Dysregulation of the BRCA1/long non-coding RNA NEAT1 signaling axis contributes to breast tumorigenesis

Dysregulation of long non-codng RNA (lncRNA) expression has been found to contribute to tumorigenesis. However, the roles of lncRNAs in BRCA1-related breast cancer remain largely unknown. In this study, we delineate the role of the novel BRCA1/lncRNA NEAT1 signaling axis in breast tumorigenesis. BRCA1 inhibits NEAT1 expression potentially through binding to its genomic binding site upstream of the NEAT1 gene. BRCA1 deficiency in human normal/cancerous breast cells and mouse mammary glands leads to NEAT1 overexpression. Our studies show that NEAT1 upregulation resulting from BRCA1 deficiency stimulates in vitro and in vivo breast tumorigenicity. We have further identified molecular mediators downstream of the BRCA1/NEAT1 axis. NEAT1 epigenetically silences miR-129-5p expression by promoting the DNA methylation of the CpG island in the miR-129 gene. Silencing of miR-129-5p expression by NEAT1 results in upregulation of WNT4 expression, a target of miR-129-5p, which leads to activation of oncogenic WNT signaling. Our functional studies indicate that this NEAT1/miR-129-5p/WNT4 axis contributes to the tumorigenic effects of BRCA1 deficiency. Finally our in silico expression correlation analysis suggests the existence of the BRCA1/NEAT1/miR-129-5p axis in breast cancer. Our findings, taken together, suggest that the dysregulation of the BRCA1/NEAT1/miR-129-5p/WNT4 signaling axis is involved in promoting breast tumorigenesis.


Quantitative RT-PCR analysis
qRT-PCR analysis of mRNA/miRNA/lncRNA expression was performed as described previously with normalization to either GAPDH or β-actin for mRNAs/ lncRNAs and to U6 small nuclear RNA for miRNAs [3]. Human NEAT1 gene is transcribed into two RNA isoforms, NEAT1-1 (~3.7 kb) and NEAT1-2 (~23 kb). Given that only NEAT1-1 contains a poly(A) tail, we used both oligo-dT and random hexamer primers to prepare two kinds of reverse transcription (RT) mixtures for allowing us to detect each NEAT1 RNA isoform level and also the total level of both isoforms. To measure the total level of both NEAT1 RNA isoforms, qRT-PCR was performed on RT mixtures generated from random hexamer primers using NEAT1-specific primers that can detect both NEAT1 isoforms (forward primer: 5′-TTTGTGCTTGGAACCTTGCT-3′; reverse primer: 5′-TCAACGCCCCAAGTTATTTC-3′). To quantify the expression level of NEAT1-1, qRT-PCR was performed on RT mixtures generated from oligo-dT primers using NEAT1-specific primers shown above. For the measurement of the NEAT1-2 expression level, qRT-PCR was performed on RT mixtures generated from random hexamer primers using NEAT1-2-specific primers (forward primer: 5′-TCTCCATTTCCCCATCTGAG-3′; reverse primer: 5′-CAGCCACAGAAAAGGGAGAG-3′). For miR-129-5p expression measurement, we used miScript primers and miScript-related reagents (Qiagen, Chatsworth, CA, USA) for RT and qRT-PCR assays.

DIG labeling of the ISH probe
To prepare the digoxigenin (DIG)-labeled probe for in situ hybridization analysis of mouse Neat1 RNA expression in mouse tissue, we performed PCR on mouse tail genomic DNA using a Neat1-specific primer pair (forward: 5′-CGGCTGTGAATGTTCCAGATG-3′; reverse: 5′-TCA ACCACCCAGTATCA AATCCA-3′) to amplify a 574-bp DNA probe. The PCR-amplified Neat1 DNA probe was subcloned into the pGEM-T-Easy vector (Promega). In vitro transcription was performed on pGEM-T-Neat1 using T7 or SP6 RNA polymerase based on the orientation of the inserted DNA to synthesize the DIG-labeled antisense Neat1 RNA probe. DIG RNA Labeling Kit (SP6/T7) (Roche, Indianapolis, IN, USA) and manufacturer's methods were used for the in vitro transcription synthesis of the DIG-labeled RNA probe.

