PF580 PIWIL1 PROMOTES DRUG RESISTANCE IN MULTIPLE MYELOMA THROUGH MODULATING MITOPHAGY

Background: PIWI‐like protein 1 (PIWIL1) is an important member of the Argonaute protein family and plays a key role in tumor cell viability, migration and invasion, and its expression is associated with the maintenance of stem‐like characteristics of tumors. Aims: We aim to determine the role of PIWIL1 in the drug resistance of multiple myeloma (MM) and its mechanism. Methods: RT‐PCR and Western blot were used to detect the expression of PIWIL1 in bone marrow‐derived CD138+ MM cells from multiple myeloma (MM) patients, MM cell lines including RMPI8226, NCL‐H929,U266 and ARH‐77 cells and normal controls. PIWIL1‐siRNA lentivirus, PIWIL1‐cNDA lentivirus and NC were transfected into MM cells to knockdown and knockin PIWIL1 gene. CCK‐8 was used to compare the sensitivity of different group of MM cells to dexamethason. By Annexin V‐fluorescein isothiocyanate/propidium iodide flow cytometry analysis, the apoptosis and cell cycle of PIWIL1 knockin, PIWIL1 knockout and control MM cells were compared. Transmission electron microscope was used to observe the autophagy in MM cells. The expression of autophagy related protein Beclin1, LC3II, p‐AKT, p‐mTOR and P62 and mitophagy related protein BNIP3, optineurin, p‐TBK‐1 was detected by Western blot. PIWIL1 knockin MM cells, PIWIL1 knockout MM cells and control cells were injected subcutaneously into BALB/C nude mice. Tumor growth was monitored every 3 days for about 3 weeks. Tumor volume was compared among different group and immunohistochemical staining was used to detect the expression of P62, p‐mTOR and optineurin expression in tumor mass. Results: In this study, we found that PIWIL1 mRNA and protein were upregulated in bone marrow‐derived CD138+ MM cells from multiple myeloma (MM) patients and MM cell lines including RMPI8226, NCL‐H929,U266 and ARH‐77 cells and that PIWIL1 mRNA level positively correlated with disease stage. PIWIL1 gene knockin promoted the proliferation of RMPI8226 and NCL‐H929 MM cells and decreased its sensitivity to dexamethasone compared with controls. Conversely, PIWIL1 gene knockout inhibited the proliferation of MM cells and increased its sensitivity to dexamethasone. Neither PIWIL1 gene knockin nor knockout could affect the apoptosis and cell cycle in MM cells. But the percentage of autophagosome, especially mitophagy was significantly increased in PIWIL1 overexpressed MM cells by transmission electron microscope detection in comparison to control cells. The expression of autophagy marker protein Beclin1, LC3II, p‐AKT and mitophagy related protein BNIP3, optineurin, p‐TBK‐1 was significantly increased in PIWIL1 knockin MM cells, while the expression of p‐mTOR and P62 was significantly decreased. In contrast, PIWIL1 gene knockout in MM cells had diametrically opposite effects on autophagy related protein. PIWIL1‐cDNA lentivirus transfected‐MM cells, PIWIL1‐siRNA lentivirus transfected‐MM cells and control cells were injected subcutaneously into nude mice. Pre‐transfection of PIWIL1‐cDNA into RPMI8226 cells led to a modest increase in tumor volume compared with NC transfectant, accompanied by decreased P62, p‐mTOR expression and increased optineurin expression in tumor mass. While PIWIL1‐siRNA lentivirus pre‐transfection inhibited the growth of xenograft MM in mice. Summary/Conclusion: All together, these findings indicate that PIWIL1 may confer a survival advantage to MM cells through modulating mitophagy and that it may be a novel therapeutic target for reversing drug resistance for MM.