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The Investigation of Effect of LON on Cell Characterization by Down Regulation with RNAi in, MCF-7, Human Breast Cancer Cells

Author: ZhangYanLin
Tutor: ZhuYunFeng；GaoJunZuo
School: PLA Military Academy of Medical Sciences
Course: Internal Medicine
Keywords: RNA interference Serine protease LON Gene therapy Drug sensitivity
CLC: R73-3
Type: Master's thesis
Year: 2005
Downloads: 51
Quote: 0
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Abstract


Perform housekeeping functions of the the serine protease LON/PIM1 mitochondria and regulation functions ; , LON protease plays an important role in the regulation of apoptosis in the case of high temperature , anoxia, injury and other stress . This study attempts to use RNAi to reduce the expression of tumor cell mitochondrial serine protease LON . By RT-PCR screening of MCF-7 cells, etc. 8 representative tumor cell lines , the results showed LON gene expression in breast cancer MCF-7 cells , was chosen to study the LON gene RNA interference the target cell. SiRNA design for LON gene and recombinant expression plasmids pSilencer- LON into pSilencer2.1 carrier even instantaneous and stability in both transfected MCF-7 cells transfected . Positive clones by RT-PCR method to assess the a LON gene expression , and set up housekeeping gene β-actin as a standard control . The results show LON expression of the gene was no significant difference between the non - transfected cells and cells transfected control plasmid , but significantly decreased in the cells transfected plasmid pSilencer-LON was . The growth and activity of the transfected cells was detected by MTT , the growth activity of the control plasmid transfected cells no significant impact ( P gt ; 0.05 ) , the interference plasmid transfection able to inhibit cell growth activity ( P lt; 0.05 ) . Mapping MCF-7 cell growth curve and flow cytometry cell growth cycle , the results showed no statistically significant differences between groups untransfected and transfected . The study results suggest LON gene down did not significantly affect the cell cycle , but the results of MTT assay analysis of the down-regulation of the gene may affect the cell 's ability to metabolize further explore to contribute to the interpretation of its regulation mechanism .

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