Xianwei Cui

Adipogenic miRNA and meta-signature miRNAs involved in human adipocyte differentiation and obesity

MicroRNAs (miRNAs) have been identified as a new class of regulatory molecules that influence many biological functions, including metabolism, adipocyte differentiation. To determine the role of adipogenic miRNAs in the adipocyte differentiation process, we used microarray technology to monitor miRNA levels in human adipose-derived mesenchymal stem cells (hMSCs-Ad), human stromal vascular cells (SVCs) and differentiated adipocytes. 79 miRNAs were found to be differentially expressed, most of which are located in obesity related chromosomal regions but have not been previously linked to adipocyte differentiation process. A systematic search was made for relevant studies in academic data bases, involving the Gene Expression Omnibus (GEO) ArrayExpress, Pubmed and Embase database. Eight studies on human adipocyte differentiation or obesity were included in the final analysis. After combining our microarray data with meta-analysis of published microarray data, we detected 42 differently expressed miRNAs (meta-signature miRNAs) in mature adipocytes compared to SVCs or hMSCs-Ad. Our study shows meta-signature miRNAs specific for adipogenesis, several of which are correlated with key gene targets demonstrating functional relationships to pathways in BMP signaling pathway, Cell differentiation, Wnt signaling, insulin receptor signaling pathway, MAPK signaling, Cell cycle and lipid metabolic process. Our study shows that the first evidence of hsa-let-7 family, hsa-miR-15a-5p, hsa-miR-27a-3p, hsa-miR-106b-5p, hsa-miR-148a-3p and hsa-miR-26b-5p got a great weight in adipogenesis. We concluded that meta-signature miRNAs involved in adipocyte differentiation and provided pathophysiological roles and novel insight into obesity and its related metabolic diseases.

Distinct expression profiles of LncRNAs between brown adipose tissue and skeletal muscle

Both brown adipose tissue and skeletalmuscle have abundant mitochondria and energy consumption capacity. They are similar in origin and gain different potential of energy metabolism after differentiation and maturation. The mechanism that cause the difference is not yet fully understood. Long non-coding RNAs (lncRNAs) which comprise the bulk of the human non-coding transcriptome have been proved to play key roles in various biological processes. Whether they will have a function on the differentiation and energy metabolism between BAT and skeletalmuscle is still unknown. To identify the cellular long noncoding RNAs (lncRNAs) involved in the progress, we used the next generation transcriptome sequencing and microarray techniques, and investigated 704 up-regulated and 896 down-regulated lncRNAs (fold-change >3.0) in BAT by comparing the expression profile. Furthermore, we reported AK003288 associated with junctophilin 2 (Jph2) gene which may affect energy metabolism. This study show distinct expression profiles of LncRNAs between brown adipose tissue and skeletal muscle which provide information for further research on differentiation of adipocyte and transdifferentiation between BAT and skeletalmuscle that will be helpful to find a new therapeutic target for combatting obesity.

Differential lncRNA expression profiles in brown and white adipose tissues

Long non-coding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. It can serve as key co-activators of proteins involved in transcriptional regulation. Studies have found that white and brown adipocytes both originate from the mesoderm. However, it remains unclear whether lncRNAs function during adipogenesis or in energy metabolism in brown adipose tissue (BAT) and white adipose tissue (WAT). In this study, we used lncRNA microarray technology to evaluate differences in the lncRNA expression profiles of WAT and BAT. We observed 735 up-regulated and 877 down-regulated lncRNAs (fold change >4.0). To reveal the potential functions of these lncRNAs, we applied GO and pathway analyses to study the differentially expressed lncRNAs. We found that AK142386 and AK133540 may affect adipogenesis and metabolism. Our data indicate that AK142386 and AK133540 may be involved in BAT and WAT development through their target genes Hoxa3 and Acad10. Together, we have identified numerous lncRNAs and these lncRNAs can potentially serve as a required component for proper adipogenesis.

