Expression Analysis And Epigenetic Study Of MicroRNA-28-5p In Multiple Myeloma

Objective:miR-28-5p is a cancer-associated tumor suppressor microRNA.The significance of this study is to investigate the expression,mechanism of action and methylation level of miR-28-5p in multiple myeloma(MM)in MM diagnosis.Application,providing a basis for seeking new targeted therapies.Methods:Firstly,Real-time RT-PCR was used to detect the expression levels of miR-28-5p and potential target CCND1 in CD138+cells of the patients with MM and bone marrow mononuclear cells of patients with non-hematologic malignancies as control,Methylation-specific PCR(MSP)was used to detect CpG island methylation levels in LPP/miR-28-5p promoter region and to correlate with other clinical indicators.Then5-aza-2'-deoxycytidine(5-Aza-dC,DAC)was used to treat MM cell line U266;after drug treatment,MSP was used to analyze the methylation status of the CpG islands in LPP/miR-28-5p promoter;the qPCR was used to detect the expression levels of miR-28-5p,and further explore the regulatory mechanism of miR-28-5p expression.Results:The methylation level of CpG island in the LPP/miR-28-5p promoter region of MM patients was significantly higher than that of healthy controls.The relative expression of miR-28-5p in MM patients(0.26±0.16)was significantly lower than that of healthy controls.(1.48±0.45),the difference was very significant(P<0.001);Compared with theCCRD1 expression of the healthy control group(0.86±0.16),the miR-28-5p target CCND1 was significantly increased in the MM group(5.84±3.96),and the difference was very significant(P=0.027).The relative expression level of miR-28-5p in newly diagnosed patients was higher than that in relapsed/progressive patients(t=2.522,P=0.065).The relative expression level of CCND1 in newly diagnosed patients was lower than that in relapsed/progressive patients(t=-1.654,P=0.174).This result is still subject to further clinical validation due to fewer cases.The relative expression of miR-28-5p in MM patients was significantly correlated with ?2-MG(r=-0.913,P<0.05).The promoter region of LPP/miR-28-5p was completely methylated in U266 cell line,and the expression level of miR-28-5p was increased after demethylation treatment.5-aza-dC can significantly inhibit the growth of U266 cell line,arrest the cell cycle in G1 phase,inhibit the biosynthesis of cellular RNA and protein,and promote early apoptosis of cells.Conclusion:The expression of miR-28-5p in MM patients is regulated by methylation of CpG islands in the promoter region of the genome.miR-28-5p may act as a tumor suppressor gene,and its low expression may be involved in the occurrence and development of MM,suggesting that miR-28-5p may become a new target for the treatment of MM.