Study On Inhibitory Effects Of HTERT-siRNA To U251 Cells (One Kind Of Human Glioma Cells) Mediated By FA-PAMAM In Vitro

Glioma is the most common type of primary intracranial tumors, with 60%are malignant ones.Advances in the conventional treatment of gliomas,including surgery,radiation therapy and chemotherapy,have not yet get the satisfied therapeutic efficacy and improved the prognosis of patients with gliomas.Therefore,clear-cut its etiopathogenesis and searching other utility therapy is emergency problem in treatment with glioma.With the development of molecular biology and its application in study of tumorrigenesis,it has been demonstrated that the development of malignant tumors inculde gliomas is due to the activation of protooncogenes and inactivation of tumor suppressor genes leading to loss of control on cell proliferation and apoptosis,and deficit of normal limited life span of cells resulting in cellular immortality and tumorigenesis.Recently studies discovered that cellular immortality was caused by the activated telomerase.The role of telomerase is to manitain telomeric length and promotes cell proliferation potential.The activated telomerase is believed to be a critical event in cellular immortility.Telomere is the component of chromosome ends of eukaryotic cells, just as the hat.It consists of species specific tandem repeats of simple sequences and binding proteins.Human and other mammals contain 5'-TTAGGG-3' repeats.In 1984,Greider and Blackburn found the activity of telomerase in Blackburn laboratory.In 1989,Morin found the telomerase in human breast cancer cell "Hela".Telomere length is progressively shortened in normal cells due to "end replicative problem" untill senescence,while it is short and stable in tumor cells.Telomerase is a special reverse transcriptase which contains both essential proteins and RNA component.The RNA component is used as the template for synthesizing the telomeric repeats to lengthen telomere.Telomerase expression has been detected in more then 90%of tumors,but it is absent in most normal somatic tissues.The higher the degree of malignancy of tumors is,the higher the telomerase activity is.Human telomerase contains three major subunits,RNA(hTR),human telomerase catalytic subunits(hTERT),and telomerase-associated protein(TP1),have been identified recently,hTERT was cloned in 1997, its template contains 11 nucleotides.In 1999,the hTERT gene promoter was cloned,its template contains 1.7bp nucleotides,with its core is -211～+40.Recently researches found that hTERT mRNA had no expressed in most normal tissue(heart,brain,breast,liver,skeletal muscle,placenta and placenta)except thymus,testis and small intestine et al.But some researches also found that hTERT mRNA was expressed in nearly all neuroepithelial tumors and meningiomas as well as other cancers.And the level of hTERT mRNA was higher in malignant tumors than in normal brain tissues,and correlated positively with the degree of malignancy of tumors,hTERT is similar with elementary eukaryotic telomerase reverse transcriptase in sequence similarity.The hTERT is the catalytic subunit of telomerase.Its expression is usually parallel with telomerase activity.The hTERT is the telomerase proteinum catalytic subunit,which is expressed in proliferative cells,also is the rate-limiting step of telomerase activation and cell cancerization.To restrain the expression of hTERT could effectively shutdown the growth of glioma cells and deduce its apoptosis.So the hTERT has been the ideal target of the gene therapy to the glioma cell.Currently treatment perscription in gene therapy for glioma include immunopotentiation therapy,gene mediated pre-enzyme drug therapy, gene replacement and anti-sense gene therapy.These methods mainly use exogenous gene which was imported into target cells to rectification cell gene deviancy in order to achieve goals of treatment.Some researches found that a few dsRNA(double-stranded RNA)can specifically inhibit the expression of target gene efficiently and degraded mRNA.It is named as RNAi(RNA interference),belonging to PTGS (post-transcriptional gene silencing).This phenomenon generally emerges in eukaryotic cells and vitro cells,for successful target validation in therapeutic approaches in gene silencing.The key point of gene therapy is make the gene high expression in target cell mediated by ideal carrier efficiently.So there is a high requirement for gene carrier.Currently carriers include viral vector and non viral vector.The former one has a high efficiently transfect rate,with a low safety disadvantage.PAMAM(polyamidoamine dendrimer)is a high efficient transfect rate non viral vector,which is modified by FA (folic acid)has a tragedy character.We construct the gene vector FA-PAMAM to transfect U251 cell in vitro,through the RNAi,to study the inhibitory effects of hTERT-siRNA to U251 cells(one kind of human glioma cells)mediated by it.PARTⅠ:construction of new gene vector FA-PAMAM and detection of its transfect efficiencyBecause of the unfavorable outcome