Genome-wide target interactome profiling reveals a novel EEF1A1 epigenetic pathway for oncogenic lncRNA MALAT1 in breast cancer

Abstract

Breast cancer is the most common cancer in women worldwide, accounting for approximately 500,000 deaths each year. MALAT1 is a highly conserved long noncoding RNA (lncRNA), and its increased expression is associated with relapse and metastatic progression in breast cancer. We performed RNA reverse transcription-associated trap sequencing (RAT-seq) to characterize the genome-wide target interaction network for MALAT1 and showed that MALAT1 interacted with multiple pathway target genes that are closely related to tumor progression and metastasis. Notably, MALAT1 bound to the promoter regulatory element of the translation elongation factor 1-alpha 1 gene EEF1A1. Knockdown of MALAT1 by shRNA caused significant downregulation of EEF1A1 in breast cancer MDA-MB231 and SKRB3 cells. Using a luciferase reporter assay, we showed that knockdown of MALAT1 reduced the promoter activity of EEF1A1 in these two breast cancer cells. Chromatin immunoprecipitation (ChIP) assay indicated that MALAT1 regulated EEF1A1 by altering the histone 3 lysine 4 (H3K4) epigenotype in the gene promoter. MALAT1 was overexpressed in breast cancer tissues and breast cancer cells. Knockdown of MALAT1 reduced cell proliferation and invasion by arresting cells at the G0/G1 phase. Ectopic overexpression of EEF1A1 reversed the altered tumor phenotypes induced by MALAT1 shRNA treatment. These data suggest an epigenetic mechanism by which MALAT1 lncRNA facilitates a pro-metastatic phenotype in breast cancer by trans-regulating EEF1A1.

Keywords: EEF1A1; LncRNA; MALAT1; breast cancer; epigenetics; histone methylation; interactome.

Figure 1

Interactome of MALAT1 lncRNA by RAT-seq. A. RAT-seq assay. MALAT1 lncRNA was in situ reverse transcribed using MALAT1-specific complementary primers at 60°C with biotin-dCTP. The biotin-MALAT1 cDNA chromatin complex was isolated by streptavidin beads and cDNAs were isolated for Illumina library sequencing. RAT-seq will generate a genome-wide target interaction network for MALAT1 lncRNA in breast cancer cells. B. Gene ontology enrichment pathway analysis of the MALAT1 RAT-Seq data. GO enrichment was analyzed with Cytoscape software. C. The MALAT1 RAT-seq interactome. The MALAT1 interactome was drawn based on the enrichment fold of the top RAT-Seq pathway target genes.

Figure 2

MALAT1 binds to the EEF1A1 promoter and epigenetically regulates its activity. A. The RAT-seq IGV binding of MALAT1 lncRNA at the EEF1A1 locus. MALAT1-RAT: the RAT-seq library created by the MALAT1-specific complementary primers; RC-RAT: the RAT-seq control library created by random oligonucleotide primers; pEEF1A1: EEF1A1 promoter; 3’-CT, 5’-CT: the 3’- and 5’-control sites; E1-E8: EEF1A1 exons. B. Quantitation of EEF1A1 binding in the MALAT1-specific RAT-seq products and the negative control RAT-seq products. C. EEF1A1 expression levels by Q-PCR in MALAT1-knockdown cells. β-Actin was used as an internal control. **P < 0.01 as compared with the control groups. D. Western blot of eEF1A1. Note the reduced expression of eEF1A1 in MALAT1-knockdown breast cancer cells. GAPDH was used as control.

Figure 3

MALAT1 epigenetically regulates EF1A1. A. pEEF1A1-luciferase assay. The EEF1A1 promoter (pEEF1A1) sequence was cloned into the upstream of luciferase gene. Luciferase reporter assay was performed in CTL group, shNC group and shMALAT1 group by co-transfecting respectively with pGL3-Basic vector or luciferase reporter vector. Data were adjusted over the negative control (CTL) and were represented as means ± SD. **P < 0.01 as compared with the control groups. B. Quantitation of Histone 3-K4 (H3K4) trimethylation. All data are presented as the relative values after normalization over the input DNA. *P < 0.05 as compared with control.

Figure 4

MALAT1 is dysregulated in breast cancer. A. Unsupervised hierarchical clustering analysis of the significantly differentially expressed genes in paracancer tissues and tumor tissues. Data from 64 mammary tissues was downloaded from TCGA database. In the heatmap, the normalized expression values are represented in shades of green and red, indicating the expression being above and below the median expression value across the samples. B. The normalized expression level of MALAT1 in 64 mammary tissues (32 paracancer tissues and 32 tumor tissues). ***P < 0.01 as compared with the paracancer group. C. Q-PCR quantitation of MALAT1 expression in breast cancer cell lines.

Figure 5

The role of MALAT1 in cell proliferation and cell cycle. A. MALAT1 shRNA knockdown in two breast cancer cell lines. MALAT1 expression was examined by Q-PCR in CTL (non-transfected control), shNC (random shRNA non-targeting control), shMALAT1 (MALAT1 shRNA-1 transfected cells), and shMALAT1-2 (MALAT1 shRNA-2 transfected cells). β-Actin was used as an internal control. **P < 0.01 as compared with CTL and shNC controls. B. Cell Proliferation. CCK-8 assay was used to determine cell growth viability at 0, 24, 48, 72 and 96 hour time points. C. Cell cycle. Flow cytometry was used to measure cell cycle profile with propidium iodide staining. Cell numbers were counted according to DNA content of G0/G1, S and G2/M phases. The statistical results are shown on the right panel. *P < 0.05 as compared with the control groups.

Figure 6

MALAT1 knockdown inhibits cell invasion in breast cancer cells. Representative images of invading MDA-MB231 cells (A) and SKBR3 cells (B) are showed on the left panel. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. Quantitation of invaded cells is shown in the right panel, mean ± SD, **P < 0.01 as compared with the control groups.

Figure 7

EEF1A1 rescues the effect induced by MALAT1 knockdown. A. Overexpression of EEF1A1 in breast cancer cells. The expression of EEF1A1 was quantitated by Q-PCR. shNC: random shRNA control; shMALAT1: Cells that were transfected with MALAT1 shRNA-1. β-Actin was used as an internal control. ***P < 0.001 as compared with the control groups. Overexpression of eEF1A1 in breast cancer cells was measured by Western blot. B. Cell growth viability as measured by CCK-8 assay. C. Cell invasion as examined by Transwell assay. Quantitation of invaded cells was shown as mean ± SD, **P < 0.01 as compared with the control groups.