Downregulation of BRAF activated non-coding RNA is associated with poor prognosis for non-small cell lung cancer and promotes metastasis by affecting epithelial-mesenchymal transition

Abstract

Background: Recent evidence indicates that long noncoding RNAs (lncRNAs) play a critical role in the regulation of cellular processes, such as differentiation, proliferation and metastasis. These lncRNAs are found to be dysregulated in a variety of cancers. BRAF activated non-coding RNA (BANCR) is a 693-bp transcript on chromosome 9 with a potential functional role in melanoma cell migration. The clinical significance of BANCR, and its' molecular mechanisms controlling cancer cell migration and metastasis are unclear.

Methods: Expression of BANCR was analyzed in 113 non-small cell lung cancer (NSCLC) tissues and seven NSCLC cell lines using quantitative polymerase chain reaction (qPCR) assays. Gain and loss of function approaches were used to investigate the biological role of BANCR in NSCLC cells. The effects of BANCR on cell viability were evaluated by MTT and colony formation assays. Apoptosis was evaluated by Hoechst staining and flow cytometry. Nude mice were used to examine the effects of BANCR on tumor cell metastasis in vivo. Protein levels of BANCR targets were determined by western blotting and fluorescent immunohistochemistry.

Results: BANCR expression was significantly decreased in 113 NSCLC tumor tissues compared with normal tissues. Additionally, reduced BANCR expression was associated with larger tumor size, advanced pathological stage, metastasis distance, and shorter overall survival of NSCLC patients. Reduced BANCR expression was found to be an independent prognostic factor for NSCLC. Histone deacetylation was involved in the downregulation of BANCR in NSCLC cells. Ectopic expression of BANCR impaired cell viability and invasion, leading to the inhibition of metastasis in vitro and in vivo. However, knockdown of BANCR expression promoted cell migration and invasion in vitro. Overexpression of BANCR was found to play a key role in epithelial-mesenchymal transition (EMT) through the regulation of E-cadherin, N-cadherin and Vimentin expression.

Conclusion: We determined that BANCR actively functions as a regulator of EMT during NSCLC metastasis, suggesting that BANCR could be a biomarker for poor prognosis of NSCLC.

Figure 1

Relative BANCR expression in NSCLC tissues and its clinical significance. (A) Relative expression of BANCR in NSCLC tissues (n = 113) compared with corresponding non-tumor tissues (n = 113). BANCR expression was examined by qPCR and normalized to GAPDH expression. Results were presented as the fold-change in tumor tissues relative to normal tissues. (B) BANCR expression was classified into two groups. (C, D) Kaplan–Meier disease-free survival and overall survival curves according to BANCR expression levels. *P < 0.05, **P < 0.01.

Figure 2

Histone deacetylation is involved in BANCR downregulation. (A) BANCR expression levels of NSCLC cell lines (A549, SPC-A1, H1299, H1650, H1975 and SK-MES-1) compared with that in normal human bronchial epithelial cells (16HBE). (B) qPCR analysis of BANCR expression levels following the treatment of SPC-A1 and A549 cells with TSA. (C) qPCR analysis of HDAC2 and HDAC3 expression levels following the treatment of SPC-A1 and A549 cells with si-HDAC2 or si-HDAC3.(D, E) qPCR analysis of BANCR expression levels following the treatment of SPC-A1 and A549 cells with si-HDAC1 and si-HDAC3.

Figure 3

Effects of BANCR on NSCLC cell viability and apoptosis in vitro. (A) SPC-A1 and A549 cells were transfected with pCDNA-BANCR. (B, C) MTT assays were used to determine the cell viability for pCDNA-BANCR-transfected SPC-A1 and A549 cells. Values represented the mean ± s.d. from three independent experiments. (D) Colony-forming assays were conducted to determine the proliferation of pCDNA-BANCR-transfected SPC-A1 and A549 cells. (E) Apoptosis was determined by flow cytometry. UL, necrotic cells; UR, terminal apoptotic cells; LR, early apoptotic cells. *P < 0.05 and **P < 0.01.

Figure 4

Effects of BANCR on NSCLC migration and invasion in vitro. SPC-A1 and A549 cells were transfected with pCDNA-BANCR. (A, B) Wound-healing assays were used to investigate the migratory ability of NSCLC cells. (C, D) Transwell assays were used to investigate the changes in migratory and invasive abilities of NSCLC cells. *P < 0.05 and **P < 0.01.

Figure 5

Effects of BANCR overexpression on tumor metastasis in vivo. (A) BANCR expression levels were determined by qPCR following the treatment of A549 cells with si-BANCR. (B) Transwell assays were conducted to determine the migratory and invasive abilities of si-BANCR-transfected A549 cells. Analysis of an experimental metastasis animal model was performed by injecting BANCR-overexpressing SPC-A1 cells into nude mice. (C) Lungs from mice in each experimental group, with the numbers of tumor nodules on lung surfaces were shown. (D) Visualization of the entire lung, and HE-stained lung sections. **P < 0.01.

Figure 6

BANCR overexpression suppresses NSCLC cell invasion and metastasis by affecting EMT. (A, B) Analysis of E-cadherin, N-cadherin, Vimentin, MMP-2, MMP-9, SNAIL1, SNAIL2, TWIST and SIP1 expression in A549 cells treated with pCDNA-BANCR. (C,D) Analysis of E-cadherin and Vimentin expression in A549 cells treated with pCDNA-BANCR by western blot and immunofluorescence. All experiments were performed in triplicate with three technical replicates. *P < 0.05 , **P < 0.01.