| Objective:Silenced the expression of SPAG6 in SKM-1 cell line by p GC-SPAG6-sh RNA recombinant lentiviral vector and constructed mouse xenograft tumor model,in order to clarify the influence of SPAG6 expression difference and decitabine treatment on cell apoptosis and PTEN gene promoter methylation in vitro and in vivo.Methods:1.SKM-1 cells were transfected with lentiviral vector to construct SPAG6-/-cell model: The sequence of lentiviral vector was determined according to the previous research of our group.3 days after transfection,the expression of green fluorescence was obserbed by fluorescence microscopy and the transfection rate was detected by flow cytometry.2.To investigate the effects of SPAG6-silencing on PTEN expression and promoter methylation: MSP was used to detect the promoter methylation of PTEN in five MDS/leukemia cell lines and in the SPAG6-silencing and-overexpression groups.Expression of PTEN and methylation related genes were detected by western blot and q RT-PCR in the group of SPAG6-silencing and negative control.3.To explore the effects of SPAG6-silencing and hypomethylating agent decitabine on the growth of SKM-1 cells: SKM-1 cells were treated with different concentrations of decitabine and histone deacetylase inhibitor LBH589,and then the survival rate of SKM-1 cells was detected by CCK8.the apoptosis rate of SKM-1 cells was detected by flow cytometry,and the expression of apoptosis-related proteins was detected by western blot.4.The effect of decitabine treatment and SPAG6-silencing on promoter methylation of PTEN in SKM-1 cells: Western blot was used to detect the expression level of PTEN and methylation related proteins after different concentrations of decitabine treatment,and MSP was used to detect promoter methylation of PTEN in different groups.5.To detect the antitumor and demthylation effects of SPAG6-silencing and decitabine treatment in vivo: Constructed NOD/SCID xenograft tumor model and observe the tumor growth three times a week.Immunohistochemical was used to detect the expression of SPAG6,PTEN and DNMT1,and western blot was used to detect the expression of PTEN,DNMT1,DNMT3 A,DNMT3B,Caspase3 and Cleaved Caspase3.The methylation level of PTEN promoter was detected by MSP.Results:1.SKM-1 cell model of SPAG6-/-was successfully constructed,and a large number of green fluorescence expressions were observed under fluorescence microscopy 3 days after transfection.Meanwhile,the transfection rate of NC-sh RNA group and SPAG6-sh RNA group was more than 80% according to flow cytometry.Western blot and q RT-PCR results showed that the protein and m RNA levels of SPAG6 in the interference group were lower than those in the control group,indicating that SPAG6 was successfully silenced.2.The expressions of PTEN protein and m RNA levels in the interference group in SKM-1 cells were up-regulated in the SPAG6-sh RNA group,and the expressions of DNMT1、DNMT3A、DNMT3B and MBD2 were also decreased.MSP results suggested that PTEN promoter was hypermethylated in K562,THP-1,HL60,HEL and SKM-1 cell lines,and SPAG6 silencing in SKM-1 could reduce PTEN promoter methylation to a certain extent.3.DAC and LBH589 were used to treat SKM-1 cells at different concentrations,CCK8 results suggested that DAC and LBH589 could reduce the cell survival rate.Flow cytometry was used to detect the cell apoptosis rate,and DAC and LBH589 could induce cell apoptosis.Besides,apoptosis rate induced by DAC/LBH589 may increase in a concentration dependent manner.Western blot results suggest DAC treatment could increase the expressions of Caspase-3,Cleaved Caspase-3,Cleaved PARP and Bad,whereas the expression of Bcl2 was downregulated,and PARP expression showed no difference.Moreover,SPAG6-silencing could increase the apoptosis-inducing effect of DAC.4.SKM-1 cell was treated with DAC at different concentrations,and western blot was used to analyse the expression levels of proteins.The results indicated that PTEN expression was increase whereas the expressions of DNMT1,DNMT3 A and DNMT3 B were reduced.MSP showed that LBH589 treatment has no effect on PTEN promoter methylation while DAC treatment alone or in combination with LBH589 could reduce the methylation of PTEN promoter obviously.At the same time,SPAG6-silencing and LBH589 could increase the effec of DAC in reducing DNMT expression.Taken together,these results showed that LBH589 and SPAG6 knockdown could enhance DAC-induced demethylation on PTEN promoter.5.The tumor tissue of xenograft mice was isolated and the tumor volume was calculated.The results indicated that the tumor volume of the interference group was smaller than that of the negative control group,while the tumor volume of the DAC-treated group was smaller than that of the saline group.Immunohistochemical results indicated that the expression of PTEN was increased while DNMT1 was decreased in the decitabine treatment and interference groups compared with the control group.TUNEL suggested that the decitabine treatment and interference groups could increase the rate of apoptosis in situ in tumor tissues.However,the results of Estern Blot showed that the expressions of Caspase3,Cleaved Caspase3 and PTEN were increased,while the expressions of DNMT1,DNMT3 A and DNMT3 B were decreased in the DAC treatment group.MSP results in mice tumor tissue suggested that methylation level of PTEN promoter could be reduced by DAC,and SPAG6-silencing could increase demethylation effect of DAC in mice.Conclusion: SPAG6-silencing could increase the expression of tumor suppressor gene PTEN,and DAC treatment could increase the apoptosis of SKM-1 cells and reduce the promoter methylation level of PTEN.However,the pro-apoptosis and PTEN demethylation effects of SPAG6 combined with DAC treatment were significantly better than those of the two treatments alone.Moreover,SPAG6-silencing combined with DAC treatment showed stronger anti-tumor properties in vivo.The results suggested that SPAG6-silencing combined with DAC may provide a new idea for the treatment of MDS. |
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