocyte differentiation. RESULTS: The analysis demonstrated that during preadipocyte differentiation, miR-9-5p was up-regulated and the fat formation related biomarkers, i.e., fatty acid-binding protein 4 (FABP4), CCAAT-enhancer binding protein α (C/EBPα), and peroxisome proliferator activated receptor γ (PPARγ) were also up-regulated. Meanwhile, the oil red-O staining assay revealed that the accumulation of lipid droplets increased. We also explored the expression pattern and role of miR-9-5p in adipogenesis using white pre-adipocytes. The results showed that miR-9-5p was up-regulated during preadipocyte differentiation, and overexpression of miR-9-5p enhanced lipid accumulation. Furthermore, we found that the overexpression of miR-9-5p significantly up- regulated the expression of marker genes, PPARγ, C/EBPα and FABP4, and increased the protein levels of PPARγ and triglyceride content. The results suggest that miR-9-5p might be involved in the regulation of rabbit preadipocyte differentiation. We predicted that leptin is the target gene of miR-9-5p, by using bioinformatics tools and the conclusion was validated by a luciferase reporter assay. Finally, we verified that the knock-down of leptin by si-leptin promoted preadipocyte differentiation in rabbits. CONCLUSION: The results of the present study indicate that miR-9-5p regulates white preadipocyte differentiation in rabbits by targeting the leptin gene.