BACKGROUND: Previous studies demonstrated that hypoxia preconditioning promotes bone marrow mesenchymal stem cell survival and angiogenesis. However, specific mechanism remained unclear. Both RNA-MALAT1 (MALAT1) and microRNA-195 (miR-195) are associated with bone marrow mesenchymal stem cell survival and angiogenesis. MALAT1 promotes bone marrow mesenchymal stem cell survival and angiogenesis, whereas miR-195 is a negative regulator of bone marrow mesenchymal stem cell survival and angiogenesis.
OBJECTIVE: To investigate whether hypoxia preconditioning could activate MALAT1 to target and downregulate miR-195 to further promote bone marrow mesenchymal stem cell survival and pro-angiogenic potential in vitro.
METHODS: Bone marrow mesenchymal stem cells cultured in vitro were divided into six groups: normoxia (20% O2), hypoxia preconditioning (1% O2), hypoxia preconditioning+si-MALAT1, hypoxia preconditioning+si-MALAT1 negative control, hypoxia preconditioning+miR-195 and hypoxia preconditioning+miR-195 negative control. MALAT1 siRNA and relevant scramble were transfected into the hypoxia preconditioning+si-MALAT1 and hypoxia preconditioning+si-MALAT1 negative control groups respectively prior to hypoxia preconditioning, and miR-195 mimics and relevant negative control were transfected into the hypoxia preconditioning+miR-195 and hypoxia preconditioning+miR-195 negative control groups respectively prior to hypoxia preconditioning. Mesenchymal stem cells in different groups were cultured for 24 hours and cell proliferation, apoptosis and vascularization were evaluated. The expression levels of MALAT1 and miR-195 were detected using qRT-PCR. The potential complementary binding sites of MALAT1 and miR-195 were predicted by RegRNA 2.0. miR-195 mimics and miR-195 mimics negative control were co-transfected into 293T cells with the luciferase reporters containing MALAT1 or MALAT1-mut. Luciferase activity was detected in different groups of cells in order to verify the relationship between MALAT1 and miR-195.
RESULTS AND CONCLUSION: (1) Compared with the normoxia group, the hypoxia group presented more rapid growth; cells apoptosis percentage was significantly declined; and number of vascular lumen like structures increased (P < 0.01). The expression of MALAT1 was upregulated, while the level of miR-195 was significantly decreased in the hypoxia group (P < 0.01). (2) Compared with the hypoxia preconditioning and hypoxia preconditioning+si-MALAT1 negative control groups, hypoxia preconditioning+si-MALAT1 group presented much lower growth rate, and cells apoptosis percentage was significantly higher; number of vascular lumen like structures decreased (P < 0.01). The expression of MALAT1 was decreased, while the level of miR-195 was increased (P < 0.01). Compared with the hypoxia preconditioning and hypoxia preconditioning+miR-195 negative control groups, hypoxia preconditioning+miR-195 group also presented much lower growth rate, and cells apoptosis percentage was significantly higher; number of vascular lumen like structures decreased (P < 0.01). The expression of MALAT1 was decreased, while the level of miR-195 was increased (P < 0.01). (3) The dual luciferase reporter assay indicated that compared with miR-195 control and blank control groups, the MALAT1 reporter gene luciferase activity was decreased significantly in miR-195 mimics group, down-regulating about 63% (P < 0.01). However, miR-195 mimics, miR-195 mimics negative control and blank control showed no effect on MALAT1-mut reporter gene (P > 0.05). (4) Hypoxia preconditioning could activate MALAT1 to target and downregulate miR-195 to further promote bone marrow mesenchymal stem cell survival and pro-angiogenic potential.