Tumor-suppressive microRNA-29 family inhibits cancer cell migration and invasion directly targeting LOXL2 in lung squamous cell carcinoma

Abstract

Lung cancer remains the most frequent cause of cancer-related death in developed countries. A recent molecular-targeted strategy has contributed to improvement of the remarkable effect of adenocarcinoma of the lung. However, such treatment has not been developed for squamous cell carcinoma (SCC) of the disease. Our recent studies of microRNA (miRNA) expression signatures of human cancers showed that the microRNA-29 family (miR‑29a, miR‑29b and miR‑29c) significantly reduced cancer tissues compared to normal tissues. These findings suggest that miR‑29s act as tumor-suppressors by targeting several oncogenic genes. The aim of the study was to investigate the functional significance of miR‑29s in lung SCC and to identify miR‑29s modulating molecular targets in lung SCC cells. Restoration of all mature members of the miR‑29s inhibited cancer cell migration and invasion. Gene expression data combined in silico analysis and luciferase reporter assays demonstrated that the lysyl oxidase-like 2 (LOXL2) gene was a direct regulator of tumor‑suppressive miR‑29s. Moreover, overexpressed LOXL2 was confirmed in lung SCC clinical specimens, and silencing of LOXL2 inhibited cancer cell migration and invasion in lung SCC cell lines. Our present data suggested that loss of tumor-suppressive miR‑29s enhanced cancer cell invasion in lung SCC through direct regulation of oncogenic LOXL2. Elucidation of the novel lung SCC molecular pathways and targets regulated by tumor-suppressive miR‑29s will provide new insights into the potential mechanisms of oncogenesis and metastasis of the disease.

Figure 1

The expression levels of miR-29a, miR-29b and miR-29c in clinical specimens and cell lines. Real-time PCR showed that the expression levels of (A) miR-29a, (B) miR-29b and (C) miR-29c were significantly lower in lung SCC tissues than in normal lung tissues (P<0.0001, P=0.0031 and P<0.0001, respectively). RNU48 was used as an internal control. Correlations between (D) miR-29a-miR-29b, (E) miR-29b-miR-29c and (F) miR-29c-miR-29a were determined in lung SCC clinical specimens.

Figure 2

Effects of miR-29a, miR-29b and miR-29c transfection on SK-MES-1 and EBC-1 cells. (A) Cell proliferation was determined with XTT assays 96 h after transfection with 10 nM miR-29s, miR-control, or mock transfection. (B) Cell migration activity was determined by migration assay 48 h after transfection. (C) Cell invasion activity was determined by Matrigel invasion assay 72 h after transfection. ^*P<0.0001.

Figure 3

Flow chart of the strategy for identification of miR-29 target genes. In total, 2,627 genes were putative targets of miR-29s according to the TargetScan database. We merged the expression analysis data of downregulated genes in miR-29a-transfected EBC-1 cells (Log₂ ratio <-2.0). Upregulated genes were determined according to the gene expression data set of lung SCC clinical specimens according to the GEO database (accession no. GSE 11117). From this selection, 7 candidate genes were identified as targets of the miR-29s.

Figure 4

Direct regulation of LOXL2 by miR-29s in SK-MES-1 and EBC-1 cells. (A) LOXL2 mRNA expression was evaluated by qRT-PCR 72 h after transfection with miR-29s. GUSB was used as an internal control. (B) LOXL2 protein expression was evaluated by western blotting 96 h after transfection with miR-29s. GAPDH was used as a loading control.

Figure 5

Direct regulation of LOXL2 by miR-29s in lung SCC cells. A luciferase reporter assay using vectors encoding putative miR-29 target sites at positions (A) 555–561 and (B) 757–763 for both wild-types and deletion types, respectively. Renilla luciferase values were normalized to Firefly luciferase values. ^*P<0.0001.

Figure 6

Silencing of LOXL2 by using si-LOXL2 in lung SCC cells. Silencing of LOXL2 mRNA and protein expression by si-LOXL2 transfection and the effects of the silencing of LOXL2 on SK-MES-1 and EBC-1 cell activities. (A) LOXL2 mRNA expression was evaluated by qRT-PCR 72 h after transfection with miR-29s. GUSB was used as an internal control. ^*P<0.0001. (B) LOXL2 protein expression was evaluated by western blotting 96 h after transfection. GAPDH was used as a loading control.

Figure 7

Effects of si-LOXL2 transfection on lung SCC cell lines. (A) Cell proliferation was determined using XTT assays 96 h after transfection with 10 nM si-LOXL2, miR-control, or mock transfection. (B) Cell migration activity was determined by migration assay 48 h after transfection. (C) Cell invasion activity was determined by Matrigel invasion assay 72 h after transfection. ^*P<0.0001.

Figure 8

Immunohistochemical staining of LOXL2 in lung SCC specimens. Differences in LOXL2 expression are observed in cancer lesions and adjacent non-cancerous tissues in the same fields. Normal lung specimens stained negatively.