| BackgroundMultiple myeloma (MM) is a malignant neoplasm showing abnormal plasma cells proliferation with bone lesions, kidney damage, anemia and other clinical symptoms. It is known to be the second common hematologic malignancy worldwide and there is an upward trend in its incidence in China. Proteasome inhibitor bortezomib is an effective approach to the treatment of MM, the efficiency of bortezomib based chemotherapy regimens is as high as80-90%, but drug resistance emerges and limits its retreatment while the mechanisms are not fully understood. The mechanism of bortezomib resistance and the solution to overcome it is a vital clinical issue. Ubiquitin proteasome pathway is the main way of protein degradation in cells while E3ubiquitin ligase determine the specificity of protein degradation closely related to cancer occurrence, development, transfer, and drug resistance. Several lines of evidence link E3ligase to myeloma and its drug resistance. Pirh2(p53-induced RING-H2) is a new discovery E3ubiquitin ligase induced by p53activation with intrinsic ubiquitin-protein ligase activity for polyubiquitination and subsequently proteasomal degradation, first thought to be coordinated with HDM2.In recent years, substrate protein for Pirh2was gradually found to confirm Pirh2being associated with malignant cancer occurrence and prognosis. HDM2/p53was predisposed for plasma cell hyperplasia, tumorigenesis and bortezomib resistance as reported. Here we further investigate the role of E3ligase Pirh2in MM bortezomib resistance to obtain a better understanding,which could facilitate botezomib retreatment as a more effective therapy and a rational therapetic stratege clinically.Objective1. To clarify the role of Pirh2in bortezomib resistance, establish bortezomib-resistant cell line and identify the expression of Pirh2changes.2. To develop Pirh2knock out and overexpression MM cells, to explore Pirh2’s effect on the MM cell biology behavior and to identify some of the mechanisms involved. To study Pirh2effect on myeloma cells in vivo by transplantation myeloma model in NOD/SCID mice.3. To explore the substrate protein of Pirh2in MM cells may interact with bortezomib resistance.Methods1. Study the expression of Pirh2on MM cell lines RPMI8226, ARP-1, ARK, MM IS, MM1R, NCI-H929, OPM-2and LP-1by immunofluorescence, Western blot, RT-PCR technique. Compare the expression of Pirh2in newly diagnosed patients with recurrent and refractory MM by RT-PCR. Develop bortezomib-resistant cell lines by continuous culture in sub-lethal concentrations of bortezomib.2. Develop Pirh2knock out and overexpression slow virus vector transfection MM cells, to explore Pirh2on the MM cell biology behavior and to identify some of the mechanisms involved by CCK8, flow cytometry and Western blot.3. The immune coprecipitation technology (Co-IP) combined with mass spectrometry for seeking substrate protein of Pirh2in MM cells.4. Use the gene expression profile for bortezomib-resistant cell and parental cells, at the same time with Pirh2knockout and overexpression cell lines, combined with the Co-IP and the result of mass spectrometry to explore the substrate protein of Pirh2in bortezomib-resistant MM cells.5. Subcutaneous injection of transfection MM cells to study Pirh2effect on myeloma cells in vivo by transplantation myeloma model in NOD/SCID mice.Results1. We confirmed generally Pirh2mRNA and protein expressions in MM cell lines RPMI8226, ARP-1, ARK, MM1S, MM1R, NCI-H929, OPM-2and LP-1and primary MM cells. ARK, ARP-1, LP-1cell lines is relatively low expressed, immunofluorescence showed Pirh2in MM cells mainly expressed in