Inhibition of LIN28B impairs leukemia cell growth and metabolism in acute myeloid leukemia

Abstract

Background: Current conventional chemotherapy for acute myeloid leukemia (AML) can achieve remission in over 70% of patients, but a majority of them will relapse within 5 years despite continued treatment. The relapse is postulated to be due to leukemia stem cells (LSCs), which are different from normal hematopoietic stem cells (HSCs). LIN28B is microRNA regulator and stem cell reprogramming factor. Overexpression of LIN28B has been associated with advance human malignancies and cancer stem cells (CSCs), including AML. However, the molecular mechanism by which LIN28B contributes to the development of AML remains largely elusive.

Methods: We modulated LIN28B expression in AML and non-leukemic cells and investigated functional consequences in cell proliferation, cell cycle, and colony-forming assays. We performed a microarray-based analysis for LIN28B-silencing cells and interrogated gene expression data with different bioinformatic tools. AML mouse xenograft model was used to examine the in vivo function of LIN28B.

Results: We demonstrated that targeting LIN28B in AML cells resulted in cell cycle arrest, inhibition of cell proliferation and colony formation, which was induced by de-repression of let-7a miRNA. On the other hand, overexpression of LIN28B promoted cell proliferation. Data point to a mechanism where that inhibition of LIN28B induces metabolic changes in AML cells. IGF2BP1 was confirmed to be a novel downstream target of LIN28B via let-7 miRNA in AML. Notably, ectopic expression of LIN28B increased tumorigenicity, while silencing LIN28B led to slow tumor growth in vivo.

Conclusions: In sum, these results uncover a novel mechanism of an important regulatory signaling, LIN28B/let-7/IGF2BP1, in leukemogenesis and provide a rationale to target this pathway as effective therapeutic strategy.

Keywords: Acute myeloid leukemia (AML); Cancer metabolism; IGF2BP1; LIN28B; Stem cell; let-7 microRNA.

Fig. 1

The effect of silencing LIN28B in AML. a Lentiviral LIN28B shRNA 1, 2, 3, 4, 5 and Scramble-shRNA were transduced in TF-1a cells. Proteins of the knockdown cell lines were harvested at 24, 48, and 90 h time points for Western blot analysis. β-actin was used as a loading control. b RNAs of shRNA 3 and 5 transduced cells were extracted after 1 week of drug selection. qRT-PCR was performed to compare LIN28B transcription level. The expression of LIN28B in each sample was normalized with GAPDH, respectively (n = 3, mean ± SD). *p < 0.01. c Cell viability assay of TF-1a cells transduced with Scramble shRNA or LIN28B shRNA 3, 5. Eight repeats of 10,000 cells in 100 μl of medium each per sample were cultured at various time points: 0, 24, 48, and 96 h by CTG assays. Measurements taken from 24 h onwards were normalized with their respective values obtained at 0 h (n = 8, mean ± SD). n.s. not significant; *p < 0.05; **p < 0.01. d qRT-PCR and Western blot analysis validation of overexpression of LIN28B in TF-1 and HEK293T cells, followed by cell viability assays at 0, 24, and 48 h times. Measurements taken from 24 h onwards were normalized with their respective values obtained at 0 h (n = 8, mean ± SD). n.s. not significant; *p < 0.05; **p < 0.01. e Colony-forming assay of TF1-pEGFP and TF1-LIN28B. The experiments were duplicated and representative pictures were presented (left panel). qRT-PCR analysis confirmed decreased LIN28B expression in LIN28B shRNA3, 5 treated THP-1 cells (middle panel). Quantification of colonies of indicated cell lines (n = 3, mean ± SD). The colony number of LIN28B-shRNAs expression THP-1 cells was significantly reduced than those of Scramble shRNA expressing cells (right panel, *p < 0.01). f Flow cytometric detection of the CD34⁺CD38⁻ population in pairs of LIN28B knockdown and overexpression cell lines. The representative FACS images were shown in the left panel and bar graphs of the percentages of CD34⁺CD38⁻ cells were listed in the right panel (n = 2, mean ± SD) (*p < 0.05, **p < 0.01)

