稳定转染MiR-499对P19CL6细胞向心肌细胞分化的影响

小鼠miR-499基因包含在心肌重链肌球蛋白Myh7b基因的第19内含子中,并且在心肌细胞中特异表达,然而其在心肌细胞中表达的生物学功能和意义尚不清楚.利用可体外分化为心肌细胞的P19CL6细胞建立稳定表达miR-499的细胞株对研究miR-499的生物学功能具有重要意义.根据小鼠miR-499基因序列,设计PCR引物...     

Autophagy eliminates cytoplasmic β-catenin and NICD to promote the cardiac differentiation of P19CL6 cells

Autophagy plays important roles in adipogenesis and neuron development. However, how autophagy contributes to cardiac development is not well understood. The main aim of our study was to determine the association between autophagy and myocardial differentiation and its roles in this process. Using a well-established in vitro cardiomyocyte differentiation system, P19CL6 cells, we found that autophagy occurred from the early stage of cardiac differentiation. Blocking autophagy by knocking-down of autophagy-related gene Atg7 or Atg5 inhibited the cardiac differentiation of P19CL6 cells. Further investigation demonstrated that LC3 and P62 could form a complex with β-catenin and NICD, respectively, and promoted the degradation of β-catenin and NICD. Enhancing autophagy promoted the formation of complex, whereas blocking autophagy attenuated the degradation of β-catenin and NICD. Taken together, autophagy could facilitate P19CL6 cells to complete the cardiac differentiation process through blocking Wnt and Notch signaling pathways.     

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose‐dependent manner

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real‐time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α‐MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL‐2‐antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA‐6 or peroxisome proliferator‐activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α‐MHC, and GATA‐4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.     

Cyclosporin A Induces Cardiac Differentiation but Inhibits Hemato-Endothelial Differentiation of P19 Cells

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.     

Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6

Background

Regulating cardiac differentiation to maintain normal heart development and function is very important. At present, biological functions of H19 in cardiac differentiation is not completely clear.

Methods

To explore the functional effect of H19 during cardiac differentiation. Expression levels of early cardiac-specific markers Nkx-2.5 and GATA4, cardiac contractile protein genes α-MHC and MLC-2v were determined by qRT-PCR and western lot. The levels of lncRNA H19 and miR-19b were detected by qRT-PCR. We further predicted the binding sequence of H19 and miR-19b by online softwares starBase v2.0 and TargetScan. The biological functions of H19 and Sox6 were evaluated by CCK-8 kit, cell cycle and apoptosis assay and caspase-3 activity.

Results

The expression levels of α-MHC, MLC-2v and H19 were upregulated, and miR-19b was downregulated significantly in mouse P19CL6 cells at the late stage of cardiac differentiation. Biological function analysis showed that knockdown of H19 promoted cell proliferation and inhibits cell apoptosis. H19 suppressed miR-19b expression and miR-19b targeted Sox6, which inhibited cell proliferation and promoted apoptosis in P19CL6 cells during late-stage cardiac differentiation. Importantly, Sox6 overexpression could reverse the positive effects of H19 knockdown on P19CL6 cells.

Conclusion

Downregulation of H19 promoted cell proliferation and inhibited cell apoptosis during late-stage cardiac differentiation by regulating the negative role of miR-19b in Sox6 expression, which suggested that the manipulation of H19 expression could serve as a potential strategy for heart disease.

     

Upregulations of Gata4 and oxytocin receptor are important in cardiomyocyte differentiation processes of P19CL6 cells

Oxytocin induces P19 cells to differentiate into cardiomyocytes possibly through the oxytocin/oxytocin receptor system. We added oxytocin to the growth medium of P19CL6, a subline of P19, but they did not differentiate into cardiomyocytes as indicated by RT-PCR and Western blotting results. During the cardiac commitment time of P19CL6 cells, the mRNA expression levels of the oxytocin receptor were upregulated by the addition of oxytocin as well as DMSO, but an upregulation of Gata4 expression levels was only observed for the cells induced by DMSO. The in silico analysis of the upstream sequence of the oxytocin receptor predicted putative binding sites for Gata4 and Nkx2.5. These results suggest that upregulations of the oxytocin receptor and Gata4 are important for cardiomyocyte differentiation processes.     

Nulp1基因抑制P19CL6细胞向心肌细胞分化

Nulp1基因是已克隆的一个新的bHLH转录因子亚家族成员.前期的研究表明：Nulp1蛋白作为一个转录抑制子对SRF信号途径有极强的抑制作用.为了进一步研究Nulp1基因在心肌分化中的作用,在能够高效分化为心肌细胞的P19CL6细胞系中过表达Nulp1基因,发现心肌分化标志基因的表达被抑制;用RNA干扰技术,使Nulp1基因在P19CL6细胞系中的内源表达降低,发现心肌分化标志基因的表达提高,说明Nulp1基因能够抑制P19CL6细胞系向心肌细胞的分化.     

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ(1)

Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a β-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However, Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/β-catenin induced gene in myoblast cell fate determination.     

In vitro manipulation of human anti-DNA antibody production by anti-idiotypic antibodies conjugated with neocarzinostatin

Anti-DNA Id, 0-81, consist of 5 to 51% of Id in human anti-ssDNA antibodies; NE-1-Id shares 2 to 20% of those in anti-dsDNA antibodies. Thus, both 0-81-Id and NE-1-Id are of the cross-reactive Id that are commonly present among anti-DNA antibodies. In order to manipulate the production of anti-DNA antibodies by human PBL, we used mouse antiidiotypic mAb or those conjugated with a cytotoxic agent, neocarzinostatin. Treatment with the conjugates caused profound suppression of anti-ssDNA and anti-dsDNA antibody synthesis related to 0-81- and NE-1-Id. This was attributed to the specific killing of the clones bearing anti-DNA Id among the lymphocytes, evidenced by the indirect rosette formation tests. The Id-mediated suppression was not solely due to selective elimination of Id-positive B cells, because 50 to 92% of anti-DNA antibodies were suppressed by treatment with the conjugates. This was supported by flow cytometry analysis that showed a decrease of anti-Id-reactive cells when T cells were treated with the conjugates. This method, then, will permit an analysis of the question as to whether T cells reactive to anti-idiotypic antibodies might participate in the regulatory mechanism for anti-DNA production and, in addition, may lead to a new therapy for SLE.