BACKGROUND: Some studies suggest that histone deacetylase is closely related to miRNA-1 and moreover influences muscle differentiation and proliferation. However, its specific regulatory mechanism is still unclear.
OBJECTIVE: To analyze the effect of miRNA-1 overexpression on the proliferation and differentiation of L6 myoblasts as well as histone deacetylase expression.
METHODS: A recombinant lentiviral vector expressing miRNA-1 was constructed, sequenced and enzymatically identified, by which L6 myoblasts were stably transfected. The expression level of miRNA-1 was detected by RT-PCR. L6 myoblasts stably transfected with miRNA-1 recombinant lentiviral vector (miRNA-1 group), L6 myoblasts transfected with empty plasmid recombinant lentiviral vector (empty vector group) and L6 myoblasts non-transfected with empty plasmid recombinant lentiviral vector (blank group) were cultured for 24, 48, 72, and 96 hours. We then detected cell proliferation by cell counting kit-8 method, observed cell morphological changes under fluorescence microscope, and detected skeletalα-actin expression by western blot after 72 hours of culture. After 24, 72, and 120 hours of culture, western blot and RT-PCR were used to detect histone deacetylase protein and gene expression in the miRNA-1 group and empty vector group.
RESULTS AND CONCLUSION: (1) miRNA-1 lentivirus vector was correctly identified by enzyme digestion and sequencing. After 48 hours of transfection, miRNA-1 gene expression in L6 myoblasts was significantly higher than that in the empty vector and blank groups (P < 0.01 ), thus realizing miRNA-1 overexpression in L6 myoblasts. (2)After 24, 48, 72, and 96 hours of culture, there was no difference in cell proliferation ability among the three groups (P > 0.05). (3) There was no significant difference in cell morphology between miRNA-1, blank and empty vector groups when cultured for 24 and 48 hours, whereas after 72 hours, mature muscle cells with spindle shape in the miRNA-1 group increased significantly in number, and the expression of skeletal α-actin in the cells was significantly higher than that in the empty vector and blank groups, confirming that miRNA-1 overexpression promotes the differentiation of L6 myoblasts. (4) Histone deacetylase bands in the miRNA-1 group were faded over time and became thinner than those in the empty vector group. The expression of histone deacetylase gene in the miRNA-1 group was lower than that in the empty vector group at different time points of culture (P < 0.01). To conclude, the over-expression of miRNA-1 can improve the differentiation ability of L6 myoblasts, and its mechanism is related to the inhibition of histone deacetylase expression.