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The Influence of Smad4 on the Migration of Hepatocarcinoma Cell by Regulating fascin and Cortactin

Author: HanXiang
Tutor: JiGuoZhong
School: Nanjing Medical University
Course: Internal Medicine
Keywords: Smad4 Fascin Cortactin Hepatoma cells Migrate
CLC: R735.7
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 0
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Abstract


Purposes: 1. Construct lentiviral expression vector for Smad4 gene RNA interference and stable low expression in hepatoma cells SMMC-7721 in Smad4. Observed Smad4 hepatoma SMMC-7721 cells migration. 3 observed Smad4 cytoskeleton-associated protein of fascin in hepatoma cells SMMC-7721 / of cortactin expression, and to explore the mechanism of Smad4 impact of liver cancer cell migration. Method: 1. Design 16 Smad4 gene of RNA interference target sequence and encoded short hairpin RNA structures designed and synthesized corresponding to the DNA template strand to form a double-stranded DNA after amplification, after annealing, with Age I and EcoRI connection the digested pMAGic1.0 carrier, transformation, and PCR, DNA sequencing. 2 will slow virus vector plasmid and endotoxin-free lentivirus packaging system co-transfected 293T cells, packaging produce virus particles, and after concentration of the virus titer was measured. 3 packaging lentiviral infection hepatoma cell SMMC-7721, and the efficiency of its interference is detected by Western blot. According to interfere with the efficiency of subsequent experiments, filter out interference, high efficiency two groups of cells. Hepatoma cell SMMC-7721 hepatoma cells without infection and infection lentiviral empty vector (RNAi-NC) as a follow-up experiment control group. 4 cell scratch assay of Smad4 interference group and control group cells at 0 h, 12 h, 24 h, 48 h scratches healing. 5 three-dimensional culture detection Smad4 interfering group and the control group cells, the morphological changes of the cells in the three-dimensional culture conditions. Western blot detection Smad4 interference group and a control group of cells, the cytoskeleton-associated protein fascin / cortactin expression changes. Results: 1. PCR identification results show positive clones PCR fragment ligated into the double-stranded DNA fragment size of 343 bp; not connected into a PCR fragment size of 306 bp. DNA sequencing results show that the sequence is consistent with the design sequence. Will slow virus vector plasmid and endotoxin-free lentivirus packaging system co-transfected 293T cells, generated packaged lentivirus particles After concentration, its virus titer of 2 x 108 TU / mL, the success of lentiviral packaging. 3. Packaging lentiviral infection hepatoma cell SMMC-7721 detects interference efficiency, filter out interference, high efficiency two groups the RNAi-group of Smad4-2 and RNAi-Smad4-12 group. Western blot showed that Smad4 protein expression levels in the two groups of cells, liver cancer cells SMMC-7721 group and RNAi-NC group reduction compared. 4. Cells scratches experimental results show that, compared to hepatoma cells SMMC-7721 group and RNAi-NC group, the low expression of Smad4 RNAi-Smad4-2 group and RNAi-of Smad4-12 group of cells scratches heal faster. 3D culture results showed low expression of Smad4 RNAi-Smad4-2 group and RNAi-Smad4-12 group of cells, its edges irregular, gradually penetrate to the surrounding gel in three-dimensional culture process. SMMC-7721 group and RNAi-NC cells maintained the original form. Group compared to the group with liver cancer SMMC-7721 cells and RNAi-NC, low expression of Smad4 RNAi-of Smad4-group and RNAi-Smad4-12 cells cytoskeleton-associated protein of fascin / of cortactin expression levels. Conclusion: Smad4 can affect the migration of hepatoma SMMC-7721 cells, this effect may be related to regulation of cytoskeleton-associated protein of fascin / of cortactin expression related to the research and development of targeted drugs will early clinical predictors of liver cancer metastasis and tumor metastasis theoretical basis.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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