Role and mechanism of miR-101 in proliferation and apoptosis of osteosarcoma cell line MG-63

OBJECTIVE:To investigate the role and mechanism of miR-101in proliferation and apoptosis of osteosarcoma cells by regulating the expression of miR-101in osteosarcoma cell line MG-63.METHODS:The expression level of miR-101in MG63cells and osteoblasts were detected by qRT-PCR.Synthetic miR-101sequence(mimics)or negative control sequence(NC)was transiently transfected into MG63cells with lipofectamine 2000vector.Meanwhile,non-transfected MG-63cells acted as a blank control.The expression of miR-101mRNA in three groups was detected by qRT-PCR after transfection.The effects of miR-101on MG-63cell proliferation,apoptosis and cell cycle were evaluated by MTT assay and flow cytometry,respectively.The expression of Beclin1and LC3Bprotein were analyzed by Western-blot.RESULTS: qRT-PCR results revealed that the expression of miR-101in osteoblasts(2.56±0.27)was 1.68times of that in MG63 cells(1.52±0.20,P=0.003)and miR-101in the mimics group(3.86±0.28)was increased by 2.95fold and 2.86times compared with the blank group(1.31±0.32)and the negative control group(1.35±0.31),respectively(F=70.28,P 0.001).The MTT assay suggested that the rate of cell proliferation of MG-63was declined significantly in the mimics group as compared with that in the blank group and the NC group(P0.01).Cell cycle analysis showed that transfection of miR-101mimics led to a lower proportion of cells in S phase and a higher proportion(13.02±0.14)%in G0/G1phase compared with the blank groups(3.15±0.98)% and the NC groups(3.26±0.94)%,indicating that growth of MG63 cells was inhibited by miR-101due to G0/G1cell cycle arrest(P0.001).In addition,the percentage of apoptosis cells was increased significantly(11.28±1.45)%in miR-101mimics group than that in the blank group(3.02±0.96)%and the NC group(3.16±0.78)%(P0.001).Western blot analysis suggested that the miR-101expression showed an inverse correlation with Beclin1and LC3Bprotein expression.Beclin1and LC3Bexpression in the miR-101transfected group were down-regulated compared with that in the blank groups.CONCLUSIONS:The expression of miR-101is low in MG-63 cells which may be involved in the tumorigenesis of osteosarcoma.MiR-101can inhibit proliferation and autophagy of osteosarcoma cells,inducing cells apoptosis and cell cycle arrest.One of the mechanisms that miR-101inhibits proliferation and autophagy of osteosarcoma cells may be probably by regulating autophagy.