Exosome-transmitted lncRNA UFC1 promotes non-small-cell lung cancer progression by EZH2-mediated epigenetic silencing of PTEN expression

Abstract

Long non-coding RNAs (LncRNAs) have been suggested as important regulators of cancer development and progression in non-small cell lung cancer (NSCLC). Nevertheless, the biological roles and clinical significance of lncRNA UFC1 in NSCLC remain unclear. We detected the expression of UFC1 in tumor tissues, serum, and serum exosomes of NSCLC patients by qRT-PCR. Gene overexpression or silencing were used to examine the biological roles of UFC1 in NSCLC. RNA immunoprecipitation and ChIP assays were performed to evaluate the interaction between UFC1 and enhancer of zeste homolog 2 (EZH2) and the binding of EZH2 to PTEN gene promoter. Rescue study was used to access the importance of PTEN regulation by UFC1 in NSCLC progression. UFC1 expression was upregulated in tumor tissues, serum, and serum exosomes of NSCLC patients and high level of UFC1 was associated with tumor infiltration. UFC1 knockdown inhibited NSCLC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. UFC1 overexpression led to the opposite effects. Mechanistically, UFC1 bound to EZH2 and mediated its accumulation at the promoter region of PTEN gene, resulting in the trimethylation of H3K27 and the inhibition of PTEN expression. UFC1 knockdown inhibited NSCLC growth in mouse xenograft tumor models while the simultaneous depletion of PTEN reversed this effect. NSCLC cells derived exosomes could promote NSCLC cell proliferation, migration and invasion through the transfer of UFC1. Moreover, Exosome-transmitted UFC1 promotes NSCLC progression by inhibiting PTEN expression via EZH2-mediated epigenetic silencing. Exosome-mediated transmit of UFC1 may represent a new mechanism for NSCLC progression and provide a potential marker for NSCLC diagnosis.

Fig. 1. UFC1 is upregulated in the tumor tissues and serum of NSCLC patients and NSCLC cell lines.

a UFC1 expression levels in tumor tissues and adjacent normal tissues were detected by qRT-PCR (n = 66). b UFC1 expression levels in serum of NSCLC patients, pneumonia patients, and healthy controls were detected by qRT-PCR. c ROC curves for the diagnostic value of serum UFC1 in NSCLC. d UFC1 expression levels in serum exosomes of NSCLC patients, pneumonia patients, and healthy controls. e ROC curves for the diagnostic value of serum exosomal UFC1 in NSCLC. f UFC1 expression levels in NSCLC cell lines (A549, H1299, H446, and H460) and normal embryonic lung fibroblast cells (MRC-5). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. UFC1 knockdown inhibits proliferation, migration and invasion of NSCLC cells.

a QRT-PCR analysis of UFC1 knockdown efficiency in A549 cells. b Cell counting assays for sh-Ctrl and sh-UFC1 transfected A549 cells. c Colony formation assays for sh-Ctrl and sh-UFC1 transfected A549 cells. d The percentage of apoptotic cells in sh-Ctrl and sh-UFC1 groups was detected by flow cytometry after Annexin V/PI staining. e Flow cytometric analysis of cell cycle distribution in sh-Ctrl and sh-UFC1 transfected A549 cells. f, g QRT-PCR (f) and western blot (g) analysis of cyclin D1, Bcl-2 and Bax expression in sh-Ctrl and sh-UFC1 transfected A549 cells. h Transwell migration and matrigel invasion assays for sh-Ctrl and sh-UFC1 transfected A549 cells. i QRT-PCR analysis of E-cadherin, N-cadherin, vimentin, slug, snail, and twist expression in sh-Ctrl and sh-UFC1 transfected A549 cells. j Western blot analysis of E-cadherin, N-cadherin, vimentin, twist, slug, snail, β-catenin, and c-Myc expression in sh-Ctrl and sh-UFC1 transfected A549 cells. The experiments were repeated for three times. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. UFC1 overexpression promotes NSCLC cell proliferation, migration and invasion.