of conventional therapy for malignant gliomas,it is top priority to develop new therapeutic strategy for malignant gliomas.Gene therapy has been studied for the treatment of malignant gliomas.The application of gene therapy has been limited due to selection of suitable gene transfer vector and transfective efficiency of it.So,approach some high efficiency and hypotoxic vector to mediate gene therapy is another hot spot.Since the effectiveness of cancer gene therapy is mainly determined by vector selectivity,the majority of the research in this area has been focused on designing an efficient and safe vector system.At present, cationic polymers,especially PAMAM appear to be a promising solution for cancer gene therapy.FR(folate receptor)is a cell factor which has a higher expression in cancer cells than normal cells,including gliomas. Other more,folate is a small molecular substance,which can covalent bonding with PAMAM,without influencing the morphological character of the PAMAM.Thus,FA-PAMAM has the target character to tumor cell.FAM reporter gene,which is linked to the hTERT-siRNA,was transfected into U251 cells using the FA-PAMAM.No-load group and blank group are determined as control.The transfect rate was detected by FCM method.The transfect rate was 67.36%.The transfect rate is nearly twice time as oligofectamine which is widely used nowadays,it was demonstrated that FA-PAMAM can be used as the transfect vector in gene therapy. PARTⅡ:study on inhibitory effects of hTERT-siRNA to U251 cells mediated by FA-PAMAM in vitroThe hTERT(human telomerase reverse transcriptase)is the telomerase proteinum catalytic subunit,which is expressed in proliferative cells,also is the rate-limiting step of telomerase activation and cell cancerization.To restrain the expression of hTERT could effectively shutdown the growth of glioma cells and deduce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the glioma cell.RNAi(RNA interference),for successful target validation in therapeutic approaches in cancer and many other diseases,it has emerged as a useful tool to specifically inhibit the expression of a selected factor and study its function in the disease process.Gene silencing by RNA interference requires the application of double-stranded short inhibitory RNA(siRNA)that is designed to bind a selected mRNA sequence after intracellular complexation with the RNA-induced silencing complex (RISC).Upon binding of the specific sequence the RISC complex cleaves the targeted mRNA which is subsequently degraded and ultimately results in highly efficient and selective protein synthesis inhibition.Only very few siRNA copies per cell are required to mediate efficient gene silencing which has raised this technique as a widely approached attractive therapeutic strategy for various diseases including cancer.Use 21nt siRNA can silence the expression ofhTERT efficiently.In part I,it was demonstrated that we have constructed the gene therapy vactor FA-PAMAM which had a high efficient transfaction. Telomerase activity and hTERT were highly expressed in malignant gliomas.It suggests that hTERT maybe used as a target for RNAi gene therapy,hTERT-siRNA was transfected into human U251 glioma cells. The telomerase activity and the expression of its related genes were examined using the same method mentioned above;the cell proliferation and apoptosis were detected by MTT assay,RT-PCR,TRAP method and Transwell assay.The results were as follows:Proliferation activity of U251 cells transfected with hTERT-siRNA was inhibited significantly and the inhibition rate was dose time dependent.The inhibitory effect was most significant at 48 hour after transfection with hTERT-siRNA,the survival rate was 68.97%(F=234.35,P＜0.05).Which as compared to that of control and no-load group.The telomerase activity and the expression of hTERT mRNA were lowered significantly in U251 cells transfected with hTERT-siRNA.But there was no change in no-load group and control groups.After transfection with hTERT-siRNA,the ability of its ability of invasion had decrease significantly,and there was no change in the control group no-load one.In conclusion,after transfection with hTERT-siRNA,telomerase activity and expression of hTERT mRNA were inhibited,and proliferation of tumor cells was decreased,moreover apoptosis of tumor cells was induced.There was no difference between in control group and no-load group.In summary,these data demonstrate that hTERT is a promising molecular target for glioma gene therapy.The ability of FA-PAMAM based protocol to transfect cells efficiently in the glioma in vitro,coupled with the effectiveness of siRNA targeted for hTERT demonstrate here, will extend the application of siRNA to nano-based therapies and to basic research,including novel expressed sequences to define gene function. Meanwhile,demonstrated potential of targeted inhibition of hTERT as a putative therapeutic strategy in this study necessitates further study to elucidate the intricacies linking genetic and epigenetic modulations of the gene.