LncRNA NEAT1 sponges miR-214 to regulate M2 macrophage polarization by regulation of B7-H3 in multiple myeloma.

BACKGROUND: LncRNA NEAT1 was associated with the tumorigenesis of multiple myeloma (MM). However, the mechanisms of M2 macrophage polarization involved with NEAT1 in MM are still unknown. METHODS: Bone marrow samples, multiple myeloma cells RPMI 8226 and monocyte cell line THP-1 were used in this study. The expression of NEAT1 and miR-214 was modified by transfection with the shNEAT1 or miR-214 inhibitor. The expression of NEAT1, miR-214 and B7-H3 in MM patient tissues and cells was analyzed by RT-qPCR. ELISA assay was used to determine the release of B7-H3 in the supernatant of cell culture. The patient survival curve was analyzed using Kaplan-Meier method. The macrophage polarization markers were examined by RT-qPCR and western blotting. The interaction between NEAT1, miR-214 and B7-H3 was analyzed by Dual-Luciferase reporter and RIP assays. AG490 was used to block the JAK2/STAT3 signaling. Co-culture of THP-1 and RPMI 8226 cells was used for macrophage polarization. RESULTS: NEAT1 and B7-H3 were up-regulated, but miR-214 was obviously down-regulated in MM patients. B7-H3, NEAT1 and miR-214 were associated with overall survival time of MM patients. NEAT1 silencing induced miR-214 and inhibited the expression and release of B7-H3 and then suppressed M2 macrophage polarization via inhibiting the JAK2/STAT3 signaling. NEAT1 directly targeted miR-214, and miR-214 directly bound to B7-H3. MiR-214 inhibitor reversed the down-regulation and release of B7-H3 and M2 macrophage polarization caused by shNEAT1. The specific JAK2/STAT3 signaling inhibitor AG490 abrogated M2 macrophage polarization. CONCLUSION: NEAT1 promoted M2 macrophage polarization by sponging miR-214 and then regulating B7-H3, thus accelerating MM progression via the JAK2/STAT3 signaling pathway. Our study revealed novel mechanisms of M2 macrophage polarization and provided new potential clinical therapeutic targets for MM.

[Effect of all-trans retinoid acid and G-CSF on growth, differentiation and RARα2 expression of myeloma cells].

OBJECTIVE: To investigate the effect of all-trans retinoid acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) on the growth, apoptosis, differentiation and expression of RARα2 of myeloma cells. METHODS: Myeloma cell lines OPM2 (RARα2 positive) and U266 (RARα2 negative) were treated with ATRA in the presence or absence of G-CSF. The cells were divided into 6 groups: control groups, G-CSF groups (treated with 1000 U/ml and 2000 U/ml), ATRA groups (treated with 1.0 µmol/L ATRA) and combined groups (treated with 1000 U/mL or 2000 U/mL G-CSF plus 1.0 µmol/L ATRA). The cell viability, growth and apoptosis were examined by MTT method, inverted microscopy and Annexin-V/PI staining, respectively; RARα2 expression was detected by reverse transcription PCR; morphology change was evaluated by Wright-Giemsa staining; CD49e expression were analyzed by flow cytometry. RESULTS: The proliferation of OPM2 cells was inhibited by ATRA treatment (P<0.05) . The growth inhibition rates in combined groups were higher than corresponding single ATRA groups (P<0.05). However, the above effects in U266 cells were not significant (P >0.05). The OPM2 cell stained by Wright-Giemsa in ATRA groups showed that the cell nucleus became smaller, chromatin condensed, number of nucleolus reduced, the volume of cytoplasm increased and the cytoplasm became dark blue. Expression rates of CD49e were low in both U266 and OPM2 cells. Expression of RARα2 in OPM2 cells of combination groups were higher than those of control group and corresponding single groups (P<0.05); and there was no significant difference between control group and G-CSF groups (P>0.05). Expression of RARα2 in U266 cells of control group and G-CSF groups was not detected; and ATRA groups and combination groups had weak expression. CONCLUSION: ATRA can induce proliferation inhibition in RARα2-expressing myeloma cells, and it may also play a certain role in promoting differentiation of RARα2 positive myeloma cells.