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Study on the Effect of Specially Down-regulated GRP94 on the Biological Properties of Human Colorectal Cancer Cell Line

Author: ChenYao
Tutor: SongJinDan
School: China Medical University
Course: Cell Biology
Keywords: Endoplasmic reticulum Glucose-regulated protein The unfolded protein response Chaperone
CLC: Q26
Type: PhD thesis
Year: 2003
Downloads: 134
Quote: 0
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Abstract


The purpose of the endoplasmic reticulum the specialized membranous organelles in eukaryotic cells, is closely related to the synthesis of proteins, lipids, but also intracellular Ca2 important reservoir. Therefore, to study the fine structure of the endoplasmic reticulum, especially ER membrane protein synthesis, secreted proteins and chaperones has become an international leading issue. The glucose-regulated protein (glucose regulated proteins, GRPs) are an important class of endoplasmic reticulum resides proteins, including GRP78 and GRP94 is. GRP94 is one of the members of the family of heat shock protein HSP90 and HSP90, with a 50% homology, is a highly conserved protein. clear a grp94 the genetics positioning encoding gene is located on chromosome 12q24.2-12q 24.3. GRP94 functionality currently known as molecular chaperones, nascent chain synthesis in the early stages, with the newborn peptide bond to form a stable complex involved in protein folding, assembly and translocation. In addition, found that it also has an antigen-presenting, misfolded protein in normal cells involved in presenting to the proteasome; as the primary tumor rejection antigens in tumor cells, lead to a specific immune response against the tumor cells. Recent studies that GRP94 may also be associated with cell cycle, cell differentiation and apoptosis there is a certain relationship. Tumor the uncontrolled proliferation, differentiation of the most serious obstacles to cell disease research GRP94 expression level of the impact of the change on the biological characteristics of the tumor cells, can Beng step to enrich the understanding of the function of GRP94. As a stress protein GRP94 transcriptional level in many physiological and pathological conditions can be significantly increased, the related signal transduction pathway from the endoplasmic reticulum to the nucleus is the unfolded protein response (the unfolded protein response, UPR) pathway . UPR pathway in mammalian cells involves series folding ability gene upregulated, protein \u0026 P enlightenment translate to decline, the GHOP mediated programmed cell death and endoplasmic reticulum related protein degradation more than one branch. These branches have some degree of autonomy, but there are inextricably linked, a branch road effects ingredient change, you might be interested in the entire UPR pathway impact, and even affect the fate of the cell. Research GRP94 expression level changes of each branch of the UPR, not only help to understand the GRP94 functionality, but also helps to understand the changes in the UPR pathway. Based on this, we have chosen the human colorectal cancer cell lines CCL229 as to study object, specific ribozyme targeting down GRP94 expression levels in cells, observe certain biological characteristics and the impact of the UPR pathway of human colorectal cancer cells deepen understanding of GRP94, UPR and tumors among relationship. Method 1. Building (1) human colon cancer cells stably down GRP94 clone plasmid pRc / RSV contain specific targeting GRP94 ribozyme the plasmid pRc/RSV-ribo1 and control group, respectively miniprep Pvu II restriction endonuclease digestion. (2) Determination pRc/RSV-ribo1 and pRc / RSV gene sequences, to a large number of plasmid DNA purification methods to obtain a purified plasmid. (3) in accordance with the Fugene TM 6 transfection reagent manual two plasmid transfection people colorectal cancer of Yichangtai on. (4), respectively, from the mRNA and protein levels by RT-PCR and Western blot method detection GRP94 expression, this identification of a positive clone transfected successfully. 2. Detection GRP94 expression levels on human colorectal biological characteristics of cancer cells (1) light microscope, observed the Gang cellular morphology, adherent state, aggregate growth capacity and growth rate. (2) the cell growth curve, calculate the cell doubling time. (3) detection of cell aggregation ability to calculate the degree of aggregation of the cells. (4) The flow cytometry cell cycle distribution. 3. Detect GRP94 expression levels related molecular chaperone GRP78 and PDI (1) mRNA and protein levels detected molecular chaperone GRP78 expression by RT-PCR and Western blot method. (2) folding enzymes PDI expression from the mRNA and protein levels detected by RT-PCR and Western blot method. 4. Detect GRP94 expression levels of UPR-CHOP pathway (1) use of the Western blot and immune cells chemical methods to detect the UPR pathway specific, closely associated with the programmed death CHOP protein levels. 5. Envy reached the level of detection of stable downward GRP94 cell lines the chemosensitivity (1) the use of fluorescence microscopy and transmission electron microscopy sewing ribozyme transfected cells and the control group from the morphological point of observation cell under different concentrations of cisplatin role withered death of Beng away. (2) using DNA agarose gel electrophoresis analysis of the sewing cellular sensitivity to the drug. (3) the use of the Western blot, caspase-3 active fragment. (4) MTT assay of cell growth inhibition rate. (5) changes in DNA content flow cytometry. Results 1. 5 stably expressing the GRP94 ribozyme clone pRc/RSV-ribo1 plasmid transfected human CCL229 cells to G418 screened 39 resistant clones were cultured for 10 days after the 24-well plates, 25 clones gradually died. The shift in the growth of small flasks, five clones adherent poor growth and death, only nine clones survived. By RT-PCR and Western blot five resistant cell clones A23187 (5μM) after 16h, the expression levels of GRP94 is significantly lower than the control group of cells in the same state, identification force positive clones. Select which one (CCL229T7) follow-up experiments. Transfected with empty vector pRc / RSV cells named CCL229C. Stress inducing cells are represented by \2. GRP94 cell clone stably down the biological characteristics of changes observed by light microscopy found no significant changes in cell morphology, but visible slow cell growth, less adherent and intensive growth of cells less able to passage of the phenomenon. Cell growth curve GRP94 expression levels of cell proliferation significantly reduce negative growth, even under stress. Cell aggregation ability detected in should?

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