The Expression And Significance Of MicroRNAs In Decitabine-resistant Myelodysplastic Syndrome Cell Lines

Objective: Hypomethylating agents are the most common and effective drugs for treating Myelodysplastic syndrome(MDS).Although hypomethylating therapy has changed the survival status of MDS patients,it has not reduced acute myeloid leukaemia(AML)conversion in high-risk MDS patients,and patients with limited treatment options after the drug resistance have a short remission time on second-line therapy and a very poor prognosis.To investigate the mechanism of drug resistance in MDS after the failure of hypomethylating therapy.In this study,the DAC-resistant MOLM-14 cell line(MOLM-14/DAC)was established by gradually increasing the concentration of decitabine(DAC)in the parental MOLM-14 cell line,and the potential mechanisms of DAC resistance were further investigated.Methods: The parental cell line MOLM-14 was induced with a start dose of 0.02 u M DAC,and the concentration of DAC was gradually increased to 3u M to establish a DAC-resistant MOLM-14 cell line(MOLM-14/DAC).Half-volume inhibitory concentrations and proliferation capacity of the MOLM-14 cell line and MOLM-14/DAC cell line were detected by the CCK-8 assay method.The cell cycle and differentiation of MOLM-14 and MOLM-14/DAC cell lines were detected by flow cytometry.Mi RNA and mRNA expression were subjected to RNA-seq analysis,and we performed gene analysis and functional enrichment of gene expression alterations in MOLM-14 and MOLM-14/DAC.Then real-time quantitative polymerase chain reaction(RQ-PCR)was performed to validate candidates.Results: 1.Cell viability: the IC50 value of the MOLM-14 cell line was 0.2875±0.03415μmol/L,and MOLM-14/DAC cell line was induced by gradually increasing the concentration of DAC from the parental MOLM-14 cell line,and it’s IC50 value was4.363±0.4437 μmol/L,the RI value was calculated >10,which proved that the drug-resistant cell line model of MOLM-14/DAC was established successfully.2.Cell morphology: compared with the MOLM-14 cell line,there are more azurophilic granules and irregular patterns of chromatin aggregation,distorted and folded karyotypes,and indistinct nuclei in the MOLM-14/DAC cell line.3.Cell cycle: compared with the parental MOLM-14 cell line,the MOLM-14/DAC cell line had a shorter G1 phase(P <0.01)and a longer S phase(P < 0.05).4.Cell differentiation: increased CD13 expression was seen in the MOLM-14/DAC cell line(P < 0.05),suggesting altered MOLM-14 differentiation.5.RNA-sequencing(RNA-seq): the levels of miRNA and mRNA expression were found to be significantly altered by sequencing between MOLM-14 and MOLM-14/DAC.KEGG functional enrichment of differential gene results revealed that the down-regulated genes were enriched in signalling pathways in the MOLM-14/DAC cell lines,such as cellular mucus molecules(CAMs)and the MAPK signalling pathway.In contrast to the MOLM-14 cell line,the upregulated genes were enriched in signalling pathways in the MOLM-14/DAC cell lines,such as MAPK signalling pathway,PI3K-AKT signalling pathway,Rap signalling pathway,etc.5.RT-qPCR: miR-484 expression was downregulated,miR-1246 expression was upregulated in agreement with the sequencing results,miR-196 expression was downregulated in agreement with the sequencing result.In contrast,the expression of Inositol polyphosphate 4-phosphatase type II(INPP4B),fibroblast growth factor receptor 3(FGFR3)and platelet-derived growth factor-D(PDGFD)was upregulated,and the expression of RAS guanyl releasing protein 1 Gene(RASGRP1)and cell adhesion molecule 1(CADM1)was downregulated,which was consistent with the sequencing results.Conclusion: In summary,we successfully established a DAC-resistant MOLM-14/DAC cell line with altered morphological features,prolonged S-phase and increased differentiation level compared to MOLM-14 cell lines.Through raw signal analysis and qPCR validation,it was hypothesized that miR-484 and its potential target genes INPP4 B,FGFR3,and PDGFD might promote the development of DAC resistance by participating in MAPK and PI3/AKT signalling pathways;miR-1246 and its potential target gene CADM1 might promote cellular DAC resistance through the biological role of cell adhesion.