Expression Of PTTG1 In Gliomas And Effect Of MicroRNA Targeting PTTG1 Gene On Biological Behavior Of Glioma Cells

Glioma is the most common type of primary intracranial tumors.It has been reported that malignant glioma accounted for about 60%of gliomas.Although the currently available multi-modalities of therapies,such as microsurgical operation,radiotherapy and chemotherapy,have been improved,the median survival time of patients with malignant glioma is only 52 weeks.So glioma is still a refractory disease in the neurosurgical field.In order to improye the present situation,the mechanism of the development and progression of gliomas as well as the new strategies for the treatment of malignant gliomas should be studied in the future.With the significant advances of molecular biology,it has been demonstrated that the development of malignant tumors including gliomas is due to the activation of proto-oncogenes and inactivation of tumor suppressor genes.PTTG was isolated from rat pituitary tumor cells in 1997 and identified as a pituitary-derived transforming gene.Recent studies have shown that PTTG1 gene is overexpressed in a variety of tumors.Forced PTTG1 expression induces cell transformation in vitro and tumor formation in nude mice.In some tumors,high PTTG1 levels correlate with invasiveness, and PTTG1 has been identified as a key signature gene associated with tumor metastasis. Increasing evidence supports a multifunctional role of PTTG1 in cell physiology and tumorigenesis.Physiological PTTG1 properties include securin activity,DNA damage/repair regulation and involvement in organ development and metabolism. Tumorigenic mechanisms for PTTG1 action involve cell transformation and aneuploidy, apoptosis,and tumorigenic microenvironment feedback.So PTTG1 gene may play an important role in the malignant progression in gliomas.Recent knowledge gained from PTTG1-null mouse models and transgenic animals and their potential application to subcellular therapeutic targeting PTTG1 are discussed.Up to date,although there have been some reports on the expression of PTTG1 genes in gliomas,but these results were obtained from a small number of brain tumors or glioma cell lines,the relationship among PTTG1 gene expression,histopathological type,proliferation activity,biological behavior of gliomas has not been reported.In the present study,the expression of PTTG1 gene in gliomas and the relationship among PTTG1 gene expression,the tumor grade and tumor proliferation were investigated in order to understand the role of PTTG1 gene in the pathogenesis of gliomas.The feasibility of using PTTG1 gene as a target for gene therapy of gliomas was also studied by in vitor and in vivo experiments.PartⅠ:Study on the gene expression of PTTG1 and its relationship to clinic histopathologic of human gliomasThe gene expression of PTTG1 in 52 human gliomas,2 malignant human glioma cell lines and 4 normal brain tissues were studied by real-time RT-PCR and western blot analysis.It was found that 4 normal brain tissues almost no expressed PTTG1 and its mRNA,and 2 malignant human glioma cell lines had PTTG1 and its mRNA overexpression.The difference of positive rate and expression level of PTTG1 and its mRNA between the low-grade and high-grade tumors is statisticly significant(P＜0.05). The results of Western blot analysis using antibody performed on 52 samples of human gliomas were coincidence with that of immunohistochemical staining(P＜0.01).The expression level of PTTG1 gene also provide us a useful parameter in evaluating the degree of malignancy in molecular level and selecting the target of gene therapy. Relationship between proliferation activity and PTTG1 gene expression in human gliomas was found that the proliferation activity of gliomas evaluated by Ki-67 LI(Ki-67 Labeling Index) is positively correlated with PTTG1 gene expression,and positively with the tumor grade.These results suggested that PTTG1 gene expression was significantly associated with the proliferation activity in human gliomas.And PTTG1 gene expression in human gliomas was found that is positively correlated with MMP2 and MMP9 gene expression.PartⅡ:Study of the effect of transfected miRNA-PTTG1 on proliferation,apoptosis and invasion of glioma cells in vitroThe pcDNA^TM 6.2-GW/EmGFPmiR-PTTG1 plasmid which contain the specific miRNA trageting PTTG1 and the negative plasmid pcDNA^TM 6.2-GW/EmGFP miR-negative were transfected respectively into human brain glioblastoma U251 