a QRT-PCR analysis of UFC1 overexpression efficiency in H1299 cells. b Cell counting assays for control and UFC1 overexpressing A549 cells. c Colony formation assays for control and UFC1 overexpressing A549 cells. d, e Flow cytometric analysis of cell apoptosis (d) and cell cycle distribution (e) in control and UFC1 overexpressing A549 cells. f, g QRT-PCR (f) and western blot (g) analysis of cyclin D1, Bcl-2 and Bax expression in control and UFC1 overexpressing A549 cells. h Transwell migration and matrigel invasion assays for control and UFC1 overexpressing A549 cells. i QRT-PCR analysis of E-cadherin, N-cadherin, vimentin, snail, slug, and twist expression in control and UFC1 overexpressing A549 cells. j Western blot analysis of E-cadherin, N-cadherin, vimentin, twist, slug, snail, β-catenin, and c-Myc expression in control and UFC1 overexpressing A549 cells.The experiments were repeated for three times. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. UFC1 interacts with EZH2 to suppress PTEN expression and activate Akt pathway.

a QRT-PCR analysis of UFC1 expression levels in different subcellular fractions of A549 cells. b RIP assays with antibodies against EZH2 and IgG control and A549 cell extracts. RNA levels in the immunoprecipitates were detected by qRT-PCR. Expression levels of UFC1 are presented as fold enrichment to input. c QRT-PCR analysis of PTEN expression in NSCLC cells with UFC1 knockdown or overexpression. d Western blot analysis of PTEN and p-Akt expression in NSCLC cells with UFC1 knockdown or overexpression. e ChIP-PCR analysis of EZH2 occupancy and H3K27me3 binding in the PTEN promoter in A549 cells transfected with sh-UFC1 and sh-Ctrl. f QRT-PCR and western blot analysis of PTEN expression in A549 cells with EZH2 knockdown. g Correlation analysis of UFC1 and PTEN expression levels in tumor tissues of NSCLC patients. The experiments were repeated for three times. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. UFC1 promotes NSCLC progression via the regulation of PTEN.

a QRT-PCR analysis of PTEN knockdown efficiency in A549 cells. b QRT-PCR analysis of UFC1 and PTEN expression in A549 cells transfected with sh-UFC1 alone or sh-UFC1+si-PTEN. c Cell counting assays for A549 cells transfected with sh-UFC1 alone or sh-UFC1+si-PTEN. d Colony formation assays for A549 cells transfected with sh-UFC1 alone or sh-UFC1+si-PTEN. e Transwell migration and matrigel invasion assays for A549 cells transfected with sh-UFC1 alone or sh-UFC1+ si-PTEN. f Tumor growth curves, tumor weights and sizes of mice injected with A549 cells transfected with sh-UFC1 alone or sh-UFC1+si-PTEN. g HE, Ki-67, and TUNEL staining in tumor tissues from mice injected with A549 cells transfected with sh-UFC1 alone or sh-UFC1+si-PTEN. The experiments were repeated for three times. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared to sh-Ctrl+si-Scr group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared to sh-Ctrl+si-Scr group.

Fig. 6. NSCLC cells derived exosomes deliver UFC1 to promote NSCLC progression by downregulating PTEN.

a QRT-PCR analysis of UFC1 expression in A549 cells treated with PBS, exosomes-depleted conditioned medium (Ex-depleted CM), exosomes from sh-Ctrl and sh-UFC1 transfected A549 cells. b–e Cell counting (b), colony formation (c), transwell migration (d), and matrigel invasion (e) assays for A549 cells treated with PBS, Ex-depleted CM, or exosomes from sh-Ctrl and sh-UFC1 transfected A549 cells. f QRT-PCR and western blot analysis of PTEN expression in A549 cells treated with PBS, Ex-depleted CM, or exosomes from sh-Ctrl and sh-UFC1 transfected A549 cells. The experiments were repeated for three times. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, compared to PBS group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared to A549 Ex group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared to sh-Ctrl Ex group.

Fig. 7. Proposed model for the oncogenic roles of UFC1 in NSCLC.

Exosome-transmitted UFC1 epigenetically silences PTEN expression via binding to EZH2, which promotes Akt pathway activation and increases cell proliferation, migration and invasion in NSCLC.