miR-124-3p enhances sensitivity of gastrointestinal stromal tumors cells to imatinib by targeting Beclin 1-mediated autophagy
Objective The aim of this study was to investi-gate the relationship between the microRNA miR-124-3p and the sen-sitivity of gastrointestinal stromal tumors(GIST)cells to imatinib(IM),and to preliminarily explore the possible underlying mecha-nisms.Methods According to the corresponding treatments,GIST cells were divided into im+GIST-T1 group,im+GIST-882 group,miR-NC group,miR-124-3p mimic group,miR-124-3p inhibitor group,NC group,im+miR-NC group,im+miR-124-3p mimic group,im+miR-124-3p inhibitor group,IM+miR-124-3p mimic+RAPA group,oe-NC group,oe-BECN1 group,oe-BECN1+miR-124-3p mimic group.Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-124-3p and Beclin-1(BECN1)mRNA in cells.Cell viability and proliferation were detected using cell counting kit-8 assay and EdU fluorescent staining.Apoptosis was detected using flow cytometry.Protein immunoblotting was used to detect protein expression.Au-tophagosome formation was detected using immunofluorescence staining.Dual luciferase reporter gene assay was used to verify the rela-tionship between miR-124-3p and BECN1.The subcutaneous inoculation method was used to construct the GIST-T1 cell transplanta-tion tumor nude mouse animal model.Results qRT-PCR showed that miR-124-3p expression was significantly elevated(P<0.01)in cells of miR-124-3p mimic group(0.351±0.014)compared to miR-NC group(0.023±0.002),while miR-124-3p inhibitor(0.011±0.002)had significantly downregulated miR-124-3p expression(P<0.01).Flow cytometry showed that transfection of miR-124-3p mimic significantly promoted apoptosis in IM-treated GIST-T1 cells(P<0.01),whereas transfection of miR-124-3 inhibitor inhibited apoptosis(P=0.004).CCK-8 results showed that RAPA treatment was able to reverse to some extent the miR-124-3p's enhancing ef-fect on IM sensitivity.Protein immunoblotting analysis showed that BECN1 protein expression and LC3II/LC3I ratio were significantly increased,while p62 protein expression was significantly decreased in cells of the IM+miR-NC group compared with the NC group(P<0.01).BECN1 protein expression and LC3II/LC3I ratio were significantly decreased,while p62 protein expression was significantly in-creased in cells of IM+miR-124-3p mimic group compared with IM+miR-NC group(P<0.01).BECN1 protein expression and LC3II/LC3I ratio were significantly increased in the IM+miR-124-3p mimic group,while p62 protein expression was significantly decreased in the IM+miR-124-3p mimic+RAPA group compared with the IM+miR-124-3p mimic group(P<0.01).Flow cytometry results showed that apoptosis was significantly increased in IM+miR-NC group compared with NC group(P<0.01)and significantly increased in IM+miR-124-3p mimic group compared with IM+miR-NC group(P<0.01).The apoptosis was significantly increased in IM+miR-124-3p mimic group compared with IM+miR-124-3p mimic group(P<0.01)and significantly decreased in IM+miR-124-3p mimic group com-pared with IM+miR-124-3p mimic group(P<0.01).3p mimic+RAPA group apoptosis was significantly decreased(P<0.01).BECN1 was the target gene of miR-124-3p,and miR-124-3p negatively regulated BECN1.miR-124-3p affected the sensitivity of GIST cells to IM by down-regulating BECN1.The results of transplantation tumor experiments in nude mice showed that miR-124-3p overexpression enhanced the inhibitory effect of imatinib on the growth of transplanted tumor cells in nude mice.Conclusion miR-124-3 enhances the sensitivity of GIST cells to IM by negatively regulating BECN1-mediated autophagy.
NETL
NSTL
万方数据
