LncCCAT1 Promotes Breast Cancer Stem Cell Function through Activating WNT/β-catenin Signaling

Background: Breast cancer stem cells (BCSCs) play an essential role in facilitating breast cancer relapse and metastasis. The underlying mechanism, however, remains incompletely understood. In the current study, we investigated the clinical significance, biological function and mechanism of a long noncoding RNA CCAT1 (LncCCAT1) in BCSCs. Methods: Firstly, lncRNAs expression in poorly differentiated breast cancer tissues and BCSCs were measured by lncRNA microarray and confirmed in breast cancer tissues and cell lines. The functional roles and mechanisms of LncCCAT1 were further investigated by gain and loss of function assays in vitro and in vivo. Results: LncCCAT1 is markedly upregulated in breast cancer tissues BCSCs and is correlated with poor outcomes in breast cancer patients. Overexpression of LncCCAT1 contributes to the proliferation, stemness, migration and invasion capacities of BCSCs. Mechanistic investigation suggests that LncCCAT1 can interact with miR-204/211, miR-148a/152 and Annexin A2(ANXA2), then upregulate T-cell factor 4 (TCF4) or promote translocation of β-catenin to the nucleus where it activates TCF4, leading to the activation of wingless/integrated (Wnt) signaling. Furthermore, TCF4 can also bind to the promoter of LncCCAT1 to promote LncCCAT1 transcription, thus forming a positive feedback regulatory circuit of LncCCAT1-TCF4-LncCCAT1 in BCSCs. Conclusions: LncCCAT1 plays an important role in breast cancer progression and may serve as a novel target for breast cancer diagnosis and therapy.

PCR amplified using primers predicted on MethPrimer website and EpiTaq™HS (Takara) was used for bisulphite PCR. The PCR products in each group were cloned into the T-Vector pMD19 (Takara), and over 10 clones were sequenced.
The separation of the nuclear and cytosolic fractions was performed using the PARIS Kit (Life Technologies, CA, USA) according to the manufacturer's instructions. Cells were incubated with hypotonic buffer (25mM Tris-HCl, pH 7.4, 1mM MgCl2 and 5mM KCl) on ice for 5min. An equal volume of hypotonic buffer containing 1% NP-40 was then added, and the sample was left on ice for another 5min. After centrifugation at 5,000g for 5min, the supernatant was collected as the cytosolic fraction. The pellets were resuspended in nuclear resuspension buffer (20 mM HEPES, pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1mM dithiothreitol and 1mM phenylmethyl sulfonyl fluoride and incubated at 4 °C for 30min. The nuclear fraction was collected after removal of insoluble membrane debris by centrifugation at 12,000g for 10min.

Western blotting analysis
Cells proteins were separated by SDS-PAGE electrophoresis and then transferred to a polyvinylidene difluoride membrane. Membranes were immunoblotted with primary antibodies and detected with horseradish peroxidaseconjugated anti-IgG. Then membranes were visualized with an enhanced chemiluminescence kit (Tanon, Shanghai, China). Antibodies used in the western blotting are provided in Supplementary Table S5.
For transient knockdown LncCCAT1, RiboTM LncRNA Smart Silencer (Ribo) was used, which is a pool containing three siRNA and three antisense oligonucleotides (Supplementary Table S6).

Proliferation, migration and Invasion assay
The capacity of cellular proliferation was measured using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) and EdU Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) according to the manufacturer's instructions. The optical density was determined with a microplate reader at a wavelength of 450 nm. To analyze the migration and invasion abilities of cells, transwell migration assay and transwell invasion assay was performed based on published method. For the cell transmembrane migration assay, all the steps were carried out similarly to those in the invasion assay except for the Matrigel (BD Biosciences, San Jose, CA, USA) coating. After incubation at 37˚C for 24 h, the filters were removed. The cells adhering to the lower surface were fixed and stained with Crystal Violet, 10 randomly selected fields in each well were counted. n=3, Magnification was 200×.

Cell transfection and Lentivirus production
Transfections were performed using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer's instructions. The double-stranded microRNA mimics and their respective negative control RNAs (GenePharma, Shanghai, China) were transfected into cells at a final concentration of 50 nM. The cells were harvested at 48h after transfection. Recombinant lentiviruses containing pre-hsa-miR-148a, pre-hsa-miR-204, hsa-miR-148a inhibitor, hsa-miR-152 inhibitor or the control were purchased from (GenePharma). For stable overexpression, the cDNA of LncCCAT1, TCF4 and ANXA2 genes were cloned into the lentiviral expression vector pLVX-IRES-ZsGreen1 (Addgene). To produce lentivirus, HEK-293T cells were co-transfected with the lentiviral vector and packaging vectors psPAX2 and pMD2.G using Lipofectamine 2000 (Invitrogen) according to the manufacturer's guidelines. Infectious lentiviruses were harvested at 48h post transfection and filtered through 0.45μm PVDF filters and the recombinant lentiviruses were concentrated 100-fold by ultracentrifugation (3 h at 50,000 g). The virus-containing pellet was dissolved in DMEM, aliquoted and stored at -80°C. Based on the shRNA sequences provided above, the most effective shRNAs were used for subsequent studies. shRNAs against ANXA2, TCF4 and control hairpins were cloned into pLVX-shRNA2 vector as described above, the virus supernatant was collected and cells were transfected with lentiviral constructs expressing as described above for 24h. The expression of LncCCAT1, ANXA2 and TCF4 in the infected cells was collected for protein and RNA analysis 96h after infection.

Fluorescence in situ hybridization (FISH) and LNA based in situ hybridization (LNA ISH)
Cy3 labeled LncCCAT1 probe was designed and synthesised by (Ribo, Guangzhou, China). For FISH assay, cells were fixed in 4% formaldehyde and permeabilized with 0.5% Triton X-100 for 5 min, washed with PBS three times and once in 2× SSC buffer. Hybridization was carried out using DNA probe sets at 37°C for 12 h.
Images were obtained with confocal laser microscope (Carl Zeiss, Oberkochen, Germany). In situ hybridization (ISH) was performed by applying the ISH Kit (Boster, Bio-Engineering Company, Wuhan, China). Formalinfixed paraffin embedded (FFPE) tissue slides were deparaffinized and deproteinated. Slides were then prehybridized in prehybridization solution for 2 h at 42 °C and incubated in DIG-labeled probe solution over night at 42°C. After stringent washing, the slides were exposed to a streptavidin-peroxidase reaction system and stained with 3, 3′diaminobenzidine (DAB, ZSGB-BIO, Beijing, China). Hematoxylin (Sigma) was used to counterstain the slides.

The copy number of LncCCAT1 and miRNAs per cell
The standard curves were formulated with limit dilution approaches using LncCCAT1 expressing vector pcDNA3.1-LncCCAT1 and reverse transcribed miR-204/211/148a/152 cDNA as standard templates, and then the exact copy numbers of LncCCAT1 and miR-204/211/148a/152 per cell were calculated according to cell counts and molecular weights.  Median expression level was used as a cutoff to divide the 80 patients into LncCCAT1 low group (n = 40) and LncCCAT1 high group (n = 40).