Hpermethylation Of MiR-335 In Human Multiple Myeloma And Promotes Tumor Migration

Objective Investigate the role of the expression and methylation of mi R-335 in the diagnosis of multiple myeloma(MM). Further investigate the effect of the methylation level of miR-335 on the migration ability of U266.Methods SYBR Green I qRT-PCR was applied to detect the relative expression of miR-335 in peripheral blood mononuclear cells(PBMC) of 43 MM patients and 30 healthy controls. Methylation specific PCR(MSP) was used to detect the promoter methylation of miR-335. Correlations with other serum clinical indicators were assessed. MM cell line U266 was treated with 5-AzadC(a demethyltion drug) and transfected with hsa-miR-335 mimics, hsa-mi R-335 inhibitor and their nagetive controls, qRT-PCR was used to detect the expression level of miR-335, and Transwell was used to detect the migration alility of U266. Matrix metalloproteinase 2(MMP-2) was detected by Western Blot after transfected miR-335 mimics and inhibitor in U266.Results The relative expression of mi R-335 in PBMC of MM patients(34.8(15.3~84.9)) was significantly lower than that in healthy controls(269.4(63.3~780.2)), Z=4.384, P<0.05. In MM patients, there were 11(25.6%, 11/43) MM patients showed completely methylated, while 23(76.7%, 23/30) healthy controls showed completely unmethylated(Uc=6.62, P<0.05). And the relative expression of miR-335 were higher in primary patients compared with relapse/progressed patients(Z=2.309, P<0.05). While promoter methylation status of primary patients(25%) were significantly lower than relapse/progressed patients(75%), T=24.5, P<0.05. The relative expression of miR-335 was significantly correlated with serum λ light chain level(r=0.51, P<0.05). The promoter region of miR-335 in U266 cell line was completely methylated. The migration ability of demethylating drug treatment group and hsa-miR-335 mimics transfection group was significantly decreased. Western Blot confirmed that hsa-mi R-335-mimics transfection reduced the expression of MMP-2.Conclusions miR-335 expression and methylation levels can be used as a potential diagnostic marker in MM, of miR-335 was silenced by hypermethylation thus promoted the migration ability of U266...