In situ hybridization analysis
In situ hybridization was performed using the DIGlabeled Neat1 RNA probe. Briefly, tissue sections were www.impactjournals.com/oncotarget/

Supplementary Materials
deparaffinized, then fixed with 4% paraformaldehyde and treated with proteinase K. After 4 hours of preincubation in a hybridization buffer at room temperature, tissue sections were hybridized with the Neat1 probe (25 ng/ml) in a hybridization buffer overnight at 55°C. After incubation in the 0.2 × SSC buffer for 1 hour at 60°C and treatment with peroxidase, tissue sections were blocked (10% FBS in 1 × PBS) for 1 hour at room temperature and then incubated with the anti-DIG antibody (1:500) overnight at 4°C. After PBST (1 × PBS with 0.05% Tween 20) wash, for the colorimetric detection reaction, tissue sections were incubated in the developer solution (NBT/BCIP, Roche) overnight at room temperature in dark. Slides were then counterstained with hematoxylin and mounted using D.P.X Mounting medium (Sigma). Stained tissue sections were visualized with Nikon Eclipse Ti microscope (Nikon Instruments Inc.; Melville, NY, USA).

MicroRNA PCR array assays
Total RNA was isolated using the RNeasy Mini kit (Qiagen, Chatsworth, CA, USA). MicroRNA expression profiling was carried out using the Breast Cancer miScript miRNA PCR Arrays according to the manufacturer's instructions (Qiagen, Chatsworth, CA, USA). Real-Time PCR was performed using the SYBR Green PCR Master Mix (Qiagen) in a BioRad CFX 1000 real time PCR machine (BioRad, Hercules, CA, USA). The exported Ct data were analyzed using Excel-based PCR Array Data Analysis Templates (Qiagen). The average Ct value of each gene obtained from duplicate experiments was used to calculate its expression value, which is expressed as 2 -∆Ct (∆Ct = Ct MOI -Ave Ct ISR ; MOI: miRNA of interest, ISR: the internal small RNA reference, Ave Ct ISR : the average Ct value of five internal miRNA RNA references).

Reporter plasmid DNA construction and dual reporter assays
To construct the human WNT4 3′-UTR reporter plasmid, a fragment (1.0 kb) of the 3′-UTR of the human WNT4 gene was amplified by PCR using primers 5′-ACG TTT GCT AGC TGG GGC TCT AAG TTT CAG GT-3′ and 5′-ACG TTT CTC GAG AGT CAC TGT CAC AAT TGC AAG A-3′ and cloned into the NheI and XhoI sites of the pSGG vector. The resulting reporter plasmid was then used as a template to generate a mutant reporter plasmid construct with mutations at the site recognized by miR-129-5p using the site-directed mutagenesis system (ThermoFisher Scientific, Grand Island, NY, USA) and primers 5′-TGC TAG TGA GGC ATA GTA TTT TAT ATC GGC TTA GTA GAC AGC TGA ACA G-3′ and 5′-CTG TTC AGC TGT CTA CTA AGC CGA TAT AAAata CTA TGC CTC ACT AGC A-3′. The mutant that contains three point mutations CTACTAA GCC(C to G)A(A to T) A(A to T)AA was confirmed by sequencing.

Bisulfite sequencing analysis
Genomic DNA was isolated using the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's user manual. Isolated genomic DNA was bisulfite-converted using the EpiTect Bisulfite kit (Qiagen) according to the protocol described in the manufacturer's user manual. For bisulfite sequencing analysis of the miR-129 gene, a 392-bp DNA fragment in the CpG-island region of miR-129 was amplified from bisulfite-converted DNA using primers: 5′-GAA TTT TGA TAG GGA GAT AGA GGG A-3′ (forward) and 5′-ACT ATT AAA TTA TAT ACA ACA AAC CCA AAC-3′ (reverse). The PCR was carried out using the PCR cycle condition: 95 o C, 30 sec; 57 o C, 30 sec; 72 o C, 60 sec (35 cycles). The PCR products were purified using the Qiagen PCR purification kit (Qiagen) and sequenced by using the sense primer with an Applied Biosystems automated fluorescent sequencer according to the manufacturer's instructions.  A, B) and MCF7 (C, D) cells were transfected with either the scramble control or BRCA1 siRNAs. 48 hours after transfection, siRNA-transfected cells were harvested and then subjected to qRT-PCR analysis of NEAT1-1 (3.7 kb isoform RNA) (A, C) and NEAT1-2 (~23 kb isoform RNA) (B, D) expression levels. *p < 0.05; **p < 0.01; ***p < 0.001.