Expression of miR-140-5p and prediction of its target gene in human mesenchymal stem cells during adipogenic differentiation / 南方医科大学学报

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>miRNA-140-5p may play an important role by regulating its target gene LIFR during adipogenic differentiation of hMSCs.</p>

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.