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. 2015 Apr 29:14:96.
doi: 10.1186/s12943-015-0344-y.

MiR-320a acts as a prognostic factor and Inhibits metastasis of salivary adenoid cystic carcinoma by targeting ITGB3

Lijuan Sun  1   2   3 , Bodu Liu  4   5 , Zhaoyu Lin  6   7 , Yandan Yao  8   9 , Yanyang Chen  10 , Yang Li  11 , Jianing Chen  12   13 , Dongsheng Yu  14 , Zhangui Tang  15 , Bosheng Wang  16 , Shuguang Zeng  17 , Song Fan  18   19 , Youyuan Wang  20   21 , Yilin Li  22 , Erwei Song  23   24   25 , Jinsong Li  26   27   28
Affiliations

MiR-320a acts as a prognostic factor and Inhibits metastasis of salivary adenoid cystic carcinoma by targeting ITGB3

Lijuan Sun et al. Mol Cancer. .

Abstract

Background: Salivary Adenoid cystic carcinoma (SACC) patients with local invasion and lung metastasis are often resistant to conventional therapy such as operation, chemotherapy and radiotherapy. To explore the underling mechanisms, we studied the roles of miRNA in regulating invasiveness of SACC cells.

Methods: MicroRNA profiling was done in SACC cells with microarray. MiRNA mimics or antisense oligonucleotide was transfected and invasiveness of SACC cells was evaluated by adhesion assay and transwell assay. The target gene of miRNA was identified by luciferase reporter assay and "rescue" experiment. Tumor metastasis was evaluated by BALB/c-nu mice xenografts. MiRNA and its target gene expression were identified by in-situ hybridization and immunohistochemistry respectively, in 302 patients from affiliated hospitals of Sun Yat-sen University and in 148 patients from affiliated hospitals of Central South University, and correlated to the clinicopathological status of the patients.

Results: MiR-320a was down-regulated in high lung metastatic ACCM and SACC-LM cells compared with the corresponding low metastatic ACC2 and SACC-83 cells, and inhibited adhesion, invasion and migration of SACC cells by targeting integrin beta 3 (ITGB3). In vivo, enforced miR-320a expression suppressed metastasis of SACC xenografts. In the two independent sets, miR-320a was downregulated in primary SACCs with metastasis compared to those without metastasis, and low expression of this miRNA predicts poor patient survival and rapid metastasis. Multivariate analysis showed that miR-320a expression was an independent indicator of lung metastasis.

Conclusions: MiR-320a inhibits metastasis in SACCs by targeting ITGB3 and may serve as a therapeutic target and prognostic marker in salivary cancers.

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Figures

Figure 1
Figure 1
Ectopic expression of miR-320a inhibits the invasiveness of salivary adenoid cystic carcinoma cells. (A) miR-320a expression in SACC cells was determined by qRT-PCR analysis. **P < 0.01 vs. parental SACC cells. ## P < 0.01 vs. mock transfection. U6 was used as an internal control. Adhesion assays (B) and transwell assays (C, D) showed that adhesion, invasion and migration of ACCM and SACC-LM cells were inhibited by miR-320a mimics (100×). (E) Quantification of adhesive, invasive and migratory cells assessed by adhesion assays and transwell assays. *P < 0.05; **P < 0.01 vs. mock.
Figure 2
Figure 2
ITGB3 is an miR-320a target gene. (A) Target sequence of miR-320a in ITGB3 3′-UTR predicted by TargetScan, as well as the mutated sequence. (B) Luciferase reporter assays were performed in ACCM and SACC-LM cells co-transfected with miR-320a-mimics and reporter vectors carrying a ITGB3 3′-UTR with a wild-type (ITGB3 3′-UTR) or mutated miR-320a (ITGB3 3′-UTR mut) response element. *P < 0.05 vs. mock transfection. (C) Integrin β3 expression, as well as FAK and Src phosphorylation, was assessed using western blot analysis. β-actin was used as an internal control. (D) Colocalization of integrin β3 and the cytoskeletal protein actin was illustrated by immunofluorescence staining. Nuclei: blue, scale bar: 10 μm.
Figure 3
Figure 3
miR-320a inhibits the invasiveness of SACC cells by targeting ITGB3. (A) Integrin β3 expression and FAK and Src phosphorylation were determined by western blot analysis in ACCM cells transfected with miR-320a mimics alone or in combination with either pcDNA 3.1+ (Vector) or pcDNA 3.1+ containing a wild-type (ITGB3) or mutant ITGB3 expression cassette of the miR-320a (ITGB3 mut) response element. β-actin was used as an internal control. Adhesion, invasion and migration of ACCM cells were assessed using adhesion assays (B) and transwell assays (C, D) (100x). (E) Quantification of adhesive, invasive and migratory ACCM cells. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vector.
Figure 4
Figure 4
Ectopic expression of miR-320a inhibits ACCM xenograft metastasis in BALB/c-nu mice. (A) Whole-body luminal imaging was performed in mice inoculated with ACCM stably expressing the luciferase gene. Luminal images (B) and HE staining (C) of paraffin sections (200×) of the lungs (upper) and livers (lower) of the tumor-bearing mice. (D) Mean ± SD wet lung weight of tumor-bearing mice (n = 8/group). (E) Expression of human HPRT mRNA relative to mouse 18S rRNA in the lungs and livers of the tumor-bearing mice was determined by qRT-PCR analysis. **P < 0.01 vs. ACC2 cells. ## P < 0.01 vs. EmGFP transfection.
Figure 5
Figure 5
miR-320a downregulation correlates with metastasis and poor patient survival in SACCs. (A) In situ hybridization for miR-320a and immunohistochemical staining for integrin β3 in primary SACCs with metastasis vs. those without metastasis (200×). Quantification of miR-320a and integrin β3 expression in primary SACC samples from the affiliated hospitals of Sun Yat-sen University (B) or from the affiliated hospitals of Central South University (C). ***P < 0.001. Kaplan-Meier curves for SACC patients from the affiliated hospitals of Sun Yat-sen University comparing miR-320a expression and metastasis (D) or miR-320a expression and survival (E) was analyzed by the log rank test. Kaplan-Meier curves for SACC patients from the affiliated hospitals of Central South University comparing miR-320a expression and metastasis (F) or miR-320a expression and survival (G). The median value was used to distinguish high expression and low expression.
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