Lin28A/CENPE Promoting the Proliferation and Chemoresistance of Acute Myeloid Leukemia

Abstract

The prognosis of chemoresistant acute myeloid leukemia (AML) is still poor, mainly owing to the sustained proliferation ability of leukemic cells, while the microtubules have a major role in sustaining the continuity of cell cycle. In the present study, we have identified CENPE, a microtubular kinesin-like motor protein that is highly expressed in the peripheral blood of patients with chemoresistant AML. In our in vitro studies, knockdown of CENPE expression resulted in the suppression of proliferation of myeloid leukemia cells and reversal of cytarabine (Ara-C) chemoresistance. Furthermore, Lin28A, one of the RNA-binding oncogene proteins that increase cell proliferation and invasion and contribute to unfavorable treatment responses in certain malignancies, was found to be remarkably correlated with CENPE expression in chemoresistance AML. Overexpression of LIN28A promoted the proliferation and Ara-C chemoresistance of leukemic cells. RIP assay, RNA pull-down, and dual luciferase reporter analyses indicated that LIN28A bound specifically to the promoter region GGAGA of CENPE. In addition, the impacts of LIN28A on cell growth, apoptosis, cell cycle progression, and Ara-C chemoresistance were reverted by the knockdown of CENPE. Hence, Lin28A/CENPE has enhanced the proliferation and chemoresistance of AML, and therefore, it could be a prospective candidate for AML treatment.

Keywords: AML; CENPE; LIN28A; cell cycle; chemoresistance.

Figure 1

Mitosis-related gene CENPE was highly expressed in chemoresistance AML patients. (A) DEGs in de novo AML patients compared with HC. (B) GSEA enrichment plots of DEGs of GO biological processes were predominantly engaged in mitotic spindle organization (GO:0007052) in de novo AML patients compared with HC. (C) Upregulated DEGs enriched KEGG pathways in de novo AML patients compared with HC. (D) DEGs in S-AML versus de novo AML patients. (E) GSEA enrichment plots of DEGs of GO biological processes were predominantly engaged in regulation of mitotic metaphase/anaphase transition (GO:0030071) in S-AML versus de novo AML patients. (F) Upregulated DEGs enriched KEGG pathways in S-AML versus de novo AML patients. (G) The DEGs in R-AML from TCGA versus primary AML from GEO. (H) Twelve targeted upregulated DEGs among R/R-AML, S-AML, R-AML, and primary/de novo AML patients. (I) CENPE in R-AML was significantly higher than that of primary AML patients. X-tile software calculated the cutoff values of CENPE in R-AML patients, and survival analysis was conducted in R-AML patients with CENPE high expression and R-AML patients with CENPE low expression.

Figure 2

CENPE interference inhibited K562 and THP-1 cell proliferation. (A, B) K562 and THP-1 cells were transfected with si-NC, si-CENPE#1, or si-CENPE#2. Knockdown efficiency of CENPE in K562 and THP-1 cells was measured by RT-qPCR. (C, D) Cell proliferation was evaluated by CCK-8 assay, and si-CENPE significantly inhibited K562 and THP-1 cell activities compared with the si-NC group. **p < 0.01. ***p < 0.001.

Figure 3

CENPE interference effected K562 and THP-1 cell apoptosis and cell cycle and drug resistance. (A, B) After 48 h of transfection, cell apoptosis was measured by flow cytometry. The cell apoptotic rates between si-NC and si-CENPE groups were analyzed in K562 and THP-1 cells. (C, D) After 48 h of transfection, cell cycle was measured by PI single-staining method. (E, F) After 48 h, Western blot analyzed the expression of cycle-related proteins Cyclin B1 and p21 in K562 and THP-1 cells. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 4

CENPE interference effected Ara-C resistance in K562 and THP-1 cells. (A, B) K562 and THP-1 cells were treated with ascending concentrations of Ara-C (0.125 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, 4 µM, and 8 µM). After 48 h, IC50 values were measured and analyzed by the CCK-8 method. The experiment was independently repeated three times and statistical differences between the si-NC and si-CENPE groups were analyzed. **p < 0.01.

