| Objective:p33ING1b比 is an importalt tumor suppressor,and its function is closely related to p53 protein.In many malignant tumors including malignant glioma,ING1 gene mutation rate is very low,but epigenetic and other mechanisms often result in lower p33ING1b expression and subcellular localization abnormal,and the intracellular content of p33ING1b negatively correlated with malignant glioma.Study confirmed that p33ING1b expression wasn’t affected by up-regulating p53 protein,and simply high expression of p53 in malignant glioma can not compensate for the lack of endogenous p33ING1b.Therefore,in the background of lack of p53 and p33ING1b,single transfection of wild-type p53 or p33ING1b can not effectively inhibit tumor cell proliferation and induce cell apoptosis,but co-transfection of p53 and p33ING1b could play an effective anti-tumor effect.PCNATAI which is modified on the basis of PCNA promoter is a promoter with highly specific proliferation.Our research aimed to study the expression efficiency of p33ING1b and p53 in malignant glioma cells which is co-transfected controlled by the promoter of PCNATAI,and the confluence on the expression of p21WAF1,cell apoptosis and migration under the up-regulation of 33ING1b and p53.Methods:① On the basis of the plasmids pEGFP-PCNATAI and pGEM-T easy p53 which were constructed previously,first amplified the full sequence of p53 coding region by PCR,then inserted the sequence into the downstream of pEGFP-PCNATAI to construct the plasmid which express p53(pPCNATAI-p53);② On the basis of the plasmids pEGFP-PCNATAI and pEGFPCI-p33ING1b which were constructed previously,first amplified the promoter region of PCNATAI by PCR,then inserted the sequence into pEGFPCl-p33ING1b to substitute the promoter of pEGFPC1 and constructed the plasmid pEGFP-PCNATAI-p33ING1b;③ Identify the recon-structed plasmids by restrict endoenzyme digestion.④ Immoral cell line HEK293 and malignant glioma cell line U251 were respectively divided into four groups,named as control group,p33 group(transfected with),p53 group(transfected with pPCNATAI-p53)and co-transfection group(transfected with pPCNATAI-p53 and pEGFP-PCNATAI-p33ING1b).⑤ Observe the expression of p33ING1b,p53 and p21WAF1 by fluorescence microscope and Western blot;⑥ Assess the degree of cell apoptosis in different groups of different cell lines by single cell gel electrophoresis and flow cytometry.⑦ The effect of different group on the migration ability is measured by scratch model.Results:① Identified by endoenymenzyme digestion,the insert site of the sequence of p53 coding region of and PCNATAI promoter of pEGFP-PCNATAI-p33ING1b is right and it is illustrated that successfully construct the two recombinant plasmids.② After single transfection by pEGFP-PCNATAI-p33ING1b and co-transfection with pPCNATAI-p53,there were high expression of fusion protein(GFP-p33ING1b)in U251,and only a little expression of fusion protein in HEK293.③ The results of western blot show that:In cell line HEK293,there is no difference in the expression of,p33ING1b and p21WAF1 among each groups;In cell line U251,the expression of p33ING1b,p53 and p21WAF1 of co-transfection group was significantly up-regulated compared with the other three groups(P<0.001);the expression of p33ING1b in p33 group is higher than p53 and co-transfection groups(P<0.05),but there was no significance between p53 and control group(P>0.05);the expression of p53 and p21WAF1 of p53 group is higher than p33 and control group(P<0.05),but there was no significance between p53 and control group(P>0.05).④ The cell apoptosis detected by single cell gel electrophoresis showed as following:In the same cell line,AI of co-transfection group is signify-cantly higher than other groups(P<0.001～0.05).In HEK293,there was no significance among p33ING1b,p53 and control group(P>0.05).In cell line U251,AI of p33ING1b and p53 groups is higher than control group(P<0.05),but there was no significance between p33ING1b and p53 group(P>0.05).In the same transfection group,AI of U251 is higher than HEK293(P<0.001～0.05).⑤ The following was the results of flow cytometry;In cell line HEK293,cell apoptosis of co-transfection group was more obvious than the other three groups and the difference was significant(P<0.05),there was no difference among the other three groups.In cell line U251,there is no obvious cell apoptosis in control group and significant cell apoptosis in the other three groups(P<0.001-0.05).Among the group of p33ING1b,p53 and co-transfection,the apoptosis cell percentage of p53 and p33ING1b group is lower than co-transfection group(P<0.001)and there was no difference in the two groups(P>0.05).In the same transfection group,the apoptosis cell percentage of U251 was obviously higher than HEK293 and the difference are significant.⑥ The distance of cell migration showed that:In the same cell line,migration distance of control、p33ING1b and p53 group was longer than co-transfection group(P<0.01～0.05).In HEK293,there was no significant difference among p33ING1b、p53 and control group.In U251,migration distance of control group was longer than p33ING1b and p53 group(P<0.05),but there was no significant difference between p33ING1b and p53wt group(P>0.05).In the same transfection group,migration distance of HEK293 was longer than U251(P<0.001～0.05).Conclusions:① Identified by endoenymenzyme digestion,sucsessfully constructed the recomminant plasmids pEGFP-PCNATAI-p33ING1b and pPCNATAI-p53 and the promoter PCNATAI inserted into the two plasmids processed high proliferting specificity.② PCNATAI could work in the way of proliferation specifity and promoted the expression of wtp53 and p33 ING1b in the malignant glioma cell line U251 which proliferate actively.③ Co-transfection of U251 with pPCNATAI-p53 and pEGFP-PCNATAI-p33INGlb could elevate the expression of exogenous p33INGlb and wtp53.④ The expression of exogenous p33INGlb and wtp53 promote apoptosis and inhibit migration of U251 cells through up-regulation the expression of p21WAF1 which is the downstream reaction factor of wtp53.⑤Co-transfection of the plasmids which respectively express exogenous p33ING1b and wtp53 controlled by the promoter PCNATAI is a effective method of malignant glioma gene therapy and has a well actually applicant perspective. |
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