cytoplasm. We found differential expression of Pirh2in bortezomib-resistant cells. Besides, Pirh2mRNA expression was also determined from bone marrow samples of MM patients. In summary, we showed that Pirh2is more highly expressed in newly diagnosed patients compare with refractory/recurrence patients (P<0.05).2. The IC50of NCI-H929and NCI-H929.BR treated with bortezomib for24h were17.62±1.92nmol/L vs234.30±6.02nmol/L by CCK8method, the resistance ratio was13.30(p<0.05). Growth curve and flow cytometry results showed no significant difference between two (P>0.05).3. Pirh2expression were decreased in bortezomib-resistant cell lines NCI-H929.BR RPMI8226.BR and OPM-2.BR.4. Growth curve and flow cytometry results showed no significant difference between Pirh2knock out, overexpression slow virus vector transfection MM cells and controls (P>0.05).5. Flow cytometry results showed Pirh2knock out reduce MM cells apoptosis and cell cycle arrest in G1phase. The percentage of G1phase in groups were RPMI8226-kd vs RPMI8226-ctl39.03±3.20%vs52.8±42.89%, OPM-2-kd vs OPM-2-ctl42.40±5.84%vs57.00±6.23%, NCI-H929-kd vs NCI-H929-ctl23.372.12%vs42.91±1.89%(p<0.05) Pirh2knock out weaken inhibition of cell proliferation ability for bortezomib by CCK-8method. The percentage of apoptosis cells in groups were RPMI8226-kd vs RPMI8226-ctl47.90±1.63%vs55.60±2.86%, OPM-2-kd vs OPM-2-ctl48.30±1.17%vs63.60±1.24%, NCI-H929-kd vs NCI-H929-ctl20.28±0.98%vs38.37±1.34%(p<0.05).6. Flow cytometry results showed Pirh2overexpression induce MM cells apoptosis and cell cycle increased in G1phase. The percentage of G1phase in groups were ARP-1-Pirh2vs ARP-1-ctl51.56±3.91%vs40.88±2.09%, ARK-Pirh2vs ARK-ctl49.10±4.32%vs31.90±3.98%, LP-1-Pirh2vs LP-1-ctl58.90±4.06%vs32.40±2.76%(p<0.05). Pirh2overexpression increased inhibition of cell proliferation ability for bortezomib by CCK-8method. The percentage of apoptosis cells in groups were ARP-1-Pirh2vs ARP-1-ctl66.90±3.73%vs41.70±1.86%, ARK-Pirh2vs ARK-ctl76.80±4.17%vs66.60±3.24%, LP-1-Pirh2vs LP-1-ctl22.02±1.23%vs15.53±2.03%(p<0.05).7. c-myc, p53level increased in Pirh2knockout cells RPMI8226-kd, OPM-2-kd and NCI-H929-kd with controls while decreased in overexpression cell lines ARP-1-Pirh2, ARK-Pirh2, LP-1-Pirh2. No changes were found in levels of HDAC1, Tip60, USP28.8. Pirh2overexpression or not did not obviously lower tumor burdens as compared with groups of controls in vivo, but c-myc decreased with controls.9. Co-IP and mass spectrometry identified Pirh2interaction protein DLAT、PDHX、 PDHA1、PDHB, TRIM21and RPL13、RPL10while TRIM21and RPL13were decreased in bortezomib-resistant cells, but whether they mediated bortezomib resistance still needs further study.Conclusions1. We confirmed generally Pirh2mRNA and protein expressions in MM cell lines RPMI8226, ARP-1, ARK, MM1S, MM1R, NCI-H929, OPM-2and LP-1and primary MM cells. We found differential expression of Pirh2in bortezomib-resistant cells. Besides, Pirh2mRNA expression was also determined from bone marrow samples of MM patients. In summary, we showed that Pirh2is more highly expressed in newly diagnosed patients compare with refractory/recurrence patients.2. Pirh2mediated the sensitivity of myeloma cells to bortezomib. Pirh2knock out reduce MM cells apoptosis and cell cycle arrest in G1phase whilePirh2overexpression induce MM cells apoptosis and cell cycle increased in G1phase.3. Pirh2can regulate c-myc, p53level to induce proliferation and apoptosis of myeloma cells.4. TRIM21, RPL13is likely to be the substrate proteins of Pirh2in MM cells, but whether they mediated Bortezomib resistance still needs further study. |
PDF Full Text Request