FOXO1-regulated lncRNA CYP1B1-AS1 suppresses breast cancer cell proliferation by inhibiting neddylation

Abstract

Purpose: Overactivated neddylation is considered to be a common event in cancer. Long non-coding RNAs (lncRNAs) can regulate cancer development by mediating post-translational modifications. However, the role of lncRNA in neddylation modification remains unclear.

Methods: LncRNA cytochrome P450 family 1 subfamily B member 1 antisense RNA 1 (CYP1B1-AS1) expression in breast cancer tissues was evaluated by RT-PCR and TCGA BRCA data. Gain and loss of function experiments were performed to explore the role of CYP1B1-AS1 in breast cancer cell proliferation and apoptosis in vitro and in vivo. Luciferase assay, CHIP-qPCR assay, transcriptome sequencing, RNA-pulldown assay, mass spectrometry, RIP-PCR and Western blot were used to investigate the regulatory factors of CYP1B1-AS1 expression and the molecular mechanism of CYP1B1-AS1 involved in neddylation modification.

Results: We found that CYP1B1-AS1 was down-regulated in breast cancer tissues and correlated with prognosis. In vivo and in vitro functional experiments confirmed that CYP1B1-AS1 inhibited cell proliferation and induced apoptosis. Mechanistically, CYP1B1-AS1 was regulated by the transcription factor, forkhead box O1 (FOXO1), and could be upregulated by inhibiting the PI3K/FOXO1 pathway. Moreover, CYP1B1-AS1 bound directly to NEDD8 activating enzyme E1 subunit 1 (NAE1) to regulate protein neddylation.

Conclusion: This study reports for the first time that CYP1B1-AS1 inhibits protein neddylation to affect breast cancer cell proliferation, which provides a new strategy for the treatment of breast cancer by lncRNA targeting neddylation modification.

Keywords: Breast cancer; FOXO1; Long noncoding RNA; NAE1; NEDD8; Neddylation.

Fig. 1

Downregulation of CYP1B1-AS1 is associated with breast cancer progression. a The Cancer Genome Atlas (TCGA) data analysis of CYP1B1-AS1 expression in each molecular subtype of breast cancer. N, non-tumor tissue. T, tumor tissue. **P < 0.01. b Quantitative real-time polymerase chain reaction (qPCR) was used to analyze the expression level of CYP1B1-AS1 in breast cancer tissues and adjacent non-tumor tissues of 44 patients. ***P < 0.001. c Receiver operating characteristic curve analysis of the diagnostic value of CYP1B1-AS1 in 44 paired breast tissue samples. P < 0.001. d Kaplan–Meier Plotter analysis of the relationship between CYP1B1-AS1 expression and patients’ overall survival. Kaplan–Meier Plotter uses data from public databases such as GEO and TCGA and provides the best available cut-off value. The cut-off value used in the analysis is 4. P < 0.001. e Kaplan–Meier Plotter analysis of the relationship between CYP1B1-AS1 expression and patients’ recurrence-free survival. Kaplan–Meier Plotter uses data from public databases such as GEO and TCGA and provides the best available cut-off value. The cut-off value used in the analysis is 1.28. P < 0.001. f qPCR was used to analyze the expression level of CYP1B1-AS1 in breast cancer cells (BT-549, SK-BR-3, MDA-MB-231, T-47D, and MCF7), and the mammary epithelial cell, MCF10A, was used as control. **P < 0.01. g Nuclear/cytoplasmic separation combined with qPCR were used to analyze the distribution of CYP1B1-AS1 in MCF10A cells. U6 and GAPDH were used as the reference for nuclear RNA extraction and cytoplasmic RNA extraction, respectively. h Fluorescence in situ hybridization (FISH) experiments combined with laser confocal microscopy showed the distribution of CYP1B1-AS1 in MCF10A cells (× 1000)

Fig. 2

The upstream regulatory mechanism of CYP1B1-AS1. a Schematic representation of the binding sites of FOXO1 predicted by JASPAR in the CYP1B1-AS1 promoter region. b The Cancer Genome Atlas (TCGA) data were analyzed to determine the expression of FOXO1 in various molecular subtypes of breast cancer. N, non-tumor tissue. T, tumor tissue. **P < 0.01. c TCGA data were analyzed to determine the correlation between CYP1B1-AS1 and FOXO1 expression in breast cancer. R, Pearson correlation coefficient. P < 0.001. d Quantitative real-time polymerase chain reaction (qPCR) analysis of the effect of interfering FOXO1 on CYP1B1-AS1 expression in MCF10A cells. FsiRNA, FOXO1 siRNA. siNC, negative control for siRNA. **P < 0.01. e qPCR analysis of the effect of up-regulated FOXO1 expression on CYP1B1-AS1 expression in MCF7 and MDA-MB-231 cells. p-FOXO1, pcDNA3.1-FOXO1. p-NC, negative control for pcDNA3.1. **P < 0.01. f Western blot results of chromatin immunoprecipitation (CHIP) assay to detect FOXO1 binding to CYP1B1-AS1 promoter fragment. Input, positive control. IgG, negative control. g Gel electropherogram of the amplified promoter fragment in CHIP-qPCR assay. Input, positive control. IgG, negative control. h Dual-luciferase assay was used to detect the downstream regulation of FOXO1 binding to the promoter fragment. p-FOXO1, pcDNA3.1-FOXO1. p-NC, negative control for pcDNA3.1. **P < 0.01. i Western blot was used to detect the changes in FOXO1 protein expression after LY294002 treatment. LY, LY294002. Solvent DMSO as blank control. j qPCR was used to detect changes in CYP1B1-AS1 expression after LY294002 treatment. LY, LY294002. Solvent DMSO as blank control. **P < 0.01

