Basic & Clinical Medicine ›› 2014, Vol. 34 ›› Issue (11): 1524-1529.
Objective We amied to identify the direct target gene of microRNA(Let-7a), as a tumor suppressor, in Ewing's Sarcoma cell lines. Methods To detect the Let-7a expression of different cell lines through Northern Blot；Constructing pMIR-reporter-CDK6 wild-type (CDK6_WT) or mutant (CDK6_MUT) and positive control (Let-7a_PC) plasmid; And then SK-ES-1 cells were transfected with Let-7a scramble, mimic and mimic control, The target gene of Let-7a was identified by using bioinformatics analysis combined with Dual-luciferase reporter and Western blot analysis. Results Northern Blot results showed the low expression of Let-7a in Ewing sarcoma cell lines, Grey mean of Let-7a /U6 snRNA maximum point：SK-ES-1 0.193±0.024(P<0.05 compared with MSCs); Restrictive enzyme digestion and DNA sequencing analysis revealed that the plasmid was constructed successfully. Let-7a was successfully overexpressed in SK-ES-1 cells (P<0.01); Compared with mutant CDK6 (CDK6_MUT) and Let-7a_PC, Wild-type CDK6 (CDK6_WT) luciferase expression levels were significantly decreased(P<0.05); over-expression of Let-7a in SK-ES-1 cell lines inhibited CDK-6 mRNA expression and protein. Conclusion The target gene of the tumor suppressor microRNA (Let-7a) was identified in Ewing sarcoma cell lines and can lay the foundation for the further experiments.
Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks
Website Copyright © Basic & Clinical Medicine