| Objective:CD 147 was originally identified as a tumor surface protein capable of inducing matrix metalloproteinase (MMP) expression in fibroblasts. It has been implicated in many tumors and may be a target protein for cancer research. However, researchers found that CD 147 expression was not limited to tumor cells and that this pleiotropic molecule is critical for retinal function, fetal development, thymic T-cell development and other normal functions. Research shows that the effectiveness of MMP inhibitors for cancer treatment is not very ideal, and with the aim to target on the MMPS inducers of CD 147 treatment may be an effective technique. This method can inhibit the expression of CD 147 to suppress the secretion of MMP and its activity, which provide a new direction and the treatment of diseases associated with MMP strategy. So, how to control and influence the expression of CD 147 became the key to solve the problem. At present, the structure, function and interaction of CD 147 protein have wider understanding, but the study of the expression regulation and biodegradation pathway was not reported. All formation and degradation can affect the level of protein. In eukaryotic cells, the ubiquitin-proteasome pathway is a main protein degradation pathway of protein degradation that mediated by regulating protein in the cell’s level and function of an important mechanism, about 80% or more of the protein in the cell degradation by this way. In adjustment, control, signal transduction and cell cycle regulation, apoptosis, UPS plays an important role and become a treatment target tumor, abnormal pathway may lead to diseases, however whether CD 147 is also associated with the degradation pathway or not is still unclear. Study the mechanism and its signal transduction process of CD147, in order to explore the blocking the conduction or the effective method to restrain the effect of its, maybe can provide us a new approach for effective suppress malignant tumor, neurodegenerative diseases.Using westernblot and in-cell western methods to investigate the effect of proteasome inhibitor MG-132 on expression of extracellular matrix metalloproteinases factor (CD 147) and preliminary discuss the degradation pathway of CD147. Provide a theoretical basis for the therapeutic strategies and directions of neoplastic disease.MG-132 is a kind of aldehyde peptide proteasome inhibitors and wide application of proteasome inhibitor. It can inhibit the 20S catalytic particle classes in chymotrypsin activity, prevent the degradation of protein of ubiquitin proteasome.Using westernblot and in-cell western methods to investigate the effect of proteasome inhibitor MG-132 on expression of CD 147 and preliminary discuss the degradation pathway of CD 147. Provide a theoretical basis for the therapeutic strategies and directions of neoplastic disease.Methods: 1 MaterialSH-SY5Y cell, cultured in 90% DMEM containing 10% fetal calf serum, 37℃,5% CO2,5-7 days. 2 WesternblotCells (SY5Y) were cultured in 6-well plates, then were treated with proteasomal inhibitors MG-132. MG-132 was used at 0,1,2.5 and 5μmol/L for 24h for the dosage assay. The cell was homogenized by an ultrasonic cell disruptor in RIPA lysis buffer. The homogenate was centrifuged at 4℃, and the supernatant was retained. The protein were boiled for 5 minutes at 95℃ before loading on a SDS-PAGE gel. Electrophoresis was performed and then the proteins were transferred onto a PVDF membrane. The membrane was blocked and then incubated with anti-CD 147 antibodyovernight at 4℃. The membrane was incubated with anti-rabbit-fluorescence secondary antibody followed by washing. The proteins were detected using the Odyssey IR fluorescence scanning imaging System, and the band density values were normalized against GAPDH.3 In-cell westernCells (SY5Y) were cultured in 96-well plates, then were treated with proteasomal inhibitors MG-132. MG-132 was used at 0,1,2.5 and 5μmol/L for 24h for the dosage assay. The proteins were detected using the Odyssey IR fluorescence scanning imaging System, and the band density values were normalized against β-actin.4 Dual-labeling immunofluorescenceSY5Y cells were cultured on glass coverslips in 24-well plates for 48h and fixed with 4% paraformaldehyde for 0.5h. After washed with PBS and incubated with blocking solution for 2h, coverslips were incubated with anti-CD 147 antibody and anti-ubiquitin antibodyin PBS containing 1% goat serum overnight at 4℃, respectively. After being washed extensively, sections were detected with a fluorescence-marked secondary antibody Dylight 488 /Dylight 594. The images were acquired with Olympus universal microscope.Results:1 Westernblot analytical data indicated that in a certain period of time, with the increase of concentration of MG-132, the relative content of CD147 relatively increased; compared with 0 μmol/L group, the content of CD 147 of5 μmol/L group increased by 151.81 ±36.26%(P<0.05)2 In-cell western analytical data indicated that in a certain period of time, with the increase of concentration of MG-132, the relative content of CD 147 relatively increased; compared with 0 μmol/L group, the content of CD 147 of 5 μmol/L group increased by 76.43±18.02%(P<0.05).3 CD 147 and ubiquitin existed co-localized characteristic in the soma.Conclusions:1 Proteasome inhibitor MG-132 can influence the expression of CD147 and there is a positive correlation. may.The content of CD 147 was increased after giving proteasomal inhibitors MG-132.2 Speculated that MG-132 could increase the expression level of CD 147 through inhibit the degradation of CD147. |
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