FFAs and adipokine-mediated regulation of hsa-miR-143 expression in human adipocytes

Accumulating evidence suggests that microRNAs (miRNAs) play an important role in regulating the pathways in adipose tissue that control processes such as adipogenesis, insulin resistance, and inflammation. MiR-143 is a well-characterized miRNA involved in adipogenesis and may be involved in regulating insulin resistance. Free fatty acids (FFAs) and adipokines, such as tumor necrosis factor-α (TNF-α), leptin, resistin, and interleukin-6 (IL-6), have already been identified as main regulators of obesity and insulin sensitivity. Therefore, we studied the effects of these inflammatory cytokines on the expression of miR-143. FFAs, resistin, and leptin downregulated miR-143 expression in human adipocytes, whereas TNF-α and IL-6 had little effect on miR-143 expression. These results suggest that the expression of miR-143 is affected by a variety of factors that are related to insulin sensitivity. Therefore, miR-143 may be an important mediator in the development of obesity-related insulin resistance.

DNA methylation profiles in placenta and its association with gestational diabetes mellitus

Emerging evidences indicate that placenta plays a critical role in gestational diabetes mellitus (GDM). DNA methylation could be associated with altered placental development and functions. This study is to uncover the genome-wide DNA methylation patterns in this disorder. DNA methylation was measured at >385,000 CpG sites using methylated DNA immunoprecipitation (MeDIP) and a huamn CpG island plus promoter microarray. We totally identified 6,641 differentially methylated regions (DMRs) targeting 3,320 genes, of which 2,729 DMRs targeting 1,399 genes, showed significant hypermethylation in GDM relative to the controls, whereas 3,912 DMRs targeting 1,970 genes showed significant hypomethylation. Functional analysis divided these genes into different functional networks, which mainly involved in the pathways of cell growth and death regulation, immune and inflammatory response and nervous system development. In addition, the methylation profiles and expressions of 4 loci (RBP4, GLUT3, Resistin and PPARα) were validated by BSP for their higher log2 ratio and potential functions with energy metabolism. This study demonstrates aberrant patterns of DNA methylation in GDM which may be involved in the pathophysiology of GDM and reflect the fetal development. Future work will assess the potential prognostic and therapeutic value for these findings in GDM.

Molecular cloning, expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442bp and contained an open reading frame of 780bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.

Peptidome analysis of human milk from women delivering macrosomic fetuses reveals multiple means of protection for infants

Breastfeeding is associated with a lower incidence of obesity, diabetes, and cardiovascular disease later in life. While macrosomic infants have a higher risk of developing obesity and other metabolic disorders. Breast milk may contain special nutrients to meet the different growth needs of different infants. Whether mothers make breast milk different to meet the requirement of macrosomic infants is still unknown. Here, we conducted a comparison between mothers delivering macrosomic and non-macrosomic infants in colostrum endogenous peptides. More than 400 peptides, originating from at least 34 protein precursors, were identified by Liquid Chromatography/Mass Spectrometry (LC/MS). Out of these, 29 peptides found to be significant differently expressed (|fold change| ≥ 3, P < 0.01). Blastp analysis revealed 41 peptides may have established biological activities, which exhibit immunomodulating, antibacterial action, antioxidation, opioid agonist and antihypertensive activity. Furthermore, we found that peptide located at β-Casein 24-38 AA has antimicrobial effect against E. coli, Y. enterocolitica and S. aureus. While, κ-Casein 89-109 AA-derived peptide plays as a regulator of preadipocyte proliferation. The profile of endogenous peptides from macrosomic term infants is different from non-macrosomic terms. This different peptide expression potentially has specific physiological function to benefit macrosomic infants. Finally, we believe that our research is a meaningfull finding which may add to the understanding of milk peptide physiological action.

