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. 2016 Sep 20;7(38):62364-62376.
doi: 10.18632/oncotarget.11634.

miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1

Wei Lu  1   2 , Yunping Hu  2 , Qiang Ma  2 , Linzhu Zhou  3 , Lin Jiang  2 , Zhizhen Li  2 , Shuai Zhao  2 , Yuzhen Xu  4 , Weibin Shi  1 , Sheng Li  5 , Yingbin Liu  1   2
Affiliations

miR-223 increases gallbladder cancer cell sensitivity to docetaxel by downregulating STMN1

Wei Lu et al. Oncotarget. .

Abstract

Background: MicroRNAs (miRs) are involved in cancer carcinogenesis, and certain regulatory miRs could provide promising therapeutic methods for refractory malignancies, such as gallbladder cancer (GBC). miR-223 was found to play a pivotal role in enhancing chemotherapeutic effects, therefore evoking interest in the role of miR-223 in GBC.

Results: miR-223 was decreased in GBC tissues and cell lines, and ectopic miR- 223 expression exhibited multiple anti-tumorigenic effects in GBC cells, including decreased proliferation, migration and invasion in vitro. However, treatment with a miR-223 inhibitor increased cell viability. We determined that STMN1 was negatively correlated with and regulated by miR-223 in GBC. miR-223 increased GBC sensitivity to docetaxel in vitro and in vivo, and the induced sensitivity to docetaxel was suppressed by the restoration of STMN1 expression.

Methods: We examined miR-223 expression in GBC tissue and GBC cell lines using qRT-PCR. The effects of modulated miR-223 expression in GBC cells were assayed using Cell Counting Kit-8 (CCK8), flow cytometry, and wound-healing and invasion assays. Susceptibility to docetaxel was evaluated in miR-223/STMN1-modulated GBC cells and xenograft tumor models. The protein expression of relevant genes was examined by Western blotting.

Conclusions: These findings indicated that miR-223 might serve as an onco-suppressor that enhances susceptibility to docetaxel by downregulating STMN1 in GBC, highlighting its promising therapeutic value.

Keywords: STMN1; gallbladder cancer; malignancy; miR-223.

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Conflict of interest statement

All authors declare no conflicts of interest. All authors declare no previous presentation of the manuscript.

