The Role Of PGC-1α In Multiple Myeloma And Its Underlying Mechanism

Part I Expression of PGC-la in multiple myeloma and its effect on cell proliferationObjective:To explore the expression of peroxisome proliferator activated receptor y coactivator-la(PGC-la) in multiple myeloma(MM) and determine the effect of PGC-la on proliferation of MM cells.Methods:MM RPMI8226and U266cells as well as normal human peripheral blood B lymphocytes from independent volunteer and lymphocytes of clinical specimens from MM patients were collected. Real-time PCR were applied to detect the expression of PGC-la and ERR-a mRNA in MM samples. After treating RPMI8226cells with small interfering RNA(siRNA) targeting PGC-la, expression of PGC-la was measured by Western blotting, and cell proliferation was checked by CCK-8.Results:Results of RT-PCR showed that the expression of PGC-la and ERR-a mRNA in RPMI8226cells were significantly elevated when compared with normal control. About55.6%of MM specimens exhibited an increased expression of PGC-la mRNA. siPGC-la inhibited the level of PGC-la. The result from CCK-8assay showed that the proliferation of RPMI8226cells was hampered after siPGC-la treatment. Conclusion:PGC-la was aberrantly expressed in MM RPMI8226cells and55.6%of clinical specimens of MM patients, and it is involved in prompting proliferation of MM cells. Part II The underlying mechanisms of PGC-1α promotes multiple myeloma cells proliferationObjective:To explore the possible mechanisms of PGC-la involved in proliferation of MM cells by changing expression of PGC-la.Methods:Inhibition of PGC-1α in RPMI-8226cells was achieved by using small interfering RNA (siRNA). RT-PCR was used to test the expression of PGC-1α, estrogen-related receptor-a (ERR-a), vascular endothelial growth factor (VEGF) and glucose transporter-4(GLUT-4) in MM. Western blotting analysis was used to detect the protein expression of PGC-la, ERR-a and GLUT-4. Transwell chambers were used to perform endothelial cell migration assay after treated RPMI8226cells with siPGC-la, and CCK-8assay was applied to evaluate the proliferation of RPMI8226cells.Results:PGC-la, ERR-a, VEGF and GLUT-4mRNA levels were decreased in RPMI8226cells after treating with siPGC-la(p<0.05), as well as the protein expression of PGC-la, ERR-a and GLUT-4. The proportion of endothelial cells successfully migrated from the chamber was significantly decreased after culturing with culture medium collected from RPMI8226cells treating with siPGC-1a. Inhibition of ERR-a also resulted in the decreased proliferation rates at24and48h.Conclusion:PGC-la affects cell proliferation of RPMI8226cells by regulation of VEGF and GLUT-4. Thus, PGC-la may be involved in metabolism and angiogenesis of myeloma. Part III Regulation of PGC-la influences sensitivity to chemotherapeutic agents in multiple myeloma cellsObjective:To explore whether PGC-la played an important role in regulation of sensitivity to chemotherapeutic agents in MM cells.Methods:The expression of PGC-la, SOD-2and GPX-1in MM RPMI8226cells after bortezomib treatment was measured by using RT-PCR. Western blotting was applied to detect the protein expression of PGC-1a in MM cells after chemotherapy. After disposing the siPGC-la treated MM RPMI8226cells with bortezomib, the level of reactive oxygen speices(ROS) and apoptosis was tested by Flow cytometry (FCM) analysis. Toxicity of bortezomib on cell proliferation after siPGC-1a treatment was assessed by CCK-8assay.Results:After treating RPMI8226cells with low dose bortezomib, the results of RT-PCR and Western blotting showed that expression of PGC-la in myeloma cells was up-regulated, as well as the mRNA expression of SOD-2and GPX-1. After inhibiting PGC-1a in MM RPMI8226cells, levels of PGC-la, SOD-2and GPX-1were all decreased. FCM assay revealed that ROS level as well as apoptosis was elevated after treatment of siPGC-la. Suppression of PGC-la by siRNA resulted in increased accumulation of ROS, and enhanced toxicity of bortezomib.Conclusion:PGC-la affects sensitivity to chemotherapeutic agents of MM cells probably via regulation of SOD-2and GPX-1that are the key factors determining the cellular ROS level, and inhibition of PGC-la could enhance the proapoptoic effect of chemotherapy in MM.