Reprogramming Of Human Glioblastoma By Synthetic Reprogramming Factor Oct4and Pluripotent Factors

Research BackgroundGlioblastoma is one of most common tumors in Neurosurgery diseases. The domestic incidence rate of brain tumor35.26%-60.96%(average44.69%), and most were malignant, patients usually dead no more than9-18months after first diagnose. It greatly threat people’s lives and health. During past10years, neurosurgery technology has rapid developed, but the prognosis of glioma has not significantly improved, the middle survival period of patients still failed to exceed one year. Therefore, a better understanding of glioma morbidity and development, Genetic and Epigenetic mechanism will help to further reveal the mechanisms of occurrence and development, and contribute to developing new treatment strategy.In2006, Yamanaka e.tl invented a new reprogramming stem cell technology, they reprogramming mouse fibroblast cell into Embryonic stem cell(ES) like pluripotent cells through retroviral transduction four factors, including Oct4、Sox2、 Klf4、c-Myc, is a milestone in stem cell field, they named such cell as Induced Pluripotent stem cell (iPSCs). iPS technique show great potent in disease diagnosis and therapy, and make it possible to generate patient-and disease-specific iPS.Nowadays, the efficiency of iPS induction is very low, usually lower than0.02%, and especially in unhealthy somatic cells, which great hamper the development of iPS application in clinic therapy.Few reports about the iPS application in anticancer therapy had been published. Only acute myelocytic leukemia (AML), mouse embryonal tumor, colorectal cancer, liver cancer, stomach cancer and pancreatic cancer cells had been reprogramed into iPS cells. But, no report about glioblastoma induced to iPS cells.Based on the hypothesis of synthetic reprogramming factor(sRF), we construct a strong transactivating potential sRF-Oct4factor, to improve the efficiency of iPS induction, and induce glioma cell into iPS cell. PurposeWe conjunct the strong transactivating domain pAD65and classic reprogramming transcription factors Oct4, to construct new reprogramming factors SRF-Oct4, and evaluate the transcriptional activity of sRR We induce glioma cells U87-MG into iPS cells through new construct sRF-Oct4factor, and explore U87-MG change of pluripotent and epigenetic after induction to further explore tumor morbidity mechanism and develop new therapy strategy.Method1) We constructed synthetic reprogramming factors (sRFs-Oct4) by linking the DNA binding domain of Oct4with pAD65domain, and transfect the re-construct plasmid into293T cell. Then, evaluate the role of synthetic reprogramming factors, we co-transfected sRF-Oct4with a reporter vector consisting of Oct4promoter (pOct4) and luciferase, and measured the activity of luciferase of firebird and renilla by the luciferase assay kit. Also, we examined the expression of Oct4-downstream target genes after transfecting293T cells with synthetic reprogramming factors by testing the activation of pOct4-EGFP fluorescence.2) We package the sRF-Oct4,Oct4,Sox2,Klf4,c-myc and EGFP retroviral system through EP2-293virus package cell line.3) We infected U87-MG cell with concentrated viral supernatant, and then transplant the target cell on Mouse Embryonic Fibroblast(MEF) feeder cells.4) We transplanted clone-like target cells into the new MEF feeder cells and continue to cultivate and stabilize passages. Then collect target cells, processing RT-PCR, Western Blot.5) We inject the target cells into immunodeficiency mice, and evaluate the generation of tumor, and making pathological sections by teratoma tissue.6) Western-blot test the expression of TIMP2and MMP-2protein in U87-MG and U87-iPS differentiate cells. Result1) We success to construct synthetic reprogramming factor sRF-Oct4, and ligation with retroviral vector. The plasmid was in accord with design gene squence.2) Synthetic reprogramming factor sRF-Oct4was stronger than wild type Oct4by Luciferase assay.3) U87-MG cell after SRF-Oct4、Sox2、Klf4and c-Myc retroviral infected, and induced by stem cell environment, iPS like clones could be seen after3weeks culture. But, U87-MG induced by Yamanaka factors could not be reprogramming to iPS cells.4) Expression of downstream target genes Oct4, Nanog were increased after transfection; Stem cell associate protein Oct4and Nanog also were increased.5) AfterU87-iPS like cell injection into immunodeficiency mice, they were capable of forming teratomas. The capable of generate tumor of differentiate iPS cell was down regulation.6) Bcl-2was down-regulated and Bax was up-regulated in U87-iPS differentiated cells.Conclusion:Re-construct reprogramming pAD-Oct4plasmid could improve the transactivation of Oct4; pAD-Oct4combination Sox2, Klf4and c-Myc could reprogramming the U87-MG into iPSCs. The capable of generate tumor of differentiateU87-iPS cell was down regulation.