ABSTRACT
Objective To investigate the effects of ZNF331 overexpression on proliferation and apoptosis of human colon cancer cell HCT116, and the relevant apoptotic mechanism.Methods The lentivirus vector of overexpressed ZNF331,Flag-pLV-Neo-ZNF331,was constructed and packaged.HCT116/p53 +/+(wild type p53)and HCT116/p53 -/-(deficient p53)cells were infected.Clones with ZNF331 overexpression were identified by Western blotting.Cell proliferation assay,colony formation assay and flow cytometry analysis were used to examine the effects of ZNF 331 on cell proliferation and apoptosis.Immunoprecipitation,luciferase reporter gene assay and real-time PCR were performed to detect interactions between ZNF331 and p53, p53 transcriptional activity and the expression of p 53 apoptotic target genes, respectively.Results The lentivirus vector of overexpressed ZNF 331 was successfully generated.Stable clones of ZNF331 overexpression were established.ZNF331 showed no significant effect on cell proliferation of HCT 116/p53 +/+, but inhibited cell proliferation of HCT116/p53 -/-(P<0.01).ZNF331 could interact with p53,dose-dependently inhibit the transcriptional activity of p53 and downregulate the mRNA levels of pro-apoptotic p53 target genes, Puma and p53AIP1 (P<0.05).ZNF331 could suppress p53-induced apoptosis(P <0.01).Conclusion The influence of ZNF331 overexpression on colon cancer cell proliferation is dependent on p 53 status.ZNF331 overexpression can suppress colon cancer cell apoptosis by interacting with p 53 and inhibiting the transcriptional activity of p 53.
ABSTRACT
Objective: To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC). Methods: The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively. The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture, and the P188 was added to induction medium, The cells were detected for morphology, surface marker, viability, and number on day 14. Results: In the two-dimensional (2D) culture, CD41(+), CD41(+)/CD61(+), CD61(+) megakaryocytic numbers increased significantly after adding P188 (all P<0.01). And in the 3D culture of adding P188, the cell volume became larger and the nuclear shape was irregular, the cytoplasm appeared magenta granules, and the megakaryocyte cells became more mature. By 3D culture, the expression of CD41/CD61 was (36.30±1.27)% vs (23.95±1.34)%, hence the differentiation for MPC was significantly higher than that in the 2D group (P<0.01). Furthermore, adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45±1.20)%]. There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188, 2D and 3D culture groups (all P>0.05). Conclusion: 3D culture was beneficial for the differentiation of MPC, but the cell viability was lower than 2D group; However, the satisfied cell growth and better induction efficiency were obtained by adding of P188, which might provide a new method of megakaryocytes production for clinical application.