Figure 4.

Correlation between the expression levels of let-7a and HMGA2 transcripts in MPN cells. (A) Scatter plot illustrating the correlation between the expression levels of let-7a and HMGA2 transcripts in the granulocytes of 151 patients with MPN (hollow circles; correlation coefficient r=−0.291, Pearson’s correlation, P=0.0002). Note that data from healthy individuals (solid gray circles) mostly fell on the y-axis, and many of them were very close to the origin. Data are shown in ΔΔCT. (B) Quantitative RT-PCR was performed to detect the levels of let-7a miRNA transcripts in parental Ba/F3, Ton.JAK2.WT and Ton.JAK2.V61F cells after doxycycline treatment (1 μg/ml) for indicated periods of time. (C) Effects on a let-7a inhibitory oligo on the expression of let-7a and Hmga2 in parental Ba/F3 cells. Two oligo concentrations (0.2 and 0.5 nM) were used for let-7a inhibition. All cells were harvested after 48 hours of transfection. Upper panel: western blot analysis of HMGA2; Lower panel: qRT-PCR analysis of let-7a and Hmga2 transcript levels. (t-test; *P<0.01; **P<0.001). (D) Effects of let-7a inhibitory and mimic oligos on Ton.JAK2.V617F cells. The working concentration was 0.5 nM for both oligos. Right panel: qRT-PCR analysis of let-7a and Hmga2 transcript levels. Note the inverse correlation between the expression levels of let-7a and Hmga2 (correlation coefficient r=−0.9281, Pearson’s correlation, P=0.002). Left panel: western blot analysis on the expression levels of JAK2, p-JAK2, PARP, cleaved PARP, BAD (Bcl-2-associated death promoter protein), p-BAD, and HMGA2. NC: scramble control; “−”: treatment with a let-7a inhibitor; “+”: treatment with a let-7a mimic. (E) The viability of parental Ba/F3 and inducible Ton.JAK2.V617F cells treated with indicated let-7a oligos. The error bars show the standard deviation of three independent experiments. Asterisks indicate statistical significance (t-test; *P<0.01; **P<0.001; ***P<0.0001).