The Expression And Functional Study Of Mir-19a、mir-19b In Children With Acute Lymphoblastic Leukemia

PART I EXPRESSION AND CLINICAL SIGNIFICANCEOF MIR-19A、MIR-19B IN CHILDREN WITH ACUTELYMPHOBLASTIC LEUKEMIAObjective To investigate the expression and clinical significance ofmiR-19a and miR-19b in children with acute lymphoblasticleukemia(ALL)．Methods The expression levels of miR-19a and miR-19b in67patients with ALL(including38newly diagnosed ALL，20completeremission(CR) ALL and9relapsed/refractory ALL) and15children withIdiopathic Thrombocytopenic Purpura(ITP)(as normal controls) wereassayed by qRT-PCR．miR-19a and miR-19b’s relationship with clinicalcharacters was analyzed．Results1．The expressions of miR-19a in newly diagnosed ALL group，CRALL group， relapsed/refractory ALL group and control group were 2.69±0.39，0.69±0.12，5.17±1.79and0.36±0.08．The expression level ofmiR-19a in all ALL groups was higher than that in the control group(P<0.05)．Among three ALL groups，miR-19a in the newly diagnosed ALLgroup and the relapsed/refractory ALL group were higher than that in theCR ALL group．The expression level of miR-19b in the newly diagnosedALL group，CR ALL group，relapsed/refractory ALL group and controlgroup were3.29±0.51，0.81±0.14，6.24±0.79和0.49±0.07．miR-19b inthe newly diagnosed ALL group and the relapsed/refractory ALL groupwere higher than that in the CR ALL group and the control group (P<0.01)．There was no statistical difference of the miR-19b expression foundbetween the CR ALL group and the control group(P>0.05)．2．The expression of miR-19a and miR-19b in6patients with ALLwere followed up in different stages．The expression of miR-19a andmiR-19b at diagnosis were1.66±0.24and2.24±0.21，and that in CRstage were0.65±0.02and0.77±0.06．The expression of miR-19a andmiR-19b in the CR ALL stage were significantly lower than that in thenewly diagnosed ALL stage．3．There was a positive correlation between the expression ofmiR-19a and miR-19b and the high white blood cell (WBC) counts(≥50×109/L)，risk classification（intermedium-risk and the high-risk group）and splenomegaly（P<0.05）．No relationship were found between theexpression of miR-19a and miR-19b and the age，gender，FAB phenotype， Immunophenotype， hepatomegaly，lymphadenectasis and the response toprednisone （P>0.05）．4. The expression level of miR-19a was not associated withBCR-ABL and MLL-AF4ALL．The expression level of miR-19b had apositive correlation with BCR-ABL ALL，but not with MLL-AF4ALL．5．miR-19a and miR-19b had similar expression level in ALL groupand the control group．There was a positive correlation between theexpression of miR-19a and miR-19b，the correlation coefficient of theirexpression was0.582(P<0.05)．Conclusion1． miR-19a and miR-19b were highly expressed in the newlydiagnosed ALL group and the relapsed/refractory ALL group， anddecreased in the CR ALL group．They might play an important role in thetumorigenesis and maintenance in childhood ALL．2．There was a positive correlation between the expression ofmiR-19a and miR-19b and the high white blood cell (WBC) counts(≥50×109/L)，risk classification（intermedium-risk and the high-risk group）and splenomegaly（P<0.05）．No relationship were found between theexpression of miR-19a and miR-19b and the age，gender，FAB phenotype，Immunophenotype，MLL-AF4ALL，hepatomegaly，lymphadenectasis andthe response to prednisone （P>0.05）． miR-19b was associated withBCR-ABL ALL．The high expression of miR-19a and miR-19b might be associated with high-risk of poor prognosis in childhood ALL．3. There was a positive correlation between the expression of miR-19aand miR-19b，which had the similer expression level and overlappingfunction. cooperativity might exist between the two miRNAs in childhoodALL． PART II THE EFFECT OF MIR-19B ON PROLIFERATIONAND EXPRESSION OF PTEN IN JURKAT CELLSObjective To investigate the effect of miR-19b specific antagomir onthe proliferation and the expression of PTEN in Jurkat cells.Methods1.The cell line overexpressing miR-19a and miR-19b wasdistinguished by qRT-PCR from the cell lines of Jurkat、HL-60、K562、HEK293.2.The miR-19b specific antagomir was transfected into Jurkatcells. Jurkat cells were divided into three groups：experimental group（miR-19b specific antagomir group），antagomir negative control group，blank control group（Jurkat group）3.The expressions of miR-19b andPTEN were detected by qRT-PCR.4. Cell proliferation was determined byMTT. Results1. The expression level miR-19a in Jurkat、K562、HL-60and HEK293were15.2±0.37，3.32±0.22，1.27±0.20and1.53±0.26；The expression levelmiR-19b in four groups were6.91±0.08，3.41±0.42，1.31±0.13and0.96±0.13；PTEN in four groups were0.39±0.01，0.67±0.007，3.62±0.41和0.94±0.20．The expression level miR-19a and miR-19b in Jurkat cellwere higher than other three cell lines (K562、HL-60、HEK293)，whilePTEN was lower than other cell lines.2. miR-19b specific antagomir was successfully transfected into Jurkatcells.The expression of miR-19b in experimental group was was1.87±0.09，which was significantly less than the antagomir negative controlgroup(6.67±0.31) and the blank control group（6.88±0.25）（P<0.01）．3. The expression of PTEN mRNA in experimental group was was2.72±0.26，which was significantly higher than the antagomir negativecontrol group(0.39±0.02) and the blank control group （0.39±0.01）（P<0.01）．4. Cell proliferation in the experimental group was significantlysuppressed by the inhibition of miR-19b.Conclusion1. The expression of miR-19a and miR-19b in Jurkat cells were higherthan that in other cell lines(HL-60、K562、HEK293).2. miR-19b specific antagomir that inhibited the expression of miR-19b was successfully transfected into Jurkat cells．3. The expression of PTEN mRNA was associatied with the decreasedexpression of miR-19b.4. Cell proliferation in the experimental group was significantlysuppressed by the inhibition of miR-19b. miR-19b might play an importantrole in the pathogenesis of ALL by inducing proliferation.