MOESM1 of Increased Th17 cells and IL-17A exist in patients with B cell acute lymphoblastic leukemia and promote proliferation and resistance to daunorubicin through activation of Akt signaling

Additional file 1: Table S1. The sequences of the primers used for real-time qPCR

Musashi-2 Silencing Exerts Potent Activity against Acute Myeloid Leukemia and Enhances Chemosensitivity to Daunorubicin

<div><p>RNA-binding protein Musashi-2 (Msi2) is known to play a critical role in leukemogenesis and contributes to poor clinical prognosis in acute myeloid leukemia (AML). However, the effect of Msi2 silencing on treatment for AML still remains poorly understood. In this study, we used lentivirus-mediated RNA interference targeting Msi2 to investigate the resulting changes in cellular processes and the underlying mechanisms in AML cell lines as well as primary AML cells isolated from AML patients. We found that Msi2 was highly expressed in AML cells, and its depletion inhibited Ki-67 expression and resulted in decreased in vitro and in vivo proliferation. Msi2 silencing induced cell cycle arrest in G0/G1 phase, with decreased Cyclin D1 and increased p21 expression. Msi2 silencing induced apoptosis through down-regulation of Bcl-2 expression and up-regulation of Bax expression. Suppression of Akt, Erk1/2 and p38 phosphorylation also contributed to apoptosis mediated by Msi2 silencing. Finally, Msi2 silencing in AML cells also enhanced their chemosensitivity to daunorubicin. Conclusively, our data suggest that Msi2 is a promising target for gene therapy to optimize conventional chemotherapeutics in AML treatment.</p></div

Msi2 silencing inhibits Akt, Erk1/2 and p38 signaling, which contributes to apoptosis mediated by Msi2 silencing in AML cells.

<p>(<b>A</b>) The phosphorylation of Akt, Erk1/2 and p38 were decreased followed by Msi2 silencing in Dami cells, HL-60 cells and primary AML cells from AML patients. Representatives and statistical data of 3 independent experiments were shown. (<b>B</b>) IGF-1 attenuated Msi2 silencing-induced apoptosis in Dami cells. Apoptotic cells were determined using Annexin V/PI double staining method in the presence or absence of IGF-1 (100 ng/ml) for 24 h (n = 3). (<b>C</b>) TPO attenuated Msi2 silencing-mediated apoptosis in Dami cells. Apoptotic cells were determined using Annexin V/PI double staining method with or without TPO (100 ng/ml) for 2 h (n = 3).</p

Msi2 expresses in leukemic cells and is down-regulated in AML cells.

<p>(<b>A</b>) The protein levels of Msi2 in leukemic cell lines and primary AML cells. Representatives and statistical data of 3 independent experiments demonstrating Msi2 expression in leukemic cell lines and primary AML cells from AML patients (n = 8) using western blot were shown. (<b>B</b>) Msi2 is down-regulated by lentivirus-mediated RNA interference in Dami cells, HL-60 cells, and primary AML cells. The levels of Msi2 were detected using western blot. Quantification of Msi2 normalized against GAPDH was shown (n = 3).</p

Msi2 silencing enhances chemosensitivity of AML cells to daunorubicin.

<p>Dami cells and primary AML cells isolated from AML patients infected with shMsi2-3 or scarmble lentivirus were untreated or treated with 200 nM daunorubicin for 48 h and cell viability (<b>A</b>) was measured using CCK-8. Data are expressed as mean ± SEM representing 3 independent experiments. Meanwhile, apoptosis was measured using Annexin V staining. Statistical data (<b>B</b>) and representatives (<b>C</b>) of 3 independent experiments were shown.</p

Msi2 silencing leads to G0/G1 cell arrest in AML cells.

<p>(<b>A</b>) Msi2 silencing increased the percentages of G0/G1 phase cells and decreased those of S phase cells in Dami cells, HL-60 cells, and primary AML cells from AML patients. Representatives and statistical data from 3 independent experiments were shown. (<b>B</b>) Msi2 silencing decreased the mRNA levels of CCND1 and Cdk2 and increased the mRNA level of p21 in AML cells. (<b>C</b>) Msi2 silencing decreased the protein levels of Cyclin D1 and Cdk2 increased the protein level of p21 in AML cells. Representatives and statistical data from 3 independent experiments were shown.</p

Msi2 silencing inhibits the proliferation of AML cells.

<p>(<b>A</b>) Msi2 silencing inhibits the proliferation of Dami cells, HL-60 cells, and primary AML cells from AML patients. Cells were plated at a density of 4 × 10³ cells/well and grown for 0 to 72 h, and cell viability was determined using CCK-8. (<b>B</b>)&(<b>C</b>) The Ki-67 expression was significantly decreased in shMsi2-3 group compared to scramble control group. Cells were harvested and fixed using 75% alcohol for 4 h at -20°C. Then, cells were washed and incubated with anti-Ki-67 Alexa Flour^®647 antibody for 30 min at room temperature and finally determined using a flow cytometry. Representatives and statistical data from 3 independent experiments were shown. (<b>D</b>) NOD/SCID mice were intravenously injected with 1 × 10⁷ HL-60 cells infected with shMsi2-3 or scramble control lentivirus. Kaplan-Meier curves for overall survival were assessed and the Log-rank test was used to determine the statistical significance.</p

Msi2 silencing induces apoptosis in AML cells.

<p>(<b>A</b>) Msi2 silencing induces apoptosis in Dami cells, HL-60 cells, and primary AML cells from AML patients. The Annexin V-APC binding and PI staining method was used to assess apoptosis, and the results shown were representatives of 3 independent experiments. (<b>B</b>) Msi2 silencing decreased the mRNA level of Bcl-2 and increased the mRNA level of Bax in AML cells. (<b>C</b>) Msi2 silencing decreased Bcl-2 expression and increased Bax and cleaved PARP expression in AML cells using western blot. Representatives and quantification of Bcl-2, Bax or cleaved PARP normalized against GAPDH were shown (n = 3).</p