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The Study on Changes of MHCC97-H Cells Biological Behaviors and KAI1 Gene Expression by Small RNA Interference of Ubiquitin Ligase (E3) gp78

Author: LiZuo
Tutor: ChenWenSheng
School: Third Military Medical University
Course: Internal Medicine
Keywords: Hepatocellular carcinoma gp78 KAI1 MHCC97-H
CLC: R735.7
Type: Master's thesis
Year: 2010
Downloads: 45
Quote: 1
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Abstract

Research background and purpose of liver cancer is a cause of human mortality high malignancy, especially its liver metastasis, is the difficulty of the clinical treatment of liver cancer. Liver cancer cells usually early after hematogenous transferred to neighboring or distant tissues and organs, and has a strong transfer of invasive ability of liver cancer cells, and this is very complicated transfer mechanism Invasion. A growing number of studies suggest that the enhancement of tumor cell migration and invasion, is one of the important mechanisms of tumor metastasis. The previous studies have been conducted in an animal cell strain showed that gp78 as an endoplasmic reticulum related protein degradation pathway (ERAD) associated ubiquitin ligase enzyme (E3), and its overexpression induced phenotypic changes of tumor cells, This change has enhanced cell viability and proliferation, cell invasion potential and ability has also been enhanced. Recent studies have reported that the level of gp78 mRNA expression in tumor tissue compared with adjacent normal tissue was significantly higher, and studies have shown that tumor metastasis suppressor factor KAI1 is the specific role of the substrate of gp78, gp78 E3 activity of osteosarcoma cells by metastasis suppressor factor the KAI1 the degradation to facilitate the transfer of osteosarcoma. But which in hepatocellular carcinoma metastasis invasion, the role played by gp78 is not yet clear, the biological characteristics of hepatocellular carcinoma cells in vitro gp78 remains unclear. In the present study, we assume that the reduced gp78 expression in hepatoma cells may lead to increased KAI1 expression levels, so that the proliferation of tumor cells to form colonies as well as the decline in migration and invasion, and then infer the transfer may be weakened liver cancer. To test this hypothesis, we used RNAi technology to silence hepatoma cells MHCC97-H the gp78 of expression in detect gp78 silence before and after liver cancer cell proliferation, migration and invasion of changes in biological activity, also detected gp78 silence hepatoma cells before and after KAI1 expressed. Research methods, according to the gp78 gene sequence (GeneBank NM ₀ 01144, siRNA design tool www.genscript.com online, design three pairs of siRNA cloned into plasmid pRNAT-U6.1/Neo, plasmid extraction and sequencing. construct a good the three gp78 interference carrier respectively named to as pRNAi-pRNAi-2 with pRNAi-3. cells were seeded in 6-well plates until cell fusion was 80% ^{9, < / sup> 0%, liposome-mediated gene transfection build good gp78 interference carrier with empty vector into MHCC97-H cells stably transfected cell lines by G418. experimental techniques to detect changes in the gp78 RNAi before and after MHCC97-H cell proliferation, colony formation ability, migration and invasion capacity. 3, using RT-PCR and Western Blot method to detect gp78 and KAI1 expression in the cells around gp78 RNAi MHCC97-H Results 1, interference constructed recombinant vector by sequencing comparison, the target sequence of the inserted oligonucleotide sequence and design exactly described interference recombinant vector was constructed successfully after G418 selection and clone selection to obtain stable transfection of cells strains, transfected cells in the laser confocal microscope fluoresce green. 2,3 siRNA eukaryotic expression plasmid two effective inhibition of gp78 expression, which is the most obvious effect of pRNAi-2. 3 MTT experiments show The interference group cell proliferative capacity was significantly lower than MHCC97-H group and empty vector cells; colony formation assay capacity MHCC97-H group and empty vector cells showed that the interference group single cells form colonies significantly reduced compared; cells scratches healing The experiments show that the interference group cell migration is significantly lower than MHCC97-H group and empty vector cells; of Transwell chamber experiments show that the interference group cell invasion with MHCC97-H group significantly decreased compared to the empty vector cells; 4, using RT- PCR and Western Blot analysis, interference cells KAI1 expression higher than MHCC97-H and empty vector cell the conclusion gp78-specific RNA interference can effectively inhibit gp78 expression MHCC97-H cells, and raised the KAI1 gene expression, thereby indirectly lower liver cancer cell proliferation, colony formation, migration and invasion, thus weakening the metastasis of liver cancer cells, reverse the malignant phenotype of hepatoma cells.}

The Study on Changes of MHCC97-H Cells Biological Behaviors and KAI1 Gene Expression by Small RNA Interference of Ubiquitin Ligase (E3) gp78

Abstract

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