The Prognostic Significance Of MiR-181a In Acute Myeloid Leukemia And Its Epigenetic Mechanism

Background:miRNAs are around 22-nucleotides long,non-coding RNAs that play important roles in the pathogenesis and prognosis of acute myeloid leukemia(AML).One microRNA,miR-181a,has been significantly associated with prognosis in cytogenetically normal AML,especially in high-risk patients with FLT3-ITD and/or NPM1 wild type.However,the association of miR-181a expression level with different FAB subtypes,different karyotypes and various genetic mutations has not been fully elucidated.The mechanism of miR-181a in AML cells,especially its role in epigenetic modification needs to be further investigated.To solve the above questions,the first part of this study investigated the associations between miR-181a expression level and the characteristics,responses to chemotherapy and prognosis of newly-diagnosed AML patients with mixed genetic backgrounds in a Chinese population.The second part studied the effects of miR-181a on protein acetylation in AML cell lines.Methods:In part one,bone marrow samples of 46 untreated AML patients were collected in Drum Tower Hospital from 2014/10 to 2015/11.Mononuclear cells were purified using density-gradient centrifugation in Ficoll,RNA was extracted using TRIZOL reagent and quantitative real-time PCR was used to detect miR-181a expression level.Statistical analysis was performed to evaluate the association between miR-181a expression level with patients' characteristic at diagnosis,responses to chemotherapy and prognosis.In part two,we established a miR-181a overexpression model in AML cell lines first,then purified our target protein by immunoprecipitation(DP)and identified it by protein mass spectrum(MS).At last,we studied the regulatory mechanism of miR-181a in a specific cell line THP-1.Results:In the clinical study,we found that the expression level of miR-181a was significantly associated with FAB subtype and granulocytic AML expressed a much higher level of miR-181 a than monocytic AML.What is more,the amount of miR-181a was significantly associated with peripheral blood white cell count,risk status,NPM1 mutation and MLL-related genetic abnormalities in patients at diagnosis.By a median follow-up of 12 months,we found that M1/2 patients with higher miR-181a expression level tended to have higher response rates to induction chemotherapy,longer overall survival and disease-free survival(OS P=0.0763,DFS P=0.0356),but M4/5 patients did not(OS P=0.6183,DFS P=0.9331).In vitro,miR-181a overexpression could significantly enhance the acetylation of overall proteins.We purified the protein with molecular weight between 85-120kD by immunoprecipitation and identified that acetylated poly[ADP-ribose]polymerase 1(PARP1)was regulated by miR-181a by protein mass spectrum.In different AML cell lines,the effects of miR-181a on PARP1 protein level were quite different.In THP-1 cell line,which harbors MLL-AF9 fusion gene,overexpression of miR-181a could cause G2/M phase arrest and increase apoptosis,which was in consistence with the effects of PARP1 inhibitor PJ34.At last,we detected the mRNA and protein level of PARP1 after miR-181a overexpression in THP-1 cells and found that miR-181a did not change its mRNA level but down-regulated its protein level.Enhancement of PARP1 acetylation might mediate the process.Conclusions:In AML patients with mixed genetic background,higher miR-181a expression level was associated with lower risk status,better responses to induction chemotherapy and higher survival rate,which was only significant in Ml/2 patients.By investigating the effects of miR-181a on protein acetylation in AML cell lines,we found that overexpression of miR-181a could significantly enhance the acetylation of overall protein.PARP1 was identified as a major target protein by mass spectrum.In THP-1 cell line,which harbors MLL-AF9 fusion gene,miR-181a could down-regulate PARP1 protein level by enhancing its acetylation.