| Background and ObjectionsAcute myeloid leukemia(AML)is the most common acute leukemia in adults and is a fatal hematological malignancy.With the genesis of the cancer,the blood progenitor cells proliferated and its proportion increased,the normal hematopoietic function is obviously insufficient,and the clinical manifestations are diverse.Despite significant advances in chemotherapy,the recurrence rate remained high in AML patients,with overall 5 year survival rates still poor,about 30%-40%.Chemotherapy resistance in leukemia cells is the main cause of unsatisfactory outcome.Therefore,the treatment of AML patients requires replacement therapies,and new therapeutic targets need to seek.microRNA(miRNA)is a non-coding short strand RNA with a size of about19-25 nucleotides,and is highly conserved between species.miRNA can target the 3’UTR of its target mRNA,and negatively regulates the expression of genes at post transcriptional levels,resulting in degradation of mRNA or inhibition of translation.A large number of studies have shown that many miRNAs are expressed abnormally in AML patients and are involved in the pathogenesis and progression of AML.The purpose of this study was to investigate the expression of miR-495 in AML patients and its effects on the proliferation and apoptosis of human leukaemia cell line HL-60.Methods1.During the period from March 2015 to April 2016,we collected the bone marrow cells of 22 cases AML patients as case group from Department of Hematology in Henan Province People’s Hospital.The umbilical cord blood specimens of 22 healthy full-term babies were collected when they were birth in the department of obstetrics and gynecology as control group.Then mononuclear cells were isolated and storage at-80℃.2.Specimens of case and control group were detected by qRT-PCR,the expression of miR-495 and Bmi-1 mRNA were observed,and SPSS 21 was used to analyze the correlation between the expression of miR-495 and Bmi-1 mRNA in the case group by Pearson x~2 test.3.miR-495 mimic and miR-495 scramble were synthesized and transfected into human leukemic cell line HL-60 with lipofectamine.The cells were then divided into three groups:miR-495 group,transfected with mi R-495 mimic;NC group,transfected with miR-495 scramble;Blank group,only added lipofectamine.4.CCK-8 method was used to detect the proliferation of cells of three groups,and the effect of up regulation of miR-495 expression on proliferation of HL-60 cells was observed.5.Flow cytometry method was used to detect the apoptosis of cells of three groups,and the effect of up regulation of miR-495 expression on the apoptosis rate of HL-60 cells was observed.6.Targetscan and MicroCosm bioinformatics websites were used to analyze and predict target genes of miR-495.7.Wild type and mutational recombinant plasmids with dual-luciferase reporter containing the Bmi-1 3’UTR region were constructed,then they were transfected respectively with the mi R-495 mimic or miR-495 scramble into human leukemia cell line HL-60 cells to detect the luciferase activity.8.si-Bmi-1 and si-NC were synthesized and transfected into HL-60 cells with lipofectamine.The si-Bmi-1 group was transfected with si-Bmi-1,and the si-NC group was transfected with si-NC.9.qRT-PCR and Western blot were used respectively to detect the relative expression of Bmi-1 mRNA and its protein in Blank group,miR-495 group,NC group,si-Bmi-1 group and si-NC group.10.The cell proliferation and apoptosis rate of the 5 groups were detected by CCK-8 and flow cytometry respectively,and the effect of mi R-495 on the regulation of Bmi-1 expression was investigated.Results1.The expression levels of mi R-495 in the bone marrow cells of AML patients were lower than in the cord blood of the control groups significantly(P<0.05),the expression levels of Bmi-1 mRNA in the case groups were significantly higher than in the control groups(P<0.05),and the expression level of miR-495 and Bmi-1mRNA in the case group was showed a significant negative correlation,the correlation coefficient is r=-0.746(t=-5.01,P<0.01).2.The result of CCK-8 method showed that the absorbance value of 450 nm of HL-60 cells in miR-495 group was significantly lower than that in Blank group and NC group after cultured for 24 h,48 h,72 h and 96 h(P<0.05).3.The result of flow cytometry method showed that the apoptosis rate of HL-60cells was increased in miR-495 group compared with the Blank group and the NC group,and the difference was statistically significant(P<0.05).4.Result of dual luciferase reporter assay showed that Bmi-1 is one of the targets of miR-495.5.The experimental results of CCK-8 method showed that the absorbance value of 450 nm of HL-60 cells in si-Bmi-1 group was lower than that in Blank group,NC group and si-NC group after cultured for 24h,48h,72h and 96h,and the difference was significant(P<0.05),while there was no significant difference between si-Bmi-1 group and miR-495 group(P>0.05).6.Flow cytometry results showed that,the apoptosis rate of HL-60 cells in si-Bmi-1 group was significantly higher than that of Blank group,NC group and si-NC group(P<0.05),and there was no significant difference between si-Bmi-1group and miR-495 group(P>0.05).Conclusions1.The expression level of miR-495 in bone marrow cells of AML patients was abnormally decreased,and the expression level of Bmi-1 was abnormally elevated.2.Up-regulation of miR-495 in human leukemia cell line HL-60 cells can target the 3’UTR region of Bmi-1 and regulate the expression of Bmi-1 negatively,which could both inhibit the growth of leukemia cells,and promote the apoptosis of leukemia cells. |
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