Study On The Effect And Mechanism Of MiR-30d In Biological Behavior Of Human Ewing's Sarcoma SK-ES-1 Cells

Objective:We aim to research the effect and underlying mechanism of miR-30 d in biological behavior of human Ewing's sarcoma(ES)SK-ES-1 cells.Methods:We conducted the CCK-8 assay to explore the effect of miR-30 d on the viability of human Ewing's sarcoma SK-ES-1 cells;Boyden chamber Transwell assay was used to evaluate the effect of miR-30 d overexpression on the invasion ability of human Ewing's sarcoma SK-ES-1 cells.Wound healing assay was performed to evaluate the effect of miR-30 d on the migration of human Ewing's sarcoma SK-ES-1cells.We performed Hoechst 33258 staining and we used fluorescence microscopy to look into the morphological changes in human Ewing's sarcoma SK-ES-1 cells.Flow cytometry analysis was carried out to evaluate the effect of miR-30 d on the apoptotic rate of SK-ES-1 cells.The expression levels of malignant phenotypic related proteins MMP-2 and MMP-9 as well as the apoptosis related proteins,such as Bcl-2 family proteins,caspase-3,and PARP were detected by western blot assay in human Ewing's sarcoma SK-ES-1 cells.Results:The results of CCK-8 assay showed that miR-30 d overexpression suppressed proliferation of human Ewing's sarcoma SK-ES-1 cells.The results of Boyden chamber Transwell assay revealed that the overexpression of mi R-30 d inhibited the invasion of SK-ES-1 cells.Wound healing assay indicated that miR-30 d overexpre-ssion inhibited the migration of human Ewing's sarcoma SK-ES-1 cells.Hoechst33258 staining assay showed that SK-ES-1 cells transfected with miR-30 d mimic revealed typical morphological characteristics of apoptotic cells,such as nucleus chromatin condensation,nucleus shrinks and fragmented nuclei.Flow cytometry analysis indicated that the apoptotic rate increased in human Ewing's sarcoma SK-ES-1 cells transfected with miR-30 d mimic.Western blot analysis showed thatmiR-30 d overexpression caused a remarkable decrease in the expression levels of MMP-2 and MMP-9.Furthermore,the ratio of Bax to Bcl-2.Caspase-3,a key executional protein in the process of apoptosis,was reduced in expression level,suggesting that caspase-3 was cleaved and therefore activated.PARP,the death substrate,was also reduced,showing the cleavage and degration of PARP.Conclusions:1.miR-30 d promotes apoptosis and restrains proliferation of Ewing's sarcoma SK-ES-1 cells.2.miR-30 d inhibits invasion and migration of human Ewing's sarcoma SK-ES-1cells.