Identification of interferon-γ-inducible-lysosomal thiol reductase (GILT) gene from Mefugu (Takifugu obscures) and its immune response to LPS challenge

Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a Mefugu cDNA (ToGILT) encodes a deduced protein of 242 amino acids with a putative molecular weight of 28.6 kDa. It contains typical features of GILT proteins including the signature sequence CQHGX2ECX2NX4C, CXXC motif and other five cysteines. Genomic analysis revealed that ToGILT gene exhibited a similar exon-intron organization to human and mouse GILT. Phylogenetic analysis showed that ToGILT derived from a common ancestor with other vertebrate GILT proteins. The ToGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in spleen and kidney after LPS induction. These results suggest that ToGILT may be involved in the immune response to bacteria challenge in Takifugu obscurus.

Molecular and biological characterization of interferon-γ-inducible-lysosomal thiol reductase gene in zebrafish (Danio rerio).

In mammals, interferon-γ-inducible-lysosomal thiol reductase (GILT) has been demonstrated to play a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction, thus unfolding native protein Ag and facilitating subsequent cleavage by proteases. Here, we reported the cloning of a GILT gene homologue from zebrafish (zGILT), a tropical freshwater fish. The full-length cDNA of zGILT gene is 768 nucleotides (nt) encoding a protein of 255 amino acids (aa), with a putative molecular weight of 28.33 kDa. The deduced protein is highly homologous to that of fish and mammalian GILTs and shares 57.1% sequence identity to that of Atlantic salmon and 55.7-21.6% sequence identity to that of various mammals. The deduced protein possesses all the main features characteristic of known GILT proteins including the signature sequence CQHGX2ECX2NX4C spanning residues 117-132, CXXC motif at residues 72-75, one potential sites for N-linked glycosylation at residual positions 54. The zGILT expression is obviously up-regulated in spleen and kidney after immunization with LPS although it also is constitutively expressed in heart, liver, muscle and intestine, suggesting that zGILT may be involved in the immune response to bacterial challenge. The soluble recombinant protein was successfully purified using Ni-nitrilotriacetic acid resin. Recombinant His-zsGILT appeared on SDS-PAGE in the ranges of their estimated size of 18.94-kDa. After purification, further study revealed that zsGILT was capable of catalyzing the reduction of the interchain disulfide bonds intact IgG. These results will allow for further investigation to unravel the role of this key enzyme in class II MHC-restricted antigen processing and to use zebrafish as an in vivo model for related studies.

Change in circulating microRNA profile of obese children indicates future risk of adult diabetes

PURPOSE Childhood obesity increases susceptibility to type 2 diabetes (T2D) in adults. Circulating microRNAs (miRNAs) in serum have been proposed as potential diagnostic biomarkers, and they may contribute to the progression toward T2D. Here, we investigated the possibility of predicting the future risk of adult T2D in obese children by using circulating miRNAs. BASIC PROCEDURES We performed miRNA high-throughput sequencing to screen relevant circulating miRNAs in obese children. The expression patterns of targeted miRNAs were further explored in obese children and adults with T2D. To investigate the underlying contributions of these miRNAs to the development of T2D, we detected the impacts of the candidate miRNAs on preadipocyte proliferation, insulin secretion by pancreatic β-cell, and glucose uptake by skeletal muscle cells. MAIN FINDINGS Three miRNAs (miR-486, miR-146b and miR-15b), whose expression in the circulation was most dramatically augmented in obese children and adult T2D patients, were selected for further investigation. Of these 3 miRNAs, miR-486 was implicated in accelerating preadipocyte proliferation and myotube glucose intolerance, miR-146b and miR-15b were engaged in the suppression of high concentration glucose-induced pancreatic insulin secretion, and they all contributed to the pathological processes of obesity and T2D. PRINCIPAL CONCLUSIONS Our results provide a better understanding of the role of circulating miRNAs, particularly miR-486, miR-146b and miR-15b, in predicting the future risk of T2D in obese children.