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Utilize siRNA Eukaryotic Expression Vector Interrupting the Gene Expressing of MGMT

Author: WangFang
Tutor: CaoJia
School: Third Military Medical University
Course: Health Toxicology
Keywords: O6-methylguanine-DNA methyltransferase tumor drug resistance Antisense RNA RNAi HeLa S3
CLC: R346
Type: Master's thesis
Year: 2006
Downloads: 69
Quote: 0
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Abstract


Of numerous forms of DNA damage which can block cell growth, O6-Alkylguanine is the major product formed in DNA by the reaction of alkylating agents. But the DNA repair protein O6-methylguanine–DNA methyltransferase (MGMT) existing in cells can revert the DNA damage induced by alkylating agents is the most important reason for the alkylating agents resistance especially for nitrosoureas and for the decreasing killing efficiency in tumor cells after treatment of alkylating agents, it’s the probably molecular mechanism of nitrosoureas resistance for many tumors.To increase tumor’s sensitivity for alkylating agents, Kreklau etcetera tried to inhibit the MGMT activity in tumor cells by inhibitor but simultaneously the MGMT activity in peripheral blood monocytes also be inhibited and the cumulation of such kind of immuno-reacted inhibition can lead to marrow toxicity. Yoshinaga etcetera reported that tumor’s resistance for alkylating agents can be reverted by inhibiting expression of MGMT gene through antisence or ribozyme strategy but also has two questions: one is the low efficiency and the other is the blindness in antisense sequence selecting. In recent years, RNA interference comes to be the most efficient manner for gene silence, siRNA has a special double string complementary structure and especially siRNA eukaryotic expression vector can express in cells during a long period of time and more stable than antisense RNA, so that the siRNA has an inimitable, possibly more perfectible effect for regulating gene expression when compared with antisense RNA or ribozyme strategy.In present study, we synthesized 3 single DNA string coding hairpin siRNA and cloned them into downstream of H1.2 promotor of pRNATin-H1.2/Neo vector to construct a recombinant vector with target gene fragment in it, then transfected the vector into HeLa S3 cell by lipid Lipofectimine 2000 mediating.Assessed the inhibiting effect of MGMT by semi-quantitative RT-PCR and real time quantitative RT-PCT methods, assessed by MTT the variation of HeLa S3 cell sensitivity to

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