BCYRN1, a c-MYC-activated long non-coding RNA, regulates cell metastasis of non-small-cell lung cancer

Abstract

Background: Long non-coding RNAs (lncRNAs) are increasingly implicated in the regulation of the progression of malignancy.

Aim: To clarify the relations among BCYRN1 (brain cytoplasmic RNA 1, a long non-coding RNA), c-MYC and cell metastasis of non-small-cell lung cancer (NSCLC).

Methods: Real-time PCR was used to measure expression of BCYRN1 in NSCLC. Knockdown and overexpression of c-MYC were respectively performed using shRNA and lentivirus to investigate its effect on BCYRN1 expression. BCYRN1 was respectively knockdown and overexpressed by siRNA and BCYRN1 mimics to investigate its role in regulating cell metastasis in vitro. ChIP (chromatin immunoprecipitation) assay was performed to confirm the binding of c-MYC to the promoter of BCYRN1. Expression levels of matrix metalloproteinases (MMP9 and MMP13) were determined using real-time PCR and Western blotting.

Results: BCYRN1 is upregulated and targeted by c-MYC in NSCLC, leading to the increase of cell motility and invasiveness. RNA interference and lentivirus infection showed a positive correlation between the expressions of c-MYC and BCYRN1. ChIP assay confirmed the binding of c-MYC to the promoter region of BCYRN1 gene. In-vitro cell metastasis experiments demonstrated that BCYRN1 was necessary in the c-MYC-regulated cell migration and invasion. The mRNA and protein expression levels of MMP9 and MMP13 descended with the decreasing BCYRN1 level and ascended with the upregulation of BCYRN1.

Conclusion: These findings uncover a regulatory mechanism in NSCLC cells involving the metastasis-promoting lncRNA BCYRN1 that improves expressions of the key metastasis-supporting proteins MMP9 and MMP13.

Keywords: BCYRN1 (brain cytoplasmic RNA 1); Metastasis; NSCLC (non-small-cell lung cancer); c-MYC; lncRNA (long non-coding RNA).

Figure 1

BCYRN1 expression level in NSCLC tissue (A) and cell lines (B), and c-MYC protein level in NSCLC cell lines (C, D). Normal: the adjacent normal tissue, used as a control; 16HBE: a normal human bronchial epithelial cell line.

Figure 2

Protein c-MYC binds to the promoter of BCYRN1. A/B. Protein (A) and mRNA (B) levels of BCYRN1 and c-MYC in the NSCLC cells infected with pWPXL. 10058-F4: a small-molecule c-MYC inhibitor; groups shRNA-c-MYC, shRNA-NC (NC: negative control), pWPXL-c-MYC and pWPXL-NC respectively represented the groups where NSCLC cells were transfected with specific shRNA against c-MYC, non-specific shRNA, and infected with pWPXL-c-MYC vector or the pWPXL vector containing non-specific sequence. (C) ChIP assay showed binding of c-Myc to the BCYRN1 promoter in NSCLC tissues. (D) ChIP assay showed binding of c-Myc to the BCYRN1 promoter in A549 cells. Soluble chromatin of A549 cells and NSCLC tissue were immunopurified using anti-c-MYC antibody and quantitative real-time PCR was used to measure the level of enrichment on BCYRN1. ** p < 0.01, indicated a very significant difference, compared with negative control.

Figure 3

The change of BCYRN1 level in A549 cells (A), and migration and invasion of A549 cells (B ~ D; B: the quantitative results for figures C and D) after siRNA interference in c-MYC overexpressed cells. Blank control: the cells alone infected with pWPXL-c-Myc. pWPXL-c-Myc + siRNA-NC: the cells jointly treated with lentivirus (pWPXL-c-Myc) infection and siRNA transfection together. pWPXL-c-Myc + siRNA-BCYRN1-1 or −2: the cells jointly treated with lentivirus (pWPXL-c-Myc) infection and siRNA transfection (the two specific siRNAs of BCYRN1were respectively named as siRNA-BCYRN1-1 and siRNA-BCYRN1-2). Magnification: 100 ×. ** p < 0.01, indicated a very significant difference, compared with control.

Figure 4

The RNA (A, C) and protein (B, D) expression levels of MMP9 and MMP13 were both downregulated by BCYRN1 inhibition, and upregulated by BCYRN1 mimics. siRNA-NC: siRNA negative control; siRNA-1: siRNA-BCYRN1-1; siRNA-2: siRNA-BCYRN1-2; NC: negative control for BCYRN1 mimics, only treated with Lipofectamine 2000. * p < 0.01, indicated a significant difference; ** p < 0.01, indicated a very significant difference, compared with negative control.