| Objective:(1)To construct the RNAi lentiviral expression vector targeting to taranscription factor protein 1(Sp1)gene and observe its inhibitory effect on Sp1 gene expression in glioma U87 cells.(2)To establish the subcutaneous glioma model in nude mice,and observe the growth of U87 cells silenced Sp1 gene in nude mice.The proliferation and apoptosis of glioma cells were detected by immunohistochemistry and flow cytometry resprectively.The expression of Sp1 and GLUT3 m RNA and protein were detected by real-time PCR and Western blot resprectively.The effect of Sp1 gene on glioma growth and its mechanism were discussed by in vivo experiment.The effect of Sp1 gene on glioma growth and its mechanism were discussed by in vivo experiment.Methods:(1)To design the sh RNA sequence of the Sp1 according to the Sp1 target gene sequence,and construct the lentiviral expression vector pLVX-sh RNA-Sp1.It was identificated by Exho I single enzyme and gene sequencing.The m RNA expression of Sp1 gene by real-time PCR.To evaluate the efficiency of interference(silencing),and select effective pLVX-sh RNA-Sp1 for transfecting into U87 cells.(2)The glioma cells were divided into three groups.The experimental group and the negative control group were transfected with the Sp1-sh RNA lentiviral vector pLVX-sh RNA-Sp1(+)and sh RNA negative control lentiviral vector-pLVX-sh RNA-Sp1(-),resprectively.The blank control group does not handle.Nude mice were divided into three groups: the experimental group,the negative control group and the blank control group,each group of 15.U87 cells infected with pLVX-sh RNA-Sp1(+),U87 cells infected with pLVX-sh RNA-Sp1(-)and uninfected U87 cells were subcutaneously inoculated subcutaneously.To establish a model of human glioma by subcutaneous implantation in nude mice,and observe the tumor growth,calculate the growth rate of the tumor.The tumor growth was measured every 4 days after the success of the tumor,and the growth curve was plotted.The nude mice were executed after 37 days,and the tumor tissue was got.(3)The expression levels of proliferation and apoptosis of tumor cells was detected by Ki-67 immunohistochemistry and Flow cytometry resprectively.The expression levels of m RNA and Protein of Sp1 and GLUT3 were measuered by Real-time PCR and Western blot resprectively.Results:(1)The results of restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid was consistent with the expected DNA sequence.The infection efficiency of U87 cells was observed in U87 cells after pLVX-sh RNA-Sp1 infection was more than 50%.The ratio of inhibition of Sp1 m RNA was 70.3% by real-time PCR.It suggested that transfection of Sp1 interference plasmid can effectively inhibit the expression of Sp1 gene in U87 cells,and the Sp1-sh RNA lentiviral expression vector,pLVX-sh RNA-Sp1,was constructed successfully.(2)The rates of tumor formation in the experimental group,the negative control group and the blank control group were 87%,93% and 100% respectively.The tumor growth curve showed that the volume of subcutaneous tumor in the experimental group was significantly smaller than that in the negative control group and the blank control group since day 13 after inoculation(P <0.05),and the volume difference between the above groups was gradually increased with the time,but it was no significant differences between the blank control group and the negative control group(p> 0.05).It suggested that Sp1 gene silencing can effectively inhibit glioma growth in nude mice.(3)Ki-67 index of tumor tissue in the experimental group,the negative control group and the blank control group were(7.359 ± 2.501)%,(37.051 ± 9.630)% and(39.498 ± 10.951)% respectively.Ki-67 index in the experimental group was significantly lower than that in the negative control group and the blank control group(p <0.05).It suggested that the proliferation of glioma cell was decreased after Sp1 gene silencing.(4)Flow cytometry showed that the apoptosis rates of tumor tissue in the experimental group,the negative control group and the blank control group were(11.23 ± 2.13)%,(4.11 ± 1.35)% and(0.12 ± 0.06)%,respectively.The apoptotic rate of the experimental group was significantly higher than that of the negative control group and the blank control group(p <0.05).It suggested that the apoptosis of glioma cells was promoted after Sp1 gene silencing.(5)Real-time PCR was used to detect the expression of Sp1 m RNA and GLUT3 m RNA.The expression levels of Sp1 m RNA and GLUT3 m RNA in the experimental group,the negative control group and the blank control group were 0.489±0.093,1.050±0.037,1.001±0.120、0.161±0.011,0.382±0.022,0.418±0.046,respectively.The expression levels of Sp1 m RNA and GLUT3 m RNA in the experimental group were lower than that in the negative control group and the blank control group(p <0.05).Western blot was used to detect the expression of Sp1 protein and GLUT3 protein.The exepression levels of Sp1 protein and GLUT3 protein in the experimental group,the negative control group and the blank control group were0.573±0.010,0.783±0.023,0.794±0.013 and 0.549±0.004,0.820±0.031,0.772±0.014,respectively.The expression levels of Sp1 protein and GLUT3 protein in the experimental group were lower than that in the negative control group and the blank control group(p <0.05).The results showed that the expression of Sp1 gene was down-regulated after Sp1 gene silencing,which resulted in the decrease of transcriptional activity of Sp1 gene and the down-regulation of GLUT3 gene expression.It suggested that Sp1 was involved in GLUT3 expression regulation and affected the energy metabolism of the tumor.Conclusion:(1)The pLVX-sh RNA-Sp1 lentivirus expression vector was successfully constructed,and confirmed that it could down-regulate the expression of Sp1 gene in U87 cells,which laid the foundation for exploring the role of Sp1 gene in glioma by RNA interference.(2)Sp1 gene silencing can effectively inhibit the growth of glioma cells in nude mice.Further experiments showed that down-regulation of Sp1 gene expression resulted in decreased proliferation,promoted apoptosis,and down-regulated the expression of GLUT3 gene of glioma cells in nude mice,which may be one of the mechanisms of its inhibition of glioma growth. |
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