miR-1248 enhances bortezomib-induced autophagy by targeting MEF2C/p38-MAPK signaling in multiple myeloma
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Objective Multiple myeloma (MM) is incurable and many patients respond poorly to bortezomib. New strategies to improve bortezomib sensitivity are needed. Methods We screened bortezomib-responsive miRNAs by RNA-seq in RPMI8226 cells (fold change >4, P<0.05). miR-1248 was measured by qRT-PCR in MM cells and in plasma from 30 patients before/after VCD therapy. MEF2C was examined by qRT-PCR and Western blot. A dual-luciferase reporter assay was performed to test miR-1248 binding to the MEF2C 3′-UTR. After miR-1248 inhibitor transfection, we detected MEF2C, p38 and autophagy markers by qRT-PCR/Western blot and assessed autophagic activity by staining. Rescue experiments overexpressed MEF2C., a xenograft model (n=6) was used to detect these markers after miR-1248 inhibition plus bortezomib(0.5 mg/kg). Results Bortezomib increased miR-1248 by 2-fold (P<0.05) and reduced MEF2C mRNA by 62% and protein by 55% (both P<0.01). Plasma miR-1248 also rose after VCD therapy (median 2.1-fold, P<0.05). Luciferase assays confirmed direct targeting of the MEF2C 3′-UTR by miR-1248. Inhibiting miR-1248 reduced bortezomib-induced autophagy markers and staining, and partially restored MEF2C and p38 (P<0.05). MEF2C overexpression weakened bortezomib-induced apoptosis (cell viability rose from 42 ± 5% to 71 ± 6%, P<0.01) and suppressed those markers. In xenografts, miR-1248 knockdown cut tumor inhibition from 58 ± 7% to 23 ± 9% (P<0.01), with partial restoration of MEF2C/p38 and blunted autophagy marke
Abstract
Objective Multiple myeloma (MM) is incurable and many patients respond poorly to bortezomib. New strategies to improve bortezomib sensitivity are needed. Methods We screened bortezomib-responsive miRNAs by RNA-seq in RPMI8226 cells (fold change >4, P<0.05). miR-1248 was measured by qRT-PCR in MM cells and in plasma from 30 patients before/after VCD therapy. MEF2C was examined by qRT-PCR and Western blot. A dual-luciferase reporter assay was performed to test miR-1248 binding to the MEF2C 3′-UTR. After miR-1248 inhibitor transfection, we detected MEF2C, p38 and autophagy markers by qRT-PCR/Western blot and assessed autophagic activity by staining. Rescue experiments overexpressed MEF2C., a xenograft model (n=6) was used to detect these markers after miR-1248 inhibition plus bortezomib(0.5 mg/kg). Results Bortezomib increased miR-1248 by 2-fold (P<0.05) and reduced MEF2C mRNA by 62% and protein by 55% (both P<0.01). Plasma miR-1248 also rose after VCD therapy (median 2.1-fold, P<0.05). Luciferase assays confirmed direct targeting of the MEF2C 3′-UTR by miR-1248. Inhibiting miR-1248 reduced bortezomib-induced autophagy markers and staining, and partially restored MEF2C and p38 (P<0.05). MEF2C overexpression weakened bortezomib-induced apoptosis (cell viability rose from 42 ± 5% to 71 ± 6%, P<0.01) and suppressed those markers. In xenografts, miR-1248 knockdown cut tumor inhibition from 58 ± 7% to 23 ± 9% (P<0.01), with partial restoration of MEF2C/p38 and blunted autophagy marker elevation. Conclusions Our findings indicate that miR-1248 enhances bortezomib sensitivity in MM cells by directly targeting MEF2C, accompanied by reduced p38 expression and autophagic activity. The coordinated changes in MEF2C and p38 define a functional axis that may be targeted to improve bortezomib response.