Figure 5

CENPE expression was highly correlated with RBP LIN28A. (A) Starbase database was used to screen out 25 RBPs, which might bind to CENPE and were differentially expressed in R-AML. (B) CENPE expression was strongly related to RBP LIN28A (r = 0.24; p < 0.05). (C) LIN28A gene expression were higher in the 151 R-AML whole blood samples from the TCGA database versus 7 primary AML samples from the GEO database. ****p < 0.0001.

Figure 6

LIN28A effected CENPE expression and mRNA stability. (A, B) Transfected K562 and THP-1 cells with si-NC or si-LIN28A, and LIN28A mRNA and protein were detected by RT-qPCR and Western blot 48 h after transfection. (C, D) Forty-eight hours after transfection of LIN28A in K562 and THP-1 cells, CENPE mRNA and protein were examined by RT-qPCR and Western blot. (E, F) The impact of the knockdown of LIN28A on the stability of CENPE mRNA was evaluated by the actinomycin D assay. *p < 0.05. **p < 0.01. ***p < 0.001.

Figure 7

LIN28A interacted with CENPE by binding to the 3’UTR region. (A) CENPE mRNA enriched by IgG or LIN28A antibodies in K562 cells were detected by RIP-conjugated RT-qPCR. (B) RNA pull-down and Western blot assays were conducted to detect the LIN28A protein levels being pulled down by biotin sense or antisense CENPE 3’UTR. (C) K562 cells were transfected with empty vector or LIN28A overexpression plasmid, and LIN28 expression was detected by RT-qPCR and Western blot. (D) The CENPE Wt and CENPE Mut were transfected in K562 cells, including CENPE Wt+Vector, CENPE Wt+LIN28A, CENPE Mut+Vector, and CENPE Mut+LIN28A, and 48 h after cell transfection, changes in luciferase activity were measured with a luciferase activity assay kit. **p < 0.01. ***p < 0.001.

Figure 8

Interaction of LIN28A and CENPE effected cell proliferation and apoptosis in K562 and THP-1 cells. (A) CCK8 assay was used to explore the effect of LIN28A overexpression and CENPE interference on cell proliferation regulated by LIN28A overexpression of K562 cells. (B) After 48 h of transfection, cell apoptosis was measured by Annexin V-FITC/PI double-staining method flow cytometry in LIN28A overexpressed and CENPE interfered LIN28A overexpressed K562 cells. The cell apoptotic rates were analyzed in K562 cells. (C) CCK8 assay was used to explore the effect of LIN28A overexpression and CENPE interference on cell proliferation regulated by LIN28A overexpression of THP-1 cells. (D) After 48 h of transfection, cell apoptosis was detected by Annexin V-FITC/PI double-staining method flow cytometry in LIN28A overexpressed and CENPE interfered LIN28A overexpressed THP-1 cells. The cell apoptotic rates were analyzed in THP-1 cells. **p < 0.01. ***p < 0.001.

Figure 9

LIN28A/CENPE inhibited the cell cycle progression in K562 and THP-1 cells. (A, B) After 48 h of transfection, cell cycle was measured by PI single-staining method in LIN28A overexpressed and CENPE interfered LIN28A overexpressed K562 and THP-1 cells. (C, D) After 48 h, Western blot analyzed the expression of cycle-related proteins Cyclin B1 and p21 in LIN28A overexpressed and CENPE interfered LIN28A overexpressed K562 and THP-1 cells. ***p < 0.001.

Figure 10

LIN28A/CENPE interaction effected Ara-C resistance in K562 and THP-1 cells. (A, B) LIN28A overexpressed and CENPE interfered LIN28A overexpressed K562 and THP-1 cells were treated with ascending concentrations of Ara-C (0.125 µM, 0.25 µM, 0.5 µM, 1 µM, 2 µM, 4 µM, and 8 µM). After 48 h, IC50 values were measured and analyzed by the CCK-8 method. The experiment was independently repeated three times and statistical differences were analyzed. *p < 0.05. **p < 0.01.