Effects of leukemia inhibitory factor receptor on the adipogenic differentiation of human bone marrow mesenchymal stem cells.

Leukemia inhibitory factor (LIF) modulates various biological processes. Although previous studies have described the effects of LIF on adipocyte differentiation, the role of LIF receptor (LIFR) on adipocyte differentiation remains unclear. Using reverse transcription­quantitative PCR (RT­qPCR), LIFR expression was demonstrated to increase during adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), indicating that LIFR may be involved in this process. To further evaluate the association between LIFR and adipogenic differentiation, lentivirus­mediated LIFR knockdown was performed in hMSCs. Cells were divided into two groups: Negative control group and LIFR­knockdown group. During the adipogenic differentiation process, intracellular lipid accumulation was assessed with Oil Red O staining at various time points (days 3, 6 and 9). Additionally, the mRNA and protein expression levels of LIF, LIFR and three molecular indicators of adipogenesis, peroxisome proliferator­activated receptor Î³ (PPARγ), CCAAT enhancer binding protein α (C/EBPα) and fatty acid binding protein 4 (FABP4/aP2), were assessed by RT­qPCR and western blotting. The culture supernatant was collected to evaluate the concentration of LIF using ELISA. The present results suggested that LIFR expression progressively increased during adipogenic differentiation of hMSCs. Conversely, LIFR knockdown significantly suppressed this process. Additionally, PPARγ, C/EBPα and aP2 were inhibited following LIFR knockdown. In contrast with LIFR, the expression levels of LIF were significantly decreased after the initiation of adipogenic differentiation. Therefore, the expression levels of LIF and LIFR exhibited opposite trends. Collectively, the present results suggested that LIFR promoted adipogenic differentiation, whereas LIF may negatively regulate this process.

Curcumin represses adipogenic differentiation of human bone marrow mesenchymal stem cells via inhibiting kruppel-like factor 15 expression.

Understanding the mechanisms of adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. The natural plant polyphenol compound curcumin can improve obesity-associated inflammation and diabetes in obese mice. The role of curcumin in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) is still unclear. We used hMSCs to investigate the details of the mechanism underlying the adipogenic effects of curcumin. At different time points (i.e., 5 days and 10 days) of hMSC adipocyte differentiation, an accumulation of large lipid droplets was analyzed in Oil Red O-stained cultured cells in two curcumin (5 µM and 10 µM) groups and the control group. The cells were also harvested for the detection of mRNA and protein expressions by quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that curcumin can suppresses adipocyte differentiation in a dose-dependent manner and inhibited the expression of PPARγ, C/EBPα, and FABP4. Importantly, curcumin can also suppress the expression of Kruppel-like factor 15, which may bind to the PPARγ promoter, resulting in downregulation of PPARγ expression to inhibit the adipogenic differentiation of hMSCs.

miR-377-3p regulates adipogenic differentiation of human bone marrow mesenchymal stem cells by regulating LIFR.

MicroRNAs are members of the family of non-coding small RNAs that regulate gene expression either by inhibiting mRNA translation or by promoting mRNA degradation at the post-transcriptional level. They play an important role in the differentiation of human bone marrow mesenchymal stem cells (hMSCs) into adipocytes. However, the role of microRNAs in this process remains to be poorly understood. Here, we observed that miR-377-3p expression was markedly decreased during adipogenic differentiation of hMSCs. Overexpression of miR-377-3p decreased adipocyte differentiation and downregulated the expression of adipogenic markers. Meanwhile, bioinformatics-based studies suggested that LIFR is a target of miR-377-3p. Further analysis confirmed that expression of LIFR present markedly increased during adipogenic differentiation of hMSCs. In addition, downregulation expression of LIFR significantly inhibited the process of adipocyte differentiation. To confirm the relation between miR-377-3p and LIFR, luciferase reporter assays were carried out. The results indicated that miR-377-3p bound directly to the 3'-untranslated region of LIFR. These data indicate that miR-377-3p suppressed adipogenesis of hMSCs by targeting LIFR, which provides novel insights into the molecular mechanism of miRNA-mediated cellular differentiation.

[Construction of Vectors Expressing Inhibiting Peptides for Nuclear Import and Its Effect on Growth and Migration of HeLa Cell].

