Yahui Shen

The role of microRNA-26b in human adipocyte differentiation and proliferation

Recent findings indicate that microRNAs (miRNAs) are involved in the regulatory network of adipogenesis and obesity. Thus far, only a few human miRNAs are known to function as adipogenic regulators, fanning interest in studies on the functional role of miRNAs during adipogenesis in humans. In a previous study, we used a microarray to assess miRNA expression during human preadipocyte differentiation. We found that expression of the miR-26b was increased in mature adipocytes. MiR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). Target prediction and Renilla luciferase analyses revealed the phosphatase and tensin homolog gene (PTEN) as a putative target gene. In this study, we found that miR-26b was gradually upregulated during adipocyte differentiation. To understand the roles of miR-26b in adipogenesis, we adopted a loss-of-function approach to silence miR-26b stably in human preadipocytes. We found that miR-26b inhibition effectively suppressed adipocyte differentiation, as evidenced by decreased lipid droplets and the ability of miR-26b to decrease mRNA levels of adipocyte-specific molecular markers and triglyceride accumulation. Furthermore, the cell growth assay revealed that miR-26b inhibition promoted proliferation. Nevertheless, it had no effect on apoptosis. Taken together, these data indicate that miR-26b may be involved in adipogenesis and could be targeted for therapeutic intervention in obesity.

Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect

Ventricular septal defects (VSD) are the most common form of congenital heart disease, which is the leading non-infectious cause of death in children; nevertheless, the exact cause of VSD is not yet fully understood. Long non-coding RNAs (lncRNAs) have been shown to play key roles in various biological processes, such as imprinting control, circuitry controlling pluripotency and differentiation, immune responses and chromosome dynamics. Notably, a growing number of lncRNAs have been implicated in disease etiology, although an association with VSD has not been reported. In the present study, we conducted an integrated analysis of dysregulated lncRNAs, focusing specifically on the identification and characterization of lncRNAs potentially involving in initiation of VSD. Comparison of the transcriptome profiles of cardiac tissues from VSD-affected and normal hearts was performed using a second-generation lncRNA microarray, which covers the vast majority of expressed RefSeq transcripts (29,241 lncRNAs and 30,215 coding transcripts). In total, 880 lncRNAs were upregulated and 628 were downregulated in VSD. Furthermore, our established filtering pipeline indicated an association of two lncRNAs, ENST00000513542 and RP11-473L15.2, with VSD. This dysregulation of the lncRNA profile provides a novel insight into the etiology of VSD and furthermore, illustrates the intricate relationship between coding and ncRNA transcripts in cardiac development. These data may offer a background/reference resource for future functional studies of lncRNAs related to VSD.

MicroRNA-375 overexpression influences P19 cell proliferation, apoptosis and differentiation through the Notch signaling pathway

Our previous study reported that microRNA-375 (miR-375) is significantly upregulated in ventricular septal myocardial tissues from 22-week-old fetuses with ventricular septal defect as compared with normal controls. In the present study, the specific effects of miR-375 on P19 cell differentiation into cardiomyocyte-like cells were investigated. Stable P19 cell lines overexpressing miR-375 or containing empty vector were established, which could be efficiently induced into cardiomyocyte-like cells in the presence of dimethyl sulfoxide in vitro. miR-375 overexpression was verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was determined according to total cell counts; cell cycle distribution and apoptosis levels were examined using flow cytometry. Apoptosis-related morphological changes were observed using Hoechst staining and fluorescence microscopy. During P19 cell differentiation, the cardiomyogenesis-related mRNAs (cardiac troponin T, GATA binding protein 4, myocyte-specific enhancer factor 2C) and mRNAs involved in the Notch signaling pathway (Notch2, Delta-like 1 and hes family bHLH transcription factor 1) were detected at days 0, 4, 6 and 10. Their differential expression was examined using RT-qPCR; the apoptosis-related genes BAX and Bcl-2 were also detected using this method. The corresponding proteins were evaluated by western blotting. Compared with the control group, miR-375 overexpression inhibited proliferation but promoted apoptosis in P19 cells, and the associated mRNAs and proteins were decreased during differentiation. miR-375 has an important role in cardiomyocyte differentiation, and can disrupt this process via the Notch signaling pathway. The present findings contribute to the understanding of the mechanisms of congenital heart disease and facilitate the development of new gene therapies.

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.

Knockdown of FABP3 impairs cardiac development in Zebrafish through the retinoic acid signaling pathway.

