| Object:Combined with bioinformatics,this study explored the role of miR-203 a regulatory network in the occurrence and development of Chronic myeloid leukemia(CML).It may provide a new theoretical basis for the molecular mechanism of CML occurrence,and provide a new intervention strategy for CML target gene therapy.Method:1.The bioinformatic prediction was used to predict the transcription factor EGR1 of miR-203 a and miR-203 a target genes WT1,Bmi-1 and XIAP;2.The q RT-PCR was employed to detected the expressions of EGR1,miR-203 a and target genes(WT1/ Bmi-1 /XIAP)in patients’ peripheral blood and K562 cells;3.The 5 ’Race assay and Chromatin Immunoprecipitation(CHIP)experiments were used to confirm the transcription starting site of miR-203 a and the binding of EGR1 to the promoter sequence of miR-203a;4.Double luciferase reporter was used to verify the interaction of miR-203 a target genes(WT1/Bmi-1 /XIAP);5.The expression regulation among EGR1,miR-203 a and target genes was studied by using RNA small-molecule mimics,interfering RNA,vector plasmid construction and other technologies.MTT and Brdu methods were used to detect the cell proliferation.q RT-PCR and Western blot were used to detect the changes of the expression levels.Result:1.The results were verified to be consistent with bioinformatics predictions in peripheral blood of patients with CML and K562 cells.Compared with the control group,the expression of transcription factors EGR1 and miR-203 a were down-regulated,and the expression of target genes(WT1/Bmi-1 /XIAP)were significantly enhanced.2.The results of bioinformatics prediction suggested that Early Growth Response1(EGR1)was the transcription factor of miR-203 a.It had an binding site of transcription factor EGR1 in the miR-203 a promoter region.The verification suggested that the transcription initiation site of miR-203 a was located at the upstream promoter region 339 bp(G).Transcription factor EGR1 would bind to the miR-203 a upstream promoter region 268 bp.3.The results of the dual luciferase reporter gene showed that compared with the control group,the luciferase activity was significantly decreased in the miR-203 a group.Mi R-203 a could bind to 3’UTR of target genes(WT1/Bmi-1/XIAP).4.K562 cells were transfected with miR-203 a minics,and overexpressed miR-203 a was significant in the cells.Compared with the control group,the expression of transcription factor EGR1 was up-regulated and the expression of target gene(WT1/ Bmi-1 /XIAP)was significantly down-regμlated in m RNA and protein levels in miR-203 a overexpressed group.5.Compared with the control group,the expressions of miR-203 a were synergistically up-regulated and the target genes expressions were significantly down-regulated,resulting from transfecting EGR1 plasmid vector into K562 cells.Compared with the control group,the expressions of miR-203 a was subsequently decreased and the target genes expressions were significantly enhanced,when the si-EGR1 was transfected into K562 cells.6.When the K562 cells was transfected with small interference RNA(si RNA)of the target gene(WT1/ Bmi-1 /XIAP),the expression of the target genes were knocked down.Compared with the control group,the effective down-regulation of the target gene expression could enhance the expression of miR-203 a.7.MTT results showed that,compared with the control group,the cell proliferation was inhibited in miR-203 a minics group;Brdu staining proliferation assay showed that the Brdu positive rate was significantly reduced in the miR-203 a minics group,which could inhibit the replication of DNA.8.MTT results showed that the cell proliferation activity could enhance in K562 cells group transfected with the plasmid vector of the target gene(WT1/ BMI1 /XIAP).However,the cell proliferation was inhibited in the co-transfection miR-203 a with target gene plasmid vector.The overexpression of target genes could offset the anti-tumor effect of miR-203 a.Conclusion:In the K562 cells,EGR1 is decreased expression and regulate the expression of target genes(WT1/Bmi-1/ XIAP)by EGR1-miR-203a-WT1/Bmi-1/XIAP signal axis,thus affecting the proliferation of the K562 cells. |
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