OBJECTIVE: To construct the expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import (Bimax),and explore the location of Bimax and its potential effects on cell proliferation and migration in HeLa cells. METHODS: Two kinds of polynucleotide encoding inhibiting peptides for nuclear import were synthesis respectively and subsequently annealed for inserting into vector pDs-Red-C1. The recombinant plasmids were transfected into competent bacterial DH-5α. After transfection,the positive bacteria were picked up for DNA sequencing. The recombinant plasmids pDs-Red-Bimax2,pDs-Red-Bimax1 and negative plasmid pDs-Red-C1 were transfected into HeLa cells respectively according to Lipofectamine2000 protocol. After transfection,the expression and location of red fluorescent protein were observed with fluorescence microscope. Furthermore,MTT assay and cell-migration assay were used to detect the proliferation and migration of Bimax transducted cells. RESULTS: DNA sequencing showed that the polynucleotides encoding Bimax1 or Bimax2 were inserted into pDs-Red-C1 vector successfully. After transfected into HeLa cells,the inhibiting peptide induced red fluorescent protein locating in nuclear. Furthermore,either the fusion protein RFP-Bimax1 or RFP-Bimax2 can suppress the proliferation and migration of HeLa cells. CONCLUSION: The expression vectors for red fluorescent protein fused with inhibiting peptides for nuclear import were successfully constructed. In addition,the fusion proteins were expressed and located in nuclear and suppressed the proliferation and migration of tumor cells.

Gene expression profiling of human bone marrow-derived mesenchymal stem cells during adipogenesis.

INTRODUCTION: Adipogenesis comprises multiple processes by which mesenchymal stem cells differentiate into adipocytes. To increase our knowledge of the mechanism underlying adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs), we performed full-genome gene expression microarray and gene ontology analyses of induced differentiation of hMSCs. MATERIAL AND METHODS: Adipogenic differentiation of hMSCs was induced by an adipogenic medium, and total RNA was extracted from undifferentiated hMSCs (day 0) and differentiated adipocytes (day 14). Then microarray hybridization of RNA samples was performed. The GeneChip Operating Software was used to analyze the hybridization data to identify differentially expressed genes, which were performed Gene Ontology categorization and pathway analysis. Pathway-act-network and genes-act-network were built according to the Kyoto Encyclopedia of Genes and Genomes database. Some differentially expressed genes were subjected to qRT-PCR to verify the microarray data. RESULTS: We detected a total of 3,821 differentially expressed genes, of which 753 were upregulated and 3,068 downregulated. These genes were well represented in a variety of functional categories, including collagen fibril organization, brown fat cell differentiation, cell division, and S phase of mitotic cell cycle. Subsequently, pathway analysis was conducted, and significant pathways (from top 50) were selected for pathway-act-network analysis, which indicated that the mitogen-activated protein kinase (MAPK) pathway and cell cycle were of high degrees (> 10). Gene-act-network analysis showed that insulin-like growth factor 1 receptor (IGF1R), histone deacetylase 1 (HDAC1), HDAC2, MAPK13, MAPK8, phosphoinositide-3-kinase regulatory subunit 1 (PI3KR1), and PI3KR2 also had high degrees (> 18). CONCLUSIONS: Collectively, these data provide novel information and could serve as a basis for future study to clarify the mechanisms underlying adipocyte differentiation of hMSCs.

[Expression of miR-140-5p and prediction of its target gene in human mesenchymal stem cells during adipogenic differentiation].

OBJECTIVE: To screen the differentially expressed miRNAs and their target genes in adipogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) to better understand the mechanism for regulating the balance between osteoblast and adipocyte differentiation. METHODS: Cultured hMSCs were induced for adipogenic differentiation, and at 0, 7, 14, and 21 days of induction, the cells were examined for miRNA and mRNA expression profiles using miRNA chip and transcriptome sequencing (RNA-seq) techniques. Correlation analysis was carried out for the miRNAs and mRNAs of potential interest. The databases including TargetScan, PicTar and miRanda were used to predict the target genes of the differentially expressed miRNA. RESULTS: The expression of miR-140-5p was down-regulated and leukemia inhibitory factor receptor (LIFR) expression increased progressively during adipogenic differentiation of hMSCs, showing a negative correlation between them. Target gene prediction using the 3 databases identified LIFR as the target gene of miR-140-5p. CONCLUSION: miRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.