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Synergetic Role of Elmo1 with Dock180 in Regulating Cell Motility and Biological Behavior of Ovarian Cancer Cell SKOV3

Author: WangZuo
Tutor: LingHuHua
School: Chongqing Medical University
Course: Obstetrics and Gynaecology
Keywords: shRNA Elmo1 recombinant plasmid DNA sequencing RNA interference Elmo1 Dock180 cell motility ovarian cancer invasion behavior
CLC: R737.31
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 0
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Abstract


Objective: To construct the recombinant eukaryotic plasmid specific to Elmo1’s coding sequence called pSUPER.Retro.-GFP/neo-Elmo1i for further study on the association of Elmo1 with Dock180 and the role of Elmo1 in the malignant biology behavior of ovarian cancer.Methods: Four pairs of 19bp coded DNA sequence specific to Elmo1 were designed, synthesized, and inserted into pSUPER.Retro.- GFP/neo (abbreviated as pSR-GFP/neo). These recombinant plasmids were named as pSR-GFP/neo - Elmo1i#1, Elmo1i #2, Elmo1i #3 and Elmo1i #4, which were testified through restriction enzyme reaction and DNA sequencing, respectively. The optimal sequences were chosen through western blot for reduced Elmo1 expression level after transiently transfecting into SKOV3.Result: Four recombinant plasmids with specific sequences targeting Elmo1 gene were subcloned successfully (designated as pSR- GFP/neo- Elmo1i#1, -Elmo1i#2, -Elmo1i#3 and -Elmo1i#4) and were confirmed by restriction enzyme/eletrophoresis analysis based on the restriction enzymes cut site on the backbone plasmid, as well as through DNA sequencing. Among these four Elmo1-shRNA, two were selected for further study due to their efficiency in silencing endogenous Elmo1 expression, which was testified through western blot after transient transfection into SKOV3.Conclusion: Construction of Elmo1 gene’s short hairpin RNA is necessary for our further experiment about its association with Dock180 and its role in the biological behavior of ovarian cancer cell SKOV3. Objective: To establish SKOV3 cells with stably endogenous Elmo1 expression knocked down (Elmo1-RNAi cells) and compare their Elmo1/ Dock180 expression and biological events with those in Dock180-RNAi cells.Methods: The recombinant Elmo1i plasmid (pSR-GFP/neo- Elmo1i#3 and -Elmo1i#4) ensured above (Part 1) were transfected into SKOV3 cells by means of Lipofectamine 2000,and two stable Elmo-RNAi cells were selected through G418 screening. Next, Elmo1/Dock180 expression and Rac1-GTP level were detected and compared with negative control (including cells transfected with empty pSR-GFP/neo, pSR-GFP/neo-NT and parental SKOV3 cells WT) and stable Dock180-RNAi cells, which was made previously. Moreover, some biologic phenotypes among each group were compared through Transwell matrigel invasion assay, cell growth curve and plate clony formation. Result: Two strain of Elmo1-RNAi cells were established and named as Elmo1i#3 and Elmo1i#4. Both Elmo1 and Dock180 expression were knocked down in Elmo1-RNAi cells, as well as in Dock180-RNAi cells. As compared with the negative control, Elmo1-RNAi cells and Dock180-RNAi cells both exhibited reduced Rac1-GTP level and thus decreased cell growth rate, plate clony formation and attenuated cell invasion through Transwell Matrigel.Conclusion: Simultaneous depletion of Elmo1 and Dock180 expression, reduced Rac1 activity, and thus frustrated cell growth and cell invasion both in Elmo1-RNAi and Dock180-RNAi cells support their bipartite role in activating Rac1 activity and promoting untoward expansion and aggressive cell motility of ovarian cancer cell SKOV3.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Female genital tumors > Ovarian tumors
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