Fig. 2

LIN28B regulates cell cycle progress of AML cells. TF-1a cells were treated either with Scramble shRNA or LIN28B shRNA3 and 5, then grown with BrdU for 12 h stained with aqua dye prior fixation and addition of anti-BrdU APC and 7-AAD as described in Materials and methods section. a Representative images of how viable cells were identified, gated and plot as pseudocolour dot-plots with APC versus 7-AAD. b Representative data showed the distribution of cells at each stage of the cell cycle (in percentage) determined by flow cytometric analysis. This experiment was duplicated. (n = 2, mean ± SD, *p < 0.05; **p < 0.01). c The cell lysates extracted from TF-1a-Scramble shRNA, TF-1a-LIN28B-shRNA3, and TF-1a-LIN28B-shRNA5 were subjected to Western blot analysis for Cyclin D1, Cyclin B1, p21waf1, and beta-actin. Beta-actin was used as internal control

Fig. 3

Microarray analysis of LIN28B knocking down TF-1a cells. a Relative expression levels for the 108 genes that changed significantly (ANOVA, p < 0.05) and more than 1.4-fold are shown in six columns. Colors indicate relative signal intensities (red: gene upregulation; blue: gene downregulation). The RNA expression profile was sorted using a hierarchical clustering method. A quantitative description of RNA expression levels is presented in Additional file 1: Table S1. Duplication was done and shown in this figure. N/A indicates unknown gene which its sequence might have potential biological effect. b GO analysis revealed that these top five most significant gene cluster variation in LIN28B knocking down TF-1a cells

Fig. 4

Networks predicted by Ingenuity Pathway Analysis in the LIN28B knocking down cells. a Top five canonical pathways affected by silencing LIN28B. b The interaction network displayed graphically as nodes (genes) was centered on JUN and LIN28B. The coding color and lines are defined by IPA software by default. The node color intensity indicates the expression of genes, with red representing upregulation and green representing downregulation. Solid lines and dotted lines indicate direct relationship and indirect relationships, respectively. c The measurements of glutamine, l-amino acid, and aspartate in TF-1a-Scramble shRNA, TF-1a-LIN28B-shRNA3, and TF-1a-LIN28B-shRNA5 cell lysates. The relative fold changes were compared to the amount of these three metabolites in TF-1a-Scramble shRNA cell lysate, which were set as 1. The experiments were triplicated (mean ± SD). *p < 0.05; **p < 0.01

Fig. 5

Characterization of IGF2BP1 as downstream target of LIN28B via let-7 microRNA. a Relative let-7a microRNA expression of TF-1a after LIN28B knockdown as quantified by qPCR. *p < 0.05; **p < 0.01. b Potential let-7 miRNA binding sequence of 3′-UTR of IGF2BP1. c HEK293T cells were co-transfected with 0.5 μg of pGL3.0, 2 μg of IGF2BP1 reporter gene, and 10 ng of Renilla vector as indicated on the x-axis in 1 ml of RPMI 1640 growth medium for 24 h prior to lysis for measuring the luminescence. All samples were normalized with renilla luciferase to ensure equal transfection efficiency. Cells transfected with control siRNA (NC siRNA) and without miRNA were used as negative controls. d let-7a and let-7b mimetic were chemically transfected into TF-1a cells and cultured for 72 h prior protein extraction for Western blot analysis. e Assessment of IGF2BP1 protein levels in HEL cells transduced with Scramble shRNA or IGF2BP1 specific shRNAs after 72 h. f Cell proliferation assays of HEL cells treated with IGF2BP1-specific shRNA3, shRNA4 (left panel) or let-7a mimic, let-7b mimics at indicated time points (n = 3, mean ± SD, *p < 0.05, **p < 0.01). g LIN28B expression primary AML cell was correlated with increased IGF2BP1 and decreased let-7a. Bone marrow samples from 17 patients with AML were collected at diagnosis and subjected to qPCR for LIN28B, IGF2BP1, and let-7a miRNA levels. The Pearson r values and p values were determined with GraphPad Prism software

Fig. 6

Mouse xenograft models of TF1-pEGFP, TF1-LIN28B, and TF1-LIN28-shRNA5 cells. Three million TF1-pEGFP, TF1-LIN28B, TF1-LIN28B-expressing LIN28B-shRNA5 (TF1-LIN28B-sh5) cells were subcutaneously injected into left inguinal regions of NOD/SCID recipient mice, respectively. The tumor volume was measured by caplier every other day. The tumor growth curves were constructed according to the average tumor volume of each group ± SD (mm³) (n = 10, p < 0.01).