Cells culture

Human breast cancer cell line MDA-MB-231 was offered by the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultivated at 37 ℃ with 5% CO2 in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA, USA) added with 10% fetal bovine serum (FBS) (Gibco), 100 ug/ml penicillin and 100 U/ml streptomycin.

Fluorescence in situ hybridization (FISH)

The specific probes for circHSDL2 labeled with Cy3 were obtained by RiboBio (Guangzhou, China). The Ribo™ FISH kit (RiboBio) was operated as instructed by the manufacturer. CircHSDL2 was hybridized using Cy3-labeled probe. Cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) [11]. A confocal microscope (Carl Zeiss, Germany) was used for observation and photo taking.

CircRNA lentivirus construction and transfection

A lentivirus carrying hsa-circHSDL2 and luciferase was constructed to establish a stable cell line (Hanbio, Shanghai, China). Cells infected with the lentivirus were selected with 2.5 ug/ml puromycin.

The miRNA mimics, siRNA and negative control were bought from RiboBio. MiRNA mimics negative control contains a sequence with no known targets. It does not bind to any known mRNA targets and does not induce any specific gene regulation changes within the cell. MDA-MB-231 cells were transfected with a Lipofectamine 8000 reagent (Beyotime, Shanghai, China) as per relevant instructions.

RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)

Total RNA of cells was isolated with a Trizol reagent (Invitrogen, CA, USA). Then RNA content and purity were measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). cDNA for circHSDL2 and miRNAs was synthesized with a PrimeScript™ RT master mix kit and a Mir-X™ miRNA first-strand preparation kit (both Takara, Japan). RT-qPCR was conducted with Taq pro universal SYBR qPCR master mix (Vazyme Biotech, China). The primers were obtained by Sangon Biotech (Shanghai, China) and listed together with siRNAs in Table S1. Relative expressions of circHSDL2 and miRNAs were analyzed with the 2−△△Ct approach.

Cell proliferation assay

The proliferating activity of the breast cancer cells was evaluated via EdU staining. Cells were grown in a medium with a 50 µM EdU reagent (RiboBio) for 2 h and fixed with 4% paraformaldehyde. Then they were stained with 1×Apollo® dyeing solution. The nuclei were stained with Hoechst33342. Datas were captured and observed under an inverted fluorescent microscope (Olympus, Japan).

Wound healing and transwell assay

Cells were planted in a 6-well plate until reaching 90% confluence and scratched with a 200 µL pipette tip in the middle of the plate. The cells were further cultivated in a serum-free medium. Cell migration was observed with the inverted microscope at both 0 and 48 h.

Cell migration and invasion assays were done with Transwell chambers (Corning, USA) without and with Matrigel (BD Science, USA) respectively. About 2 × 104 cells were suspended in 200 µL of FBS-free DMEM and seeded into the upper chambers. Then 600 µL of DMEM with 20% FBS was added into the lower chamber. After 24 h, the migrating or invading cells were fixed with 4% paraformaldehyde and dyed with crystal violet. Pictures were captured with the inverted microscope.

Dual-luciferase reporter assay

Dual-luciferase reporter assays were carried out as previously described [14]. The sequences of circHSDL2 and the mutant version without miR-7978 binding sites were amplified and cloned into a luciferase reporter vector pmiR-RB-Report™ (RiboBio), termed circHSDL2-wt and circHSDL2-mut respectively. HEK293T cells (1.5 × 104) were planted into 96-well plates and cotransfected with these plasmids and mimics or the control. Then the firefly and Renilla luciferase activity was tested using a dual-luciferase assay kit as per relevant protocols (Beyotime).

MiRNA and mRNA forecasting

The target miRNAs that may bind to circHSDL2 were predicted with CircMir (http://www.bioinf.com.cn/) and the Cancer-Specific CircRNA Database (CSCD, http://gb.whu.edu.cn/CSCD/). CircMir is based on miRanda 2010 Release (http://www.microrna.org/microrna/getDownloads.do) and RNAhybrid-2.1.2 (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) to predict circRNA-miRNA interactions. The intersection of miRNAs predicted by the three databases was used for further study.

Databases of miRDB (https://mirdb.org/), miRtarbase (https://mirtarbase.cuhk.edu.cn/), and targetscan (http://www.targetscan.org/) were used to forecast the target mRNAs of miR-7978. The target mRNAs were further intersected with upregulated proteins in the proteomics results of MDA-MB-231 cells overexpressing circHSDL2.

Label-free quantitative proteomics

Proteomics analysis of breast cancer cells was conducted by Biotree (Shanghai, China). MDA-MB-231 cells overexