| ObjectiveWe investigate the expression of microrna-637(（miR-637）in bone marrow specimens of patients with multiple myeloma（MM）and MM cell lines（U266 and RPMI8226）.Transfect miR-637 mimics and inhibitors with Lipofectamine TM 3000 liposomes to construct MM cell lines transfected with NUPR1 lentivirus,and investigate the effects and mechanism of miR-637 targeting NUPR1 on the proliferation,apoptosis and autophagy of MM cells the study.Methods1.Collect bone marrow specimens from 36 newly diagnosed MM patients and 21 healthy adults.Extract bone marrow mononuclear cells.2.In vitro cell experiment: q RT-PCR and Western blot were used to detect the expression of miR-637 in bone marrow mononuclear cells and MM cell lines（U266 and RPMI8226）.Analysis of four bioinformatics databases showed that miR-637 and NUPR1 were associated with each other.In this experiment,we examined the correlation between miR-637 and NUPR1 using dual luciferase experiments.Cell counting kit-8（CCK-8）method was used to observe the proliferation of MM cells.Apoptosis and autophagy reaction levels,flow cytometry and western blot to detect phagocytic function.Western blot detects PTEN,Phosphorylated AKT and AKT pathway proteins.Results1.Low discovery of miR-637 in bone marrow specimens of newly diagnosed MM patients: Compared with healthy adults,the discovery of miR-637 in bone marrow specimens of newly diagnosed MM patients is decreasing.2.miR-637 inhibits MM cell proliferation,autophagy,and promotes apoptosis: transfect mimics NC（nonspecific control）,miR-637 mimics,inhibitor NC and miR-637 inhibitors to MM cells respectively.To verify the transfection efficiency of miR-637 mimics and inhibitors,q RT-PCR and green fluorescence microscope were used to verify the transfection efficiency.The CCK-8 experimental method was used to detect the cell proliferation ability of miR-637 in 1 to 5 days.The results showed that the proliferation ability of miR-637 mimics was lower than that of the NC group.On the contrary,after transfection with miR-637 inhibitors,MM cells Increased proliferation ability.U266 and RPMI8226 cells were observed by V/PI staining,and the apoptosis rate of miR-637 mimics increased significantly.After transfection of miR-637 mimics,the expression of apoptosis index Bax,cleaved caspase 3 and autophagy index p62 increased,while the ratio of apoptosis index Bcl2 and autophagy index LC3 decreased.3.miR-637 targets NUPR1 in MM cells: through bioinformatics prediction analysis and dual luciferase experimental detection,the results indicate that NUPR1 is miR-637 potential targets.Protein expression level significantly reduced the expression of NUPR1 after transforming miR-637 mimics.On the contrary,after liposome transfer of inhibitors.The expression level of NUPR1 was significantly increased.Expression levels of NUPR1 in bone marrow samples of newly diagnosed MM patients were significantly higher than those of healthy adults and newly diagnosed MM patients.NUPR1 has a negative correlation with miR-637.4.NUPR1 mediates the effect of miR-637 on MM cells: NUPR1-LV is transfected into U266 and RPMI8226 cell lines,and the transfection rate of NUPR1 overexpression virus is detected by green fluorescence microscope.Combined with CCK-8,flow cytometry,Western blot and other experiments,the research results show that after transfection of NUPR1-LV,miR-637 has an inhibitory effect on the proliferation and apoptosis of MM cells,and has a promoting effect on autophagy.5.miR-637 targeting NUPR1 regulates MM cell apoptosis and autophagy through PTEN/AKT pathway: after transfection of miR-637 mimics,PTEN of MM cells decreased;phosphorylated protein expression Phosphorylated AKT increased,but total AKT expression did not change significantly.Conclusion1.miR-637 was low expressed in mononuclear cells of patients with multiple myeloma.2.miR-637 inhibits the proliferation and autophagy of MM cells by regulating NUPR1 to promote the apoptosis of MM cells.3.miR-637 is another molecular mechanism that affects the occurrence and development of MM. |
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