Fig. 3

Upregulation of CYP1B1-AS1 inhibits cell proliferation and induces apoptosis. a Quantitative real-time polymerase chain reaction (qPCR) was used to detect the changes of CYP1B1-AS1 after lentivirus infection of MCF7 and MDA-MB-231 cells. **P < 0.01. b qPCR was used to detect FOXO1 mRNA in MCF7 and MDA-MB-231 cells after up-regulation of CYP1B1-AS1. c, d Counting Kit 8 (CCK-8) was used to detect the changes in the proliferation ability of MCF7 and MDA-MB-231 cells after up-regulation of CYP1B1-AS1. **P < 0.01. e, f The clone formation assay was used to examine the effect of up-regulation of CYP1B1-AS1 on the clone formation ability of breast cancer single cells. **P < 0.01. g, h Flow cytometry was used to analyze the effect of up-regulation of CYP1B1-AS1 on breast cancer cell cycle. **P < 0.01. i, j Flow cytometry was used to analyze the effect of up-regulation of CYP1B1-AS1 on apoptosis. **P < 0.01. k, m Representative images of tumor tissue in the xenograft model and compared the volume and weight of tumor tissue in the MCF7-exp and MCF7-NC groups (n = 6). Tumor volumes shown in l were calculated every 3 days after injection. **P < 0.01. n, o Representative images of immunohistochemical staining showing the expression levels of Ki-67 in MCF7-exp and MCF7-NC transplanted tumor tissues (× 400). **P < 0.01. p, q Representative images of TUNEL fluorescence staining showing the level of apoptosis in MCF7-exp and MCF7-NC transplanted tumor tissues (× 400). **P < 0.01

Fig. 4

Transcriptome sequencing analysis of MCF7 cells following CYP1B1-AS1 upregulation. a Cluster heatmap of differential gene expression analysis between MCF7-exp and MCF7-NC groups. b Volcano plots show the distribution of differentially expressed genes between the MCF7-exp and MCF7-NC groups. Q, the corrected P-value. c Horizontal bar graph showing Gene Ontology enrichment analysis of differentially expressed genes, and the top five items with P < 0.05 are shown for each category. d Dot plot showing Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of differentially expressed genes, and the top 15 items with P < 0.05 are shown

Fig. 5

CYP1B1-AS1 binds directly to NAE1 protein. a Gene Ontology enrichment analysis of CYP1B1-AS1 pull-down proteins, each class showing the top five items of P < 0.05. b Kyoto Encyclopaedia of Genes and Genomes pathway enrichment analysis of CYP1B1-AS1 pull-down proteins, showing all items with P < 0.05. c Kaplan–Meier Plotter analysis of the relationship between NAE1 expression and overall survival in breast cancer patients. P < 0.01. d Kaplan–Meier Plotter analysis of the relationship between NAE1 expression and recurrence-free survival in breast cancer patients. P < 0.001. e Western blot was used to detect NAE1 protein in CYP1B1-AS1 sense strand pull-down protein solution. CYP1B1-AS1 antisense strand pull-down protein solution was used as a negative control, and MCF7-exp whole cell lysate was used as a positive control. f The RNA immunoprecipitation products were isolated and purified, and the amount of CYP1B1-AS1 bound to anti-NAE1 or IgG was measured by qPCR. IgG was used as a negative control. **P < 0.01

Fig. 6

CYP1B1-AS1 affects neddylation through NAE1. a, b Western blotting was used to detect neddylated proteins in MCF7 and MDA-MB-231 cells after interference with NAE1. siNAE1, NAE1 siRNA. siNC, negative control for siRNA. **P < 0.01. c, d Western blotting was used to detect NAE1 and neddylated proteins in MCF7 and MDA-MB-231 cells after CYP1B1-AS1 upregulation. *P < 0.05, **P < 0.01. e, f Representative images of immunohistochemical staining showing the expression of NEDD8 and NAE1 in xenograft tumors. **P < 0.01. g, h Western blot was used to detect the changes in cyclin D1, p21, BCL2, BAX and FOXO1 after up-regulation of CYP1B1-AS1. *P < 0.05. i Schematic diagram of lncRNA CYP1B1-AS1 inhibiting the proliferation of breast cancer by binding NAE1