Figures

Figure 1
Figure 1. Aberrant miR-223 expression in gallbladder cancer tissue samples and correlations with STMN1 upregulation
(A) qRT-PCR detection of miR-223 expression in 5 normal gallbladder tissues, 16 gallbladder cancer tissues and their matched pericarcinous gallbladder peripheral tissues, 2 gallbladder cancer cell lines. miR-223 expression was significantly higher in normal gallbladder tissues (P = 0.0002) and peripheral tissues from GBC patients (P = 0.0003) but was downregulated in GBC tissue. The data are presented as the mean ± SD from three independent experiments. (B) The inverse correlation between miR-223 and STMN1 mRNA expression in gallbladder cancer tissue samples (n = 16) by linear regression analysis. (C) A Western blot examining the protein expression level in the tissue samples of 5 gallbladder cancer samples and their peripheral tissues. (D) Sequence alignment of miR-223 with the 3′ UTR of the STMN1 gene. (E) miR-223 expression in normal (left) and cancerous (right) gallbladder tissues examined by in situ hybridization.
Figure 2
Figure 2. Modulation of miR-223 and STMN1 expression in gallbladder cancer cells by miR-223 mimics, a miR-223 inhibitor and a STMN1 overexpression plasmid
(A) miR-223 levels in GBC-SD and NOZ cell lines were significantly elevated upon transfection of a miR-223 mimics vector and decreased by a miR-223 inhibitor. (B) Both the STMN1 mRNA and protein expression levels were decreased after the transfection of miR-223 mimics in GBC-SD cells. (C) Both STMN1 mRNA and protein expression levels were increased after transfection of a miR-223 inhibitor in GBC-SD cells. (D) STMN1 expression was significantly increased after transfection of a STMN1 expression plasmid. The expression of miR-223 and STMN1 mRNA was measured by qRT-PCR and the expression of STMN1 protein by Western blotting.
Figure 3
Figure 3. The effect of miR-223 mimics and inhibitor on GBC cell proliferation
(A) Overexpression of miR-223 suppressed cell growth in GBC-SD and NOZ cells. (B) Inhibition of miR-223 stimulated cell growth in GBC-SD and NOZ cells. (C) and (D) The effect of overexpression and inhibition of miR-223 on the cell growth curve of GBC-SD and NOZ cells. At 24 h after transfection of the indicated vector, GBC-SD and NOZ cells were seeded into 96-well cell culture plates. The proliferative effects were evaluated by CCK8 assay 48 h later, as shown in (A) and (B). Cell viability was measured every 24 h using a CCK8 assay as shown in (C) and (D). The data are presented as the mean ± SD from three independent experiments.
Figure 4
Figure 4. Overexpression of miR-223 inhibits GBC cell migration and invasion
(A) The migratory ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a wound-healing migration assay. Representative phase-contrast photomicrographs and wound-closure rates are shown at 0 and 24 h after wound formation. (B) The invasive ability of GBC-SD and NOZ cells transfected with miR-223 mimics was assessed using a transwell invasion assay. Following a 24-h incubation, invasive cells that passed through the Matrigel chambers were fixed and stained; these cells and the cell migration rates are shown. The error bars represent the mean ± SD of triplicate experiments (**P < 0.01; ***P < 0.001).
Figure 5
Figure 5. GBC-SD and NOZ cells were resistant to docetaxel and sensitized by ectopic expression of miR-223
(A) GBC-SD and NOZ cells were resisted to 100 μM docetaxel (DTX). Either GBC-SD or NOZ cells were seeded into 96-well cell culture plates for 24 h, then treated with DMSO or docetaxel; the cytostatic effects were evaluated by CCK8 assay. (B) miR-223 mimics enhanced the cytotoxicity of docetaxel in GBC-SD cells and NOZ cells. After 24 h of transfection, GBC-SD or NOZ cells were seeded in 96-well cell culture plates and treated with either DMSO or 10 μM docetaxel for an additional 48 h followed by a CCK8 assay. (C) and (D) A low concentration of docetaxel was lethal to GBC-SD cells and NOZ cells overexpressing miR-223. At 24 h after transfection, GBC-SD and NOZ cells were seeded into 96-well cell culture plates with either DMSO or 10 μM docetaxel, and cell viability was measured every 24 h using a CCK8 assay. (E) (F) and (G) Overexpression of miR-223 inhibited tumor growth and induced sensitivity to docetaxel in the NOZ xenograft model. There were four groups of mice: scramble control with either PBS or DTX and miR-223 mimics with either PBS or DTX. The tumor growth curves are shown in (E). The mice were sacrificed, and the tumors were harvested and weighed 21 days after the initial injection (*P < 0.05; **P < 0.01; ***P < 0.001) as shown in (F). The harvested tumor tissues were examined for STMN1 expression by immunohistochemistry. (G) NOZ cells lentivirally transduced with scramble control (left side) or exogenous miR-223 (right side).
Figure 6
Figure 6. The cytotoxic effect of docetaxel to gallbladder cancer cells overexpressing miR-223 was suppressed by STMN1 restoration
(A) Transfection of an STMN1-expressing plasmid reversed the growth inhibition of docetaxel in gallbladder cancer cells overexpressing miR-223. GBC-SD and NOZ cells were transfected with indicated vector and seeded into 96-well cell culture plates for 24 h. The cells were then treated with either DMSO or 10 μM docetaxel and cultured for another 48 h before they were subjected to a CCK8 assay. The data are presented as the mean ± SD from three independent experiments. (B) and (C) Overexpression of miR-223 increased apoptosis of GBC-SD cells. However, this effect was suppressed by transfection of a STMN1 expression vector. GBC-SD cells were transfected with the indicated vector for 24 h, after which, the cells were seeded into 6-well cell culture plates. The following day, cells were treated with either DMSO or 10 μM docetaxel for 24 h. The extent of apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit by flow cytometry as shown in panel (B) and DAPI-stained the nuclei of GBC-SD cells as shown in panel (C). (D) Overexpression of miR-223 increased docetaxel-induced caspase 3 cleavage, whereas co-transfection of the STMN1-expressing plasmid suppressed this effect in GBC-SD cells.
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