and SHG-44 cells by lipofectin medium.The impact on proliferation and apoptosis of U251, SHG-44 cells in vitro after inhibition of PTTG1 with miRNA was investigated by methods of real-time RT-PCR,western-blot,Confocal Scanning Laser Microscope(CSLM),flow cytometry(FCM) and caspase-3 activity assay.The U251 glioblastoma cell transfectants and the SHG-44 astrocytoma cell ones resulted in dramatic down-regulation of PTTG1 mRNA and protein as demonstrated by real-time RT-PCR,western-blot analysis.Four clones with low expression of PTTG1 constructal so showed decreased proliferation in vitro as detected by MTT method.The quantitative analysis of apoptotic cells by FCM showed that the apoptotic cells increased in U251 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 20.4%compared with U251(16.9%) and U251 cells(tranfected with pcDNA^TM 6.2-GW/EmGFPmiR-negative)(14.9%).And the quantitative,analysis of apoptotic cells by FCM showed that the apoptotic cells increased in SHG-44 cells(tranfected with miRNA-PTTG1) with the apoptotic rate of 22.2%compared with SHG-44(15.2%) and SHG-44 cells(tranfected with pcDNA^TM 6.2-GW/EmGFPmiR-negative)(14%).Caspase-3 activity assay indicated that the activated caspase-3 was significantly increased(P＜0.01) in U251 and SHG-44 cells (tranfected with miRNA-PTTG1).Transient transfection of U251 and SHG-44 cells with miRNA-PTTG1 showed a significant increase in expression of p53,Bax and p21WAF1/Cip1,decrease in expression of and ki67,c-myc,p53,MMP2,MMP9 measured by western blot analysis.And the results by FCM also showed that RNA knockdown can sensitize glioma cells to p53-dependent apoptosis.The above results suggested that by miRNA can interfere with PTTG1 expression,and induce apoptotic cell death in glioma cells,and tumor suppressor protein p53 is required for miRNA-PTTG induced apoptotic cell death,and inhibition of PTTG1 could be a useful therapeutic strategy for the treatment of glioma.The effect on invasiveness of U251 cells was investigated by methods of Matrigel and wound-healing.And the results showed that RNA knockdown of PTTG1 can inhibit the invasiveness of glioma.cells.PTTG1 gene may be selected as a target for gene therapy of gilomas,and it should be studied further in vivo experiment.PartⅢ:The specific therapeutic implication of miRNA-PTTG1 in vivo for xenograft gliomasIn order to further investigate the role of PTTG1 gene in the growth and proliferation of glioma cells,and find out the feasibility of using PTTG1 gene as a target for gene therapy of gliomas as well as to observe the therapeutic effect of PTTG1 gene for gliomas in vivo and it's prospect in clinical application.15 nude mices weighing 16～20g were divided into three groups:U251 cells(A group),U251 plus negative vector-treated group(transfected with transfected with pcDNA^TM 6.2-GW/Em GFPmiR-negative)(B group),U251 plus miRNA-PTTG1-treated group(transfected with pcDNA^TM 6.2-GW/EmGFPmiR-PTTG1)(C group) were inoculated respectively in back subcutaneous tissue of nude mice to establish xenograft models of human brain glioma. The tumor growth status was observed and tumor volume was measured termly,and the tumor growth curve was drawn.The animals were killed and the tumor weight was investigated 4 weeks after inoculation.The results showed that the tumorigenesis time delayed,tumor grew slow,both tumor volume and tumor weight decreased significantly (P＜0.05) in C group as compared with those in A and B groups.HE staining for each group of tumor specimen indicated thatthe histological features of these nude mice tumor is in accordance with those of glioblastoma multiformes.The results of western blot showed that the activated caspase-3 was significantly increased in C group in comparison with those in A and B groups.The above results suggested that by disrupting PTTG1 expression,miRNA mediated RNA knockdown can antagonize the anti-apoptotic effect of PTTG1,activate caspase-3,and then induce apoptotic death of U251 cells in nude mice human brain glioma.In summary,this research suggested that PTTG1 may be a key protein in initiation and progress of human brain glioma,its mechanism may involve the proliferative and anti-apoptotic effect of PTTG1 proteins in the tumorigenesis and development of glioma. Blocking of function of PTTG1 or down regulation of its expression in tumors may result in suppression of tumor growth and metastasis.PTTG1 may be considered as a potential and promising target for gene therapy of glioma.