Expression And Clinical Significance Of MN1, P16and RARβ Genes In Acute Leukemias

Objective:①To investigate the expression level and prognostic significance of MN1gene and mcroRNA-181b (miR-181b), microRNA-20a (miR-20a) in de novo acutemyeloid leukemia (AML) patients, and analyze the relationship between MN1and clinicalcharacteristics, molecular markers in AMLs.②To determine the effect of promoter regionDNA methylation on the expression of tumor suppress genes, including p16INK4a (p16)and retinoic acid receptor β (RARβ) in AMLs and acute leukemia cell lines.③To explorethe effect of5-Aza-2’-deoxycytidine (decitabine; DAC) and all-trans retinoic acid (ATRA)on the expression of p16and RARβ, as well as their antineoplastic activities in acuteleukemia cell lines in vitro, including cells proliferation, differentiation and apoptosis. Andto reveal the action mechanism of the two drugs treatment for acute leukemia.Methods:①The expression level of MN1gene and miR-181b, miR-20a in bone marrowmononuclear cells was measured in158cases of newly diagnosed AML patients and20cases of normal healthy donors by real-time quantitative reverse transcriptase polymerasechain reaction (RT-PCR).②The expression level of p16and RARβ and the methylationstatus of its promoter were detected in mononuclear cells from bone marrow (BM) samplesof30cases of newly diagnosed AML patients and20cases of normal healthy donors, andacute leukemia cell lines U937, SHI-1, and K562by RT-PCR and Methylation-specificPCR (MSP) respectively.③RT-PCR, western blot and MSP were performed to study p16and RARβ genes mRNA and protein expression and the DNA methylation status changesin its promoter region within acute leukemia cell lines U937, SHI-1and K562after DACand ATRAtreatment for48h.④After DAC and ATRAtreatment of cell lines for24h,48h,and72h, the effect of DAC and ATRA on proliferation, differentiation, and apoptosis ofcells was evaluated by WST-1assay, flow cytometry (FCM). Results:①MN1gene and miR-181b, miR-20a expression levels in AML patients weresignificantly higher than those in normal control group (Z=-4.089，P<0.001；Z=-2.386，P=0.017； Z=-2.186， P=0.029). Patients of FAB-AML-M1, FAB-AML-M5andFAB-AML-M6exhibited significant overexpression of miR-181b, and patients withFAB-AML-M4and FAB-AML-M5exhibited significant overexpression of miR-20acompared with normal controls (P<0.05). In multivariable analyses, high MN1andmiR-181b expression were associated with lower complete remission (CR) rates (P=0.01;P=0.03), shorter relapse-free survival (RFS; P=0.02; P=0.045) and overall survival (OS;P=0.02; P=0.017), whereas high miR-20a expression was associated with higher CR rates(P=0.008) and longer OS (P=0.04). High MN1expression was associated with spleeninvolvement (χ2=4.341, P=0.037), NPM1wild-type (χ2=10.452, P=0.001), highermiR-181b expression levels (rs=0.319，P=0.035) and lower miR-20a expression levels(rs=-0.202，P=0.015).②By using RT-PCR, AMLs, U937, SHI-1and K562cell linesdemonstrated lower levels of p16and RARβ expression, while extremely higher levelswere found in normal controls. In MSP analysis, the DNA hypermethylation in p16andRARβ genes promoter region were identified in AMLs, U937and SHI-1. But no suchDNA hypermethylation were observed in the same region of normal controls. The DNAhypermethylation in p16promoter region was identified and the RARβ promoter regionwas not methylated in K562cells.③After U937, SHI-1and K562cell lines were treatedwith different concentrations of DAC [1μmol/l (1DAC),2μmol/l (2DAC),4μmol/l(4DAC）] and ATRA [0.5μmol/l (0.5RA)] alone or in combination for48h, the p16andRARβ genes mRNAand protein expression level were enhanced in U937cells treated withDAC and ATRA alone or in combination, in comparision with those without drugstreatment（P <0.05）. The RARβ gene mRNA and protein expression level wereupregulation by treatment of DAC in combination with ATRA compared with controlgroup（P <0.05）, and the two drugs had no effects on p16expression in SHI-1and K562cells. At the same time, MSP results showed DNA methylated levels decreased andunmethylated levels increased in p16and RARβ genes promoter region in cell linesfollowing treatment with DAC alone or in combination with ATRA, but treatment with ATRA alone did not change its methylation status of DNA. The effect of DAC was in aconcentration-dependent manner, and DAC at4μmol/l in combination with ATRA at0.5μmol/l treated cells had a strongest effect（P <0.05）.④After U937, SHI-1and K562cell lines were treated with different concentrations of DAC (1DAC,2DAC,4DAC） andATRA (0.5RA) alone or in combination for24h,48h, and72h, DAC and ATRA alone orin combination effectively induced cell growth inhibition, differentiation and apoptosis（P<0.05）. The effect of DAC was in a concentration-dependent manner, and DAC at4μmol/lin combination withATRAat0.5μmol/l treated cells had a strongest effec（tP <0.05）. DACcould effectively inhibit SHI-1cells proliferation, whereas ATRA had no inhibitory effect.DAC alone or in combination with ATRA effectively induced SHI-1cell differentiationand apoptosis（P <0.05）. The effect of DAC was in a concentration-and time-dependentmanner, and DAC at4μmol/l in combination with ATRA at0.5μmol/l treated cells had astrongest effect（P <0.05）. The combination of the two drugs was synergistic with respectto growth inhibition, differentiation and apoptosis of U937and SHI-1cells. DAC andATRA alone or in combination had no effect on growth inhibition, differentiation andapoptosis of K562cells（P>0.05）.Conclusion:①MN1and miR-181b, miR-20a are overexpressed in AML patients. Thehigher expression of MN1and MN1associated miRNAs are closely related to theprognosis of AML, which can be used as significant pathogenesis and prognostic markersof AML.②The combination of DAC and ATRA upregulate levels of p16and RARβ andinduce growth inhibition, differentiation and apoptosis in U937and SHI-1cells. These datasuggest that DAC used in combination with ATRA has clinical potential in the treatment ofAML.