Study On The Effect Of Knock-down HTERT By RNAi On Glioma Cell

PART ONE: Construction of siRNA expression vector targetinghTERT and its expression in 293T cell[Objective] To construct an siRNA expression vector targeting hTERT,and to investigate its inhibitory effect on the target gene expression inmammalian cells.[Methods] Three hairpin structure of siRNA transcript templatestargeting hTERT gene and a negative control were synthesized, thenligated with pRNAT-H1.1/Neo vector and sequenced. The recombinantvectors were then transfected into 293T cells and the expression ofhTERT was detected by fluorescent quantitative RT-PCR and Westernblot.[Results] The data of sequencing showed that the recombinant plasmidscontained the correct sequences of siRNA transcript templates targetinghTERT gene. The results of RT-PCR and Western blot indicated theexpression of hTERT was inhibited by 22.90% as compared with controlgroup in 293T cells transfected with the best one in the three candidates.[Conclusion] The siRNA expression vector targeting hTERT has beenconstructed successfully, and screened for the sequence with the highest inhibitory efficiency on the target gene. It set a good research platform forthe gene therapy of tumor which takes hTERT as the target.PART TWO: The construction of lentivirus-mediated RNAi vectorcontaining hTERT【Objective】To construct a recombinant lentivirus RNAi vector carryinghTERT gene to obtain the titer of the lentiviral stock for investigation ofthe expression in the eukaryotic cell and the affection of the hTERT genesilencing in the eukaryotic cells.【Methods】One hairpin structure of siRNA transcript templates targetinghTERT gene and a negative control were synthesized, then ligated withpLVTHM vector and sequenced. The recombinant vectors were thentransfected with viral packaging mix into T293 cells, viral supernatantwas harvested to determine the titer. U87 cells infected by virus wereharvested and the expression of hTERT and telomerase activity wasdetected by RT-PCR and TRAP assay.【Results】The data of sequencing showed that the reconstructiveplasmids contained the correct sequences of hTERT siRNA transcripttemplates. A vector producing cell line T293 was established, and the titerfor infection was obtained. RT-PCR and TRAP analysis demonstrated thathTERT shRNA expression construction could suppress the expression of hTERT and telomerase activity.【Conclusion】A lentivirus RNAi vector targeted hTERT gene wassuccessfully constructed, which depressed the expression of hTERT andtelomerase activity effectively. It set a good research platform for thegene therapy of tumor which takes hTERT as the target.PART THREE: Study on the effects of lentivirus delivers siRNAagainst hTERT in U87MG cells【Objective】To study on the effect of downregulation of hTERTexpression by lentivirus delivered siRAN in U87 cell and the potentialgene therapy against glioma on clinical application.【Method】The siRNA against hTERT was delivered by lentivirus: Lt-I,lentivirus: Lt-non, carrying a scrambled fragment as siRNA negativecontrol, and untreated U87 cell as negative contral. After U87 cells wereinfect Lt-I, Lt-non and PBS respectively, the expression of hTERTmRNA, telomerase activity, cell proliferation, telomere length, ceilinvasion ability were detected by RT-PCR, TRAP, MTT, FISH andTranswell assay. And the development of tumors in subcutaneouslygrafted nude mice was also observed.【Result】siRNA dramatically depressed the expression of hTERT mRNA,telomerase activity, cell invasion ability and the development of tumors in subcutaneously grafted nude mice.【Conclusion】Lentivirus delivered siRNA against hTERT can inhibitglioma cell proliferation and migration prior to its effect on telomerelength, indicating that hTERT contributes greatly to glioma developmentand could be used as a gene therapy for malignant glioma.