MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia - PubMed (original) (raw)

MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia

Zhu Xishan et al. Sci Rep. 2015.

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Abstract

Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

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Figures

Figure 1

Figure 1. Downregulation of miR-320a expression in CML cell lines and CML cancer stem cells compared with the corresponding controls.

(A). Relative expression of miR-320a in 4 CML cell lines and one normal control were detected by qRT-PCR. All experiments were repeated at least three times. Each bar represents the mean of three independent experiments. *P < 0.05. (B). Relative expression of miR-320a in 70 specimens of CML cancer stem cells and normal MSCs were carried out by qRT-PCR. Data are shown as –△△CT values. (C) The mean and standard deviation of miR-320a expression levels in 70 specimens of CML cancer stem cells and normal MSCs were shown. Data are presented as 2−△Ct values (**P < 0.01). (D). Survival analysis of CML. OS and RFS curves for 90 CML patients with high or low miR-320a expression were constructed using the Kaplan-Meier method and evaluated using the log-rank test.

Figure 2

Figure 2. miR-320a inhibits CML cell migration, invasion, proliferation and induces apoptosis.

(A) The miR-320a expression was significantly increased in K562 after infection with LV-hsa-miR-320a. (B) The BCR/ABL expression was significantly decreased in K562 after infection with LV-hsa-miR-320a. (C) K562 proliferation was significantly reduced after LV-hsa-miR-320a infection compared with cont-miR infection. (D) miR-320a overexpression significantly inhibited the colony-forming ability of K562. (E–G) K562 cell migration, invasion, proliferation and apoptosis were restored after BCR/ABL restoration. The data represent the means ± s.d.; *p < 0.001, **p < 0.05, ***p < 0.01.

Figure 3

Figure 3. Overexpression of miR-320a inhibits tumorigenicity and increases apoptosis in vivo.

(A) Photographs of tumors derived from RV-miR-320a, RV-miR-control or K562 cells in nude mice. (B) Growth kinetics of tumors in nude mice. Tumor diameters were measured every 7 days. (*p < 0.05, **p < 0.01).(C) Average weight of tumors in nude mice. (**p < 0.01). (D) Comparison of proliferation index. (*p < 0.05). (E) The percentage of apoptotic cells was counted. (**p < 0.01).

Figure 4

Figure 4. miR-320a directly targets BCR/ABL.

(A) The 3′-UTR element of BCR/ABL messenger RNA was partially complementary to miR-320a. miR-320a, anti-miR-320a or scramble control and luciferase reporter containing either a wild type or a mutant 3′-UTR were co-transfected into HEK-293T cells. And a Renilla luciferase expressing construct exerts as internal control. (B) Western blot analysis of BCR/ABL expression in K562 cells infected with miR-320a, and NC transfected with miR-320a inhibitors (Anti-miR-320a). The gels have been run under the same experimental conditions. (C). Analysis of correlation of miR-320a and BCR/ABL expression in CML cancer stem cells and normal MSCs. *p < 0.05, **p < 0.01, ***p < 0.001. The analysis indicated that BCR/ABL and miR-320a were negatively correlated. (D–F) BCR/ABL abrogated the suppressive roles of miR-320a in K562 invasion and growth. K562 cells stably expressing miR-320a or scramble were transfect with or without BCR/ABL plasmids. Invasion assays (D), apoptosis analysis (E) and cell proliferation analysis (F) were performed with the above cells as described in Materials and Methods. Data are presented as mean ± s.e.m from at least three independent experiments. (G) Spearman’s correlation scatter plot of the levels of miR-320a (determined by in situ hybridization) and BCR/ABL protein (determined by immunohistochemistry) in 90 CML specimens. Representative images of BCR/ABL expression by immunohistochemistry were shown. Original magnification: ×200.

Figure 5

Figure 5. miR-320a down-regulates the phosphorylation of PI3K, AKT and NF-κB via BCR/ABL.

(A) The phosphorylation and total expression levels of PI3K, AKT and NF-κB in K562 cells infected with LV-hsa-miR-320a or cont-miR. (B) An immunoblot analysis of BCR/ABL expression in K562 cells infected with LV-hsa-miR-320a or cont-miR, with or without BCR/ABL restoration. (C) The phosphorylation and total expression levels of PI3K, AKT and NF-κB in K562 cells infected with LV-hsa-miR-320a or cont-miR, with or without BCR/ABL restoration. The expression levels of the phosphorylated proteins were normalized to those of the respective total proteins. The data represent the means ± s.d.; *p < 0.01. All the gels have been run under the same experimental conditions.

Figure 6

Figure 6. miR-320a promotes an epithelial phenotype in K562.

(A) Right panel: BCR/ABL expression was detected by western blot in K562 cells after treatment with 3 independent siRNA sequences (siNRP1) or a control (siC). Left panel: Relative expression of BCR/ABL was shown in the histogram. (B) Right panel: An immunoblot analysis of N-cadherin, vimentin, fibronectin and E-cadherin in K562 cells transfected with siNRP1 or siC. Left panel: Relative expression of proteins was shown in the histogram. (C) An immunoblot analysis of N-cadherin, vimentin, fibronectin and Ecadherin in K562 cells infected with LV-hsa-miR-320a or cont-miR, with or without BCR/ABL restoration. The protein expression levels were normalized to Actin. The data represent the means ± s.d.; *p < 0.01. All the gels have been run under the same experimental conditions.

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