Fatty acid-binding protein 3 (FABP3) is a member of the intracellular lipid-binding protein family, and is primarily expressed in cardiac muscle tissue. Previously, we found that FABP3 is highly expressed in patients with ventricular-septal defects and is often used as a plasma biomarker in idiopathic dilated cardiomyopathy, and may play a significant role in the development of these defects in humans. In the present study, we aimed to investigate the role of FABP3 in the embryonic development of the zebrafish heart, and specifically how morpholino (MO) mediated knockdown of FABP3 would affect heart development in this species. Our results revealed that knockdown of FABP3 caused significant impairment of cardiac development observed, including developmental delay, pericardial edema, a linear heart tube phenotype, incomplete cardiac loop formation, abnormal positioning of the ventricles and atria, downregulated expression of cardiac-specific markers and decreased heart rate. Mechanistically, our data showed that the retinoic acid (RA) catabolizing enzyme Cyp26a1 was upregulated in FABP3-MO zebrafish, as indicated by in situ hybridization and real-time PCR. On the other hand, the expression level of the RA synthesizing enzyme Raldh2 did not significantly change in FABP3-MO injected zebrafish. Collectively, our results indicated that FABP3 knockdown had significant effects on cardiac development, and that dysregulated RA signaling was one of the mechanisms underlying this effect. As a result, these studies identify FABP3 as a candidate gene underlying the etiology of congenital heart defects.

Silencing of FABP3 promotes apoptosis and induces mitochondrion impairment in embryonic carcinoma cells

Fatty acid binding protein 3 (FABP3) (also known as H-FABP) is a member of the intracellular lipid-binding protein family, and is mainly expressed in cardiac muscle tissue. The in vivo function of FABP3 is proposed to be in fatty acid metabolism, trafficking, and cell signaling. Our previous study found that FABP3 is highly regulated in patients with ventricular septal defect (VSD), and may play a significant role in the development of human VSD. In the present study, we aimed to investigate the impact of FABP3 knockdown by RNA interference (RNAi) on apoptosis and mitochondrial function of embryonic carcinoma (P19) cells. The results revealed that downregulated FABP3 expression promoted apoptosis, and resulted in mitochondrial deformation, increased mitochondrial membrane potential (MMP), and decreased intracellular ATP synthesis. In addition, the knockdown of FABP3 also led to excess intracellular ROS production. However, there was no obvious influence on the amount of mitochondrial DNA. Collectively, our results indicated that FABP3 knockdown promoted apoptosis and caused mitochondrial dysfunction in P19 cells, which might be responsible for the development of human VSD.

Obesity‑associated microRNA‑26b regulates the proliferation of human preadipocytes via arrest of the G1/S transition

MicroRNAs (miRNAs) are short, 20‑24 nucleotide non‑coding RNAs, which are involved in multiple biological processes, including obesity. Our previous investigation revealed that miRNA (miR)‑26b is differentially expressed in preadipocytes and mature adipocytes in humans. However, its role in the proliferation of human preadipocytes remains to be fully elucidated. In the present study, intracellular lipid accumulation was assessed using oil red O staining and the trigycerlide (TG) content was quantified using a TG assay kit, adipogenesis associated genes and cyclin D2 were analyzed using western blotting, and the effects of miR‑26b on the proliferation of preadipocytes was investigated using Cell Counting Kit‑8 assays and cell cycle analysis. Human preadipocytes overexpressing miR‑26b exhibited increased TG content in the adipocytes. During differentiation, the protein expression levels of adipogenesis‑associated marker genes, including peroxisome proliferator‑activated receptor γ, CCAAT/enhancer‑binding protein α, fatty acid‑binding protein and hormone‑sensitive lipase were upregulated in cells overexpressing miR‑26b, compared with the negative control cells. In addition, growth of human preadipocytes overexpressing miR‑26b occurred a slower rate and more remained in the G1 phase, compared with the negative control cells. In addition, miR‑26b downregulated the protein expression of cyclin D2. These results demonstrated that miR‑26b promoted differentiation and, at least party by targeting cyclin D2, attenuated cell proliferation via arresting the G1/S transition.

Transplantation of iPSc Ameliorates Neural Remodeling and Reduces Ventricular Arrhythmias in a Post‐Infarcted Swine Model

