ABSTRACT
Purpose To analyze the effects of full length and N-terminal fragment of serum response factor (SRF-Full and SRF-N) on TGF-β1-induced differentiation in c-Kit + cardiac stem cells (CSC).Methods Rat SRF-Full and SRF-N (1-254 aa) coding sequences were obtained from cDNA library and cloned into the linearized lentviral vector GV358 (Ubi-MCS3FLAG-SV40-EGFP-IRES-puromycin) to generate the recombinant vectors,and then positive clones were selected and sequenced after transducing the competent cells with recombinant vectors.The recombinant lentvirus were packaged through transfecting the HEK293T cells with SRF-Full,SRF-N overexpressing plasmids and viral packaging plasmids.Neonatal SD rat cKit + CSCs were isolated via magnetic activated cell sorting,and TGF-β1-induced differentiation in SRF-Full and SRF-N overexpression virus-infected CSCs was assessed by quantitative PCR.Results SRF-Full and SRF-N coding sequences were successfully obtained and properly cloned into the linearized GV358.The positive clones were selected and further confirmed by sequencing.With the help of packaging plasmids,the SRFFull and SRF-N overexpressing plasmids-transfected HEK293T cells successfully produced the lentiviral particles with the titer of 2 × 108 TU/mL,and the SRF-Full-Flag and SRF-N-Flag fusion protein were detected by Western blot in virus-infected HEK293T cells.Addition of TGF-β1 significantly induced upregulated mRNAs in cardiomyocyte markers (Nkx2.5,Gata4,cTnI) and smooth muscle cell marker (SM22α) but not the epithelia cell marker (vWF) in CSCs.Overexpression of SRF-Full facilitated TGF-β1-triggered cardiomyocyte differentiation.However,SRF-N exerted anti-differentiation effects in TGF-β1-treated cells.Conclusion The SRF-Full and SRF-N overexpressing recombinant lentiviral vectors are successfully constructed.SRF-Full facilitates while SRF-N suppresses TGF-β1-induced cardiomyocyte differentiation in c-Kit + CSCs.
ABSTRACT
OBJECTIVE@#To explore the role of proto-oncogene Pim-1 in the proliferation and migration of nasopharyngeal carcinoma (NPC) cells.@*METHODS@#Pim-1 expressions in NPC cell lines CNE1, CNE1-GL, CNE-2Z and C666-1 were examined by RT-PCR, western blotting and immunoflucesence, respectively. After CNE1, CNE1-GL and C666-1 cells were treated with different concentrations of Pim-1 special inhibitor, quercetagetin, the cell viability, colony formation rate and migration ability were analyzed.@*RESULTS@#Pim-1 expression was negative in well-differentiated CNE1 cells, whereas expressed weakly positive in poor-differentiated CNE-2Z cells and strongly positive in undifferentiated C666-1 cells. Interestingly, CNE1-GL cells that derived from CNE1 transfected with an Epstein Barr virus latent membrane protein-1 over-expression plasmid displayed stronger expression of Pim-1. Treatment of CNE1-GL and C666-1 cells with quercetagetin significantly decreased the cell viability, colony formation rate and migration ability but not the CNE1 cells.@*CONCLUSIONS@#These findings suggest that Pim-1 overexpression contributes to NPC proliferation and migration, and targeting Pim-1 may be a potential treatment for anti-Pim-1-expressed NPCs.