Dissertation > Medicine, health > Oncology > Nervous system tumors

Small Interference RNA Targeting Kruppel-Kike Factor8Inhibits the Glioblastoma U251Cells Growth by Inducing Apoptosis

Author WanWeiFeng
Tutor ZhuJi
School Chongqing Medical University
Course Surgery
Keywords KLF8 glioblastoma apoptosis
CLC R739.4
Type Master's thesis
Year 2012
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Objective: Glioblastoma is primary brain tumors that arise from glialcells, and is one of the most common primary malignant brain tumor inadults. It remains formidable although treated by aggressive surgery,radiotherapy or chemotherapy. Therefore, it is necessary to further studythe intrinsic molecular mechanisms of glioblastoma in formation,proliferation, invasion and apoptosis, and we hope to establish thefoundation for the subsequent molecular biology targeted gene therapy.New studies show that Krüppel-like factor8is an important oncogene, andit widely participates in the pathophysiological processes of tumor,including the regulation of cell cycle, proliferation, differentiation, apotosis,development and tumorigenesis. And KLF8is an activator for matrixmetalloproteinase9(MMP-9), which can lead to inhibit the cell adhesionand promote the tumor invasion. The mechanisms between KLF8and theformation, invasion, and development in glioblastoma are still unknown. Itis necessary to examine the role of KLF8in glioblastoma, so we can find the mechanisms in pathophysiological process of glioblastoma. We hope toidentify whether the KLF8can act as a target for molecular targetedtherapy.Methods: RNA interference(RNAi) can specifically turn off theexpression of the specific genes, the technology has been widely used toexplore gene function and gene therapy in infectious diseases andmalignant tumors. KLF8(NM007250)siRNA or negative control siRNAwere inserted into a pGCSIL-green fluorescent protein(GFP)vector.ThesiRNA plasmids were transfected into293T cells,together with thelentiviral helper plasmids,pHelper1.0and pHelper20.,to generate therespective lentiviruses.Viral stocks were created and used to infect U251cells. So we get an experimental group of KLF8siRNA U251cells and acontrol group of negative siRNA U251cells. And we detect the cell cycle,proliferation, and apotosis in the experimental group and control group.Results: To determine the effect of RNA interference (RNAi) on theexpression of KLF8in U251cells, the mRNA levels of KLF8wereanalyzed. The cells infected with KLF8siRNA exhibited lower expressionof KLF8mRNA than those with negative control siRNA. The growthpattern observed changed visibly among transfectants from the second tothe fifth day in U251cells. Cells treated with KLF8siRNA exhibited lowergrowth rates compared with the negative control siRNA. Moreover, BrdUincorporation assay also revealed that the inhibition of KLF8expression significantly reduced the growth rate of U251cells during the4-day incu-bation period. By FACScalibur flow cytometer72h following infection, wefound that KLF8siRNA caused G2/M phase arrest and affected cell cycleprogress.KLF8siRNA enhanced the rate of apoptosis. These results suggestthat KLF8siRNA inhibited glioblastoma cell proliferation through theenhancement of cell apoptosis.Conclusions: These results suggest that Krüppel-like factor8mayplay an important role in cell cycle, proliferation and apoptosis inglioblastoma. Through the KLF8siRNA, the U251cell apoptosis wassignificantly enhanced and cell proliferation was inhibited. It is suggestedthat KLF8gene play an important role in glioma proliferation. KLF8siRNA can block KLF8gene in the downstream transduction pathway bypromoting apoptosis of glioma and inhibition of tumor proliferation, so thatKLF8siRNA can treat glioblastma. KLF8siRNA can provide a newgene-targeting therapy idea and give great help to the treatment ofglioblastoma.

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