Neural remodeling after myocardial infarction (MI) may cause malignant ventricular arrhythmia, which is the main cause of sudden cardiac death following MI. Herein, we aimed to examine whether induced pluripotent stem cells (iPSc) transplantation can ameliorate neural remodeling and reduce ventricular arrhythmias (VA) in a post‐infarcted swine model. Left anterior descending coronary arteries were balloon‐occluded to generate MI. Animals were then divided into Sham, PBS control, and iPS groups. Dynamic electrocardiography programmed electric stimulation were performed to evaluate VA. The spatial distribution of vascularization, Cx43 and autonomic nerve regeneration were evaluated by immunofluorescence staining. Associated protein expression was detected by Western blotting. Likewise, we measured the enzymatic activities of superoxide dismutase and content of malondialdehyde. Six weeks later, the number of blood vessels increased significantly in the iPSc group. The expression of vascular endothelial growth factor and connexin 43 in the iPS group was significantly higher than the PBS group; however, the levels of nerve growth factor and tyrosine hydroxylase were lower. The oxidative stress was ameliorated by iPSc transplantation. Moreover, the number of sympathetic nerves in the iPSc group was reduced, while the parasympathetic nerve fibers had no obvious change. The transplantation of iPSc also significantly decreased the low‐/high‐frequency ratio and arrhythmia score of programmed electric stimulation‐induced VA. In conclusion, iPSc intramyocardial transplantation reduces vulnerability to VAs, and the mechanism was related to the remodeling amelioration of autonomic nerves and gap junctions. Moreover, possible mechanisms of iPSc transplantation in improving neural remodeling may be related to attenuated oxidative stress and inflammatory response. J. Cell. Biochem. 115: 531–539, 2014.

Overexpression of TFAM Protects 3T3-L1 Adipocytes from NYGGF4 (PID1) Overexpression-Induced Insulin Resistance and Mitochondrial Dysfunction

NYGGF4, also known as phosphotyrosine interaction domain containing 1(PID1), is a recently discovered gene which is involved in obesity-related insulin resistance (IR) and mitochondrial dysfunction. We aimed to further elucidate the effects and mechanisms underlying NYGGF4-induced IR by investigating the effect of overexpressing mitochondrial transcription factor A (TFAM), which is essential for mitochondrial DNA transcription and replication, on NYGGF4-induced IR and mitochondrial abnormalities in 3T3-L1 adipocytes. Overexpression of TFAM increased the mitochondrial copy number and ATP content in both control 3T3-L1 adipocytes and NYGGF4-overexpressing adipocytes. Reactive oxygen species (ROS) production was enhanced in NYGGF4-overexpressing adipocytes and reduced in TFAM-overexpressing adipocytes; co-overexpression of TFAM significantly attenuated ROS production in NYGGF4-overexpressing adipocytes. However, overexpression of TFAM did not affect the mitochondrial transmembrane potential (ΔΨm) in control 3T3-L1 adipocytes or NYGGF4-overexpressing adipocytes. In addition, co-overexpression of TFAM-enhanced insulin-stimulated glucose uptake by increasing Glucose transporter type 4 (GLUT4) translocation to the PM in NYGGF4-overexpressing adipocytes. Overexpression of NYGGF4 significantly inhibited tyrosine phosphorylation of Insulin receptor substrate 1 (IRS-1) and serine phosphorylation of Akt, whereas overexpression of TFAM strongly induced phosphorylation of IRS-1 and Akt in NYGGF4-overexpressing adipocytes. This study demonstrates that NYGGF4 plays a role in IR by impairing mitochondrial function, and that overexpression of TFAM can restore mitochondrial function to normal levels in NYGGF4-overexpressing adipocytes via activation of the IRS-1/PI3K/Akt signaling pathway.

Silencing of FABP3 leads to apoptosis-induced mitochondrial dysfunction and stimulates Wnt signaling in zebrafish.

Fatty acid binding protein 3 (FABP3, also termed heart-type fatty acid binding protein) is a member of the intracellular lipid-binding protein family that may be essential in fatty acid transport, cell growth, cellular signaling and gene transcription. Previously, we demonstrated that FABP3 was involved in apoptosis-associated congenital cardiac malformations; however, its mechanism of regulation remains unclear. Apoptosis has increasingly been considered to be important in cardiac development. In the present study, a zebrafish model was used to investigate the involvement of FABP3‑morpholino (MO)-induced apoptosis and mitochondrial dysfunction in cardiac development. During the early stages of cardiac development, injection of FABP3‑MO into zebrafish resulted in significant impairment in cardiac development and promoted the rate of apoptosis which was correlated with significant dysfunction of the mitochondria. For example, the ATP content was markedly decreased at 24 and 48 h post-fertilization (pf), reactive oxygen species production was significantly enhanced at 24 and 48 h pf and the mitochondrial DNA copy number was reduced at 24, 48 and 72 h pf. Additionally, Nkx2.5 expression was upregulated in FABP3-MO zebrafish, and Wnt signaling molecules (Wnt1, Wnt5 and Wnt11) were also highly expressed in FABP3-MO zebrafish at 24, 48 and 72 h pf. In conclusion, the results indicated that FABP3 knockdown exhibited significant toxic effects on cardiac development and mitochondrial function, which may be responsible for the knockdown of FABP3-induced apoptosis. Apoptosis was one of the mechanisms underlying this effect, and was correlated with the activation of Wnt signaling. These studies identified FABP3 as a candidate gene underlying the etiology of congenital heart defects.