Abstract
To study the molecular mechanism by which miR-203a affects the development of CML, bioinformatics software was used to predict the upstream transcription factors and downstream target genes of miR-203a. A 5’-rapid amplification of cDNA ends assay was performed to detect gene transcription initiation sites. A chromatin immunoprecipitation assay was used to verify the binding of transcription factors and promoter regions. A double luciferase reporter gene vector was constructed to demonstrate the regulatory effect of miR-203a on target genes. Real-time PCR and western blotting were used to detect the relative expression levels of genes and proteins, respectively. The results showed that there was a binding site for the transcription factor EGR1 in the upstream promoter region of miR-203a. WT1, BMI1, and XIAP were identified as target genes regulated by miR-203a. EGR1 and miR-203a were downregulated in human peripheral blood mononuclear cells and the CML K562 cell line, while WT1, BMI1, and XIAP were upregulated. The transcription initiation site of miR-203a was identified in the upstream promoter region (G nucleotide at −339 bp), and the transcription factor EGR1 could bind to the promoter region (at −268 bp) of miR-203a and increase its expression. Over expression of miR-203a inhibited the proliferation of K562 cells. A rescue assay showed that overexpression of WT1, BMI1, and XIAP offset the antitumor effect of miR-203a. Conclusion, EGR1 positively regulated the expression of miR-203a, thus relieving the inhibition of miR-203a on the translation of its target genes (WT1, BMI1, and XIAP) and affecting the proliferation of K562 cells.
Highlights
Chronic myelogenous leukemia (CML) is a clonal bone-marrow proliferative disease originating from hematopoietic stem cells, which mainly involves granulocytic lineages
The results showed that Cbp/p300-interacting transactivator 2, tyrosineprotein phosphatase non-receptor type 2, Wilms tumor 1 (WT1), and 22 other target genes were involved in the occurrence and development of CML
Due to the negative regulatory relationship between miRNAs and their target genes, we screened out the target genes that were expressed at a high level in CML as candidate genes, and the WT1, BMI1, and X-linked inhibitor of apoptosis protein (XIAP) genes were selected for further study (Figure 1)
Summary
Chronic myelogenous leukemia (CML) is a clonal bone-marrow proliferative disease originating from hematopoietic stem cells, which mainly involves granulocytic lineages. The traditional chemotherapy drugs for CML are unable to target tumor tissue, which often leads to side effects and adverse reactions, so that chemotherapy is frequently interrupted, resulting in a decrease in the sensitivity of tumor cells to treatment and the formation of early resistance (Iino et al, 2019; Kouhpeikar et al, 2019). In CML, increased expression of miR-150 and miR-146a, and reduced expression of miR-142-3p and miR199b-5p was observed after 2 weeks of TKI treatment, suggesting that this drug has the ability to rearrange the miRNA (Flamant et al, 2010). MiR-150 has consistently been observed to be downregulated across multiple studies making it a promising candidate for early CML diagnosis (Fallah et al, 2015) In CML, increased expression of miR-150 and miR-146a, and reduced expression of miR-142-3p and miR199b-5p was observed after 2 weeks of TKI treatment, suggesting that this drug has the ability to rearrange the miRNA (Flamant et al, 2010). miR-150 has consistently been observed to be downregulated across multiple studies making it a promising candidate for early CML diagnosis (Fallah et al, 2015)
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