The role of DEPTOR-mTOR signaling pathway-mediated autophagy on osteoclasts differentiation and maturation in multiple myeloma
ZHANG Haoran1 QIAO Xuxu1 BI Minghong1 ZHAI Yunzhi1 QIAN Liyu2 PAN Chengwu2 ZHAO Lun1
1.Department of Medical Oncology, the First Affiliated Hospital of Bengbu Medical College, Anhui Province, Bengbu 233004, China;
2.Department of Surgical Oncology, the First Affiliated Hospital of Bengbu Medical College, Anhui Province, Bengbu 233004, China
Abstract：Objective To study the role of DEPTOR knockdown in RPMI-8226 cells on the protein expression of RANKL and differentiation of THP-1 into osteoclast-like cells in a contactless co-culture system and its possible mechanism. Methods Constructed DEPTOR shRNA expression vector GV115-shRNA was transferred into RPMI-8226 cell to produce packaged lentivirus. Western blot was applied to measure the protein levels of DEPTOR and RANKL. The expression of autophagy-associated proteins LC-3 and Atg5 were confirmed by Western blot analysis. Three groups were divided：THP-1 group, THP-1 + RPMI-8226 group and THP-1 + DEPTOR shRNA group. Osteoclast-like cells were identified by TRAP. The mRNA levels of calcitonin receptor (CTR) and Cathepsin-K were examined using RT-PCR. Results The results showed that protein expression levels of DEPTOR and RANKL were significantly lower in RPMI-8226 cells transfected with GV115 DEPTOR shRNA compared with that in untransfected cells (P < 0.05). The expression levels of autophagy-associated proteins LC-3 and Atg5 in the DEPTOR shRNA group were significantly lower than those in the control shRNA group and the parental group (P < 0.05). In the co-culture system, THP-1 cell could differentiate into TRAP positive multinuclear cells. RPMI-8226 promoted mRNA expression of CTR and Cathepsin-K (P < 0.05). DEPTOR shRNA suppressed osteoclast-like cells formation and decreased CTR and Cathepsin-K mRNA expression in co-cultures, the differences were statistically significant (P < 0.05). Conclusion In the coculture system, DEPTOR shRNA inhibits the differentiation of THP-1 cells into TRAP positive multinuclear cells, which may be due to its inhibition on RANKL expression in RPMI-8226 cells, and the inhibition of autophagy will restrain osteoclast maturation.
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2018, Vol. 15 
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