An investigation of CD147 expression in apoptotic vesicles

Apo-V;apoptotic;vesicles;CD147;CD169;cancer;melanoma;B16;EL4;tumour;metastasis;transfection;knockdown;GFP;FLAG;spleen;lymph;node;immunology;immunity;glycoproteins

Coutinho, Frazer Paul ; McLellan, Alexander Donald

University of Otago

关键词: Apo-V;    apoptotic;    vesicles;    CD147;    CD169;    cancer;    melanoma;    B16;    EL4;    tumour;    metastasis;    transfection;    knockdown;    GFP;    FLAG;    spleen;    lymph;    node;    immunology;    immunity;    glycoproteins;   

Others  :  https://ourarchive.otago.ac.nz/bitstream/10523/4607/1/CoutinhoFrazerP2014MSc.pdf

美国|英语

来源: Otago University Research Archive

【 摘 要 】

Our laboratory has recently found that glycoprotein CD147 (Basigin, EMMPRIN) is highly enriched on the surface of apoptotic vesicles (Apo-V) isolated from EL4 and B16 tumour cells. The CD147 protein can appear at a range of sizes due to the presence of multiple isoforms and glycosylation processes. CD147 has been described as an anti-apoptotic molecule and is involved in the invasion and metastasis of metastatic cancers. Apoptotic vesicles (Apo-V) have been shown to bind the CD169 sialic acid-receptor expressed by macrophages in the sub-capsular sinus of lymph nodes and the marginal zone of the spleen. It has been suggested that this interaction of vesicles with the CD169 receptor may have the potential to suppress immune responses. In this investigation, the expression and role of CD147 in Apo-V was observed. After many failed attempts to knockdown CD147 expression in EL4 cells using small hairpin RNA (shRNA) techniques, an alternate pathway was undertaken using B16 melanoma cells. The observed knockdown in cellular fractions of B16 was also replicated in corresponding Apo-V fractions. No significant changes in Apo-V production in the CD147 shRNA knock-down B16 cell line were observed, neither were there major changes in Coomassie blue bands as analysed by polyacrylamide gel electrophoresis. This suggested that CD147 knockdown did not affect apoptotic vesicle production quality. The binding of Apo-V to spleen and lymph node sections was not affected by the knockdown of CD147, suggesting that the CD147 glycoprotein is not the major ligand for CD169 in lymphatic tissues. To further investigate the potential of CD147 to bind CD169, a CD169-Fc-FLAG protein was produced. However, no specific binding of CD169-Fc-FLAG to CD147 was detected.Since CD147 has been implicated in tumour metastasis, the metastatic potential of CD147 knockdown B16 cell lines was next tested to confirm the knockdown. This resulted in functional changes in the cell line behaviour. However, no conclusive evidence for an effect of CD147 knockdown on metastasis was noted.