Loss of long noncoding RNA FOXF1-AS1 regulates epithelial-mesenchymal transition, stemness and metastasis of non-small cell lung cancer cells

Abstract

Although recent evidence shows that long noncoding RNAs (lncRNAs) are involved in the regulation of gene expression and cancer progression, the understanding of the role of lncRNAs in lung cancer metastasis is still limited. To identify novel lncRNAs in non-small cell lung cancer (NSCLC), we profile NSCLC tumor and matched normal samples using GeneChip® Human Gene 2.0 ST Array, which provides the most accurate, sensitive, and comprehensive measurement of protein coding and lncRNA transcripts. We identified a panel of key factors dysregulated in lung cancer. Among them, the expression of FOXF1-AS1 was significantly downregulated in lung cancer. Stable overexpression of FOXF1-AS1 inhibits lung cancer cell migration and invasion by regulating EMT. Meanwhile, loss of FOXF1-AS1 mediates stem-like properties of lung cancer cells. Interestingly, we found that FOXF1-AS1 physically associates with PRC2 components EZH2 and loss of FOXF1-AS1 mediates cell migration and stem-like properties require EZH2. Loss of FOXF1-AS1 is also correlated with downregulation of FOXF1 in lung cancer. These results suggested that FOXF1-AS1 might regulate EMT, stemness and metastasis of NSCLC cells via EZH2, indicating it as a therapeutic target for future treatment of NSCLC.

Keywords: EMT; FOXF1-AS1; LncRNA; lung cancer; metastasis.

Figure 1. The expression of FOXF1-AS1 was significantly downregulated in lung cancer

A. Hierarchical clustering showed the expression differences of lncRNAs between control matched normal samples (MN1, MN2, MN3) and tumor tissue samples (T1, T2, T3), of which the expression of LncRNA FOXF1-AS1 was significantly low in tumor tissue groups compared control groups. B. FOXF1-AS1 was lowly expressed in tumor tissues (n=50) than normal tissues (n=50) detected by real time qRT-PCR. p < 0.0001. C. The expression of FOXF1-AS1 mRNA had no significance between lung Adenocarcinoma (AD) (n=30) and Squamous Carcinoma (SC) (n=20) detected using real time qRT-PCR. P=0.1344. D. The expression of FOXF1-AS1 mRNA had no significance among ADs at different phases (n=10 for AD-I, AD-II and AD-III respectively).

Figure 2. Stable overexpression of FOXF1-AS1 inhibited lung cancer cell migration and invasion

A. The expression of FOXF1-AS1 was decreased variously among different lung cancer cell lines compared to normal IMR-90 cells. B. The expression of FOXF1-AS1 was assessed in CALU1 and NCIH1975 cells co-transfected with full length FOXF1-AS1 cDNA using plasmid vectors compared with control CALU1 and NCIH1975 cells. C. The representative images showed the effect of FOXF1-AS1 overexpression on migration of CALU1 and NCIH1975 cells by transwell migration assay. D. The number of migration cells was quantified among three different wells. E. The representative images showed the effect of FOXF1-AS1 overexpression on invasion of CALU1 and NCIH1975 cells by transwell matrigel invasion assay. F. The number of invasive cells was quantified among three different wells.

Figure 3. FOXF1-AS1 regulated epithelial-mesenchymal transition in lung cancer cells

A. The representative image of CALU1 and NCIH1975 cells and the stable transfected with full length FOXF1-AS1 cDNA through light microscope. The morphological changes of CALU1 and NCIH1975 cells from a fibroblastoid appearance elongated spindle shape to cobblestone shape was observed after stable overexpression FOXF1-AS1, which is like mesenchymal to epithelial transition. B. Western blot analysis of EMT markers E-Cadherin and Vimentin after transfection. Note the decreased Vimentin and increased E-Cadherin in both CALU1 and NCIH1975 cells transfected with full length FOXF1-AS1 cDNA. The expression of GAPDH was used as an internal control. C. The representative images of IF staining for E-Cadherin and Vimentin. Each protein was detected using specific antibodies. The changes in the signal of E-Cadherin and in the signal of Vimentin were clearly detected in the cells.

Figure 4. Loss of FOXF1-AS1 mediates stem-like properties of lung cancer cells

A. The representative images of stem-like spheres in CALU1, NCIH1975 and their two transfected ones using light microscope. B. Numbers of the stem-like spheres in each group. The bars were calculated as the total of three wells from each group photographed by light microscope. The overexpression of FOXF1-AS1 inhibited sphere formation of lung cancer cells to stem-like cells significantly. *p<0.05. C. The expression of CD166 and CD44 in the cells of CALU1, NCIH1975, CALU1-FOXF1-AS1 and NCIH1975-FOXF1-AS1 detected by flow cytometry analysis. Stem-like lung cancer cells tended to express CD166 and CD44. The overexpression of FOXF1-AS1 downregulates stem-like cell population in CALU1 and NCIH1975 cells.

Figure 5. FOXF1-AS1 physically associates with PRC2 components EZH2 and loss of FOXF1-AS1 mediates cell migration and stem-like properties require EZH2

A. mRNA of EZH2 was highly expressed in tumor tissues (n=50) than normal tissues (n=50) detected by real time-qPCR. p < 0.0001. B. The expression of FOXF1-AS1 and EZH2 was inversely correlated with each other in lung cancer tissues. Pearson r=−0.62. p<0.0001. C. Binding of FOXF1-AS1 to EZH2 complex in CALU1 cells, shown by RNA immunoprecipitation followed qRT-PCR (mean±SD, n=3, *p < 0.05 versus lgG). D. Relative FOXF1-AS1 expression was analyzed by qRT-PCR after CALU1-FOXF1-AS1 cell transduction with shFOXF1-AS1 transfection and was represented on the bar graphs. Data showed that the expression of FOXF1-AS1 was suppressed significantly.*p<0.05. E. The effect of shFOXF1-AS1 and co-treatment with EZH2 inhibitor Dznep on migration of CALU1-FOXF1-AS1 cells was detected by transwell assay. Blocking the function of EZH2, shFOXF1-AS1 could not induce the migration of CALU1-FOXF1-AS1 cells. F. The effect of shFOXF1-AS1 and co-treatment with EZH2 inhibitor Dznep on stem-like sphere formation and CD166+CD44+ cell population of CALU1-FOXF1-AS1 cells was detected by light microscope and flow cytometry analysis. Blocking the function of EZH2, shFOXF1-AS1 could not promotes stem-like sphere formation and CD166+CD44+ cell population of CALU1-FOXF1-AS1 cells.

Figure 6. Loss of FOXF1-AS1 is correlated with downregulation of FOXF1 in lung cancer

A. The expression of FOXF1 was assessed in CALU1 and NCIH1975 cells co-transfected with full length FOXF1-AS1 cDNA using plasmid vectors compared with control CALU1 and NCIH1975 cells by real time-qPCR. B. mRNA of FOXF1 was lowly expressed in tumor tissues (n=50) than normal tissues (n=50) detected by real time-qPCR. p < 0.0001. C. The expression of FOXF1 protein level was lowly expressed in tumor tissues than normal tissues detected by IHC. D. The expression of FOXF1-AS1 and FOXF1 was positive correlated with each other in lung cancer tissues. Pearson r=0.68. p<0.0001. E. the significantly low expression of FOXF1 in lung adeno-carcinomas (LUAD) and lung squamous carcinomas (LUSC) was also observed in The Cancer Genome Atlas (TCGA).