| Purpose:Reprinted by lentiviral gene expression of heat shock protein inhibition Mortalin and detected by western blot interrelationship with four transmembrane protein CD 151 in HCC cells, and inhibition of phosphatidylinositol 3-kinase/protein kinase B (PI3K/ AKT) signal Pathway and to study its effect on liver cancer cell migration and invasion.Methods:1. Yoshimitsu Biotechnology (Shanghai) Co., Ltd., in vitro use easy RNAi lentivirus construction techniques in order to interfere with the synthesis of labeled Mortalin TRAP1, TRAP2, three target gene sequences were TRAP3 carrier PGMLV-SC5 (2494):hU6-MCS-CMV-ZsGreen 1-PGK-Puro packaging, transfected into HepG2 cells, the transfected cells labeled TRAP1 cell group, TRAP2 cell group, TRAP3 cell group, by fluorescence expression and western blot technology, select TRAP1 cell group, TRAP2 cell group, TRAP3 cell group 3 group cells, namely Mortalin highest transfection efficiency was decreased as the next largest group of cells in cell experiments. Then using western blot technique comparable not transfected human hepatoma cell line HepG2 cells group (control group), the target sequences do not carry the virus particles transfected human hepatoma cell line HepG2 cells group (negative control group) and decreased expression of the most Mortalin human hepatoma cell line HepG2 cells group (experimental group), three groups of cells in the expression of CD 151.2. Mortalin expressed by the first part of the experiment screened largest decline in human hepatoma cell line HepG2 cells group (experimental group) and not been transfected human hepatoma cell line HepG2 cells group (control group) by western blot assay its PI3K/AKT signaling pathway key protein:protein kinase B (AKT) and the phosphorylation of protein kinase B (P-AKT) expression, discuss Mortalin downward effect on HepG2 cells PI3K/AKt signaling pathway.3. Scratch experiments were applied and Transwell assay to inhibit the expression of posterity Mortalin HepG2 cells migration and invasion ability to change the situation.Result:1. Three target sequences for inhibiting the expression of TRAP 1 Mortalin, TRAP2, TRAP3 three groups of transfected HepG2 cells, the expression TRAP2 sequence transfected human hepatoma cell HepG2 (TRAP2 cell group) is the most significant decline in Mortalin and test group the next experiment. Not been transfected human hepatoma cell HepG2 cells group (control group), the target sequences do not carry the virus particles transfected human hepatoma cell line HepG2 cells group (negative control group) and Mortalin decreased expression of the most human hepatoma cell line HepG2 cells group (experimental group), after comparing the Western blot, no significant reduction of the expression of CD151, statistically no difference.2. Mortalin decreased expression of the most human hepatoma cell line HepG2 cells group (TRAP2 cell group) and not been transfected human hepatoma cell line HepG2 cells group compared, PI3K/AKT signaling pathways AKT down slightly, but obvious, no statistically significant. P-AKT expression decreased with Mortalin have come down, there are significant differences, with statistical significance.3. Mortalin decreased expression of human hepatoma cell line HepG2 cells up group (TRAP2 cell group) as the experimental group, not been transfected human hepatoma cell line HepG2 cell group was the control groups, the target sequence transfected human viral particles do not carry HepG2 cells as the negative control group of cells. In the Transwell assay Cell invasion ability experiment:the control group and negative control group, the experimental group and negative control group, the experimental group and control group pairwise comparison, the experimental group was significantly lower invasion will. In the scratch test:In 0 hours,12 hours,24 hours,36 hours four time periods, the experimental group and control group were compared scratch width, the migration of the experimental group did not change significantly.In conclusion:1. After HepG2 cells, the inhibition Mortalin expression, CD151 expression did not appear significantly reduced, was no correlation between the two.2. After the human hepatoma HepG2 cells in Mortalin of, PI3K/AKT signaling pathways AKT no significantly reduced, indicating downward Mortalin human HepG2 cells does not affect the amount of total AKT, and P-AKT expression decreased with Mortalin have The cut, clear differences between the description of the cut will cause Mortalin the human hepatoma cell HepG2 PI3K/AKT signaling pathways AKT phosphorylation decreased, and this difference is not due to the expression of the total amount ranging AKT cause is due to inhibition of human After the expression of HepG2 cells Mortalin, inhibits AKT activation, thereby inhibiting the PI3K/AKT signaling signal transduction pathway.3. In the cell invasion experiment:Not transfection of HepG2 cells cells group (control group) and target sequences do not carry the virus particles transfected human hepatoma cell line HepG2 cells group (negative control group) invasive cells number no significant difference (p> 0.05), described the virus itself does not have a load of human hepatoma HepG2 cell invasion capability of generating change, transfected human hepatoma cell line HepG2 cells group (control group) were compared invasive cells did not express the largest decline Mortalin human hepatoma cell line HepG2 cells group (TRAP2 cell group) test cell invasion group were significantly different (p<0.05), target sequences do not carry the virus particles transfected human hepatoma cell line HepG2 cells group (negative control group) compared invasive cells Mortalin decreased expression of most human hepatoma cells HepG2 cells group (TRAP2 cell group) test cell invasion group also significantly different (p<0.05), with the blank control group and negative control group consistent results. Description Mortalin inhibit the expression in human hepatoma cells HepG2, have a significant effect on the in vitro invasiveness. In cell migration experiment: compare Mortalin decreased expression of the most human hepatoma cell line HepG2 cells group (TRAP2 cell group) test cell group and non-transfected human hepatoma cell line HepG2 cells group (control group) at 0 hours,12 hours,24 hours,36 hours scratch width, no significant differences (p> 0.05), described Mortalin of inhibiting the expression, did not inhibit HepG2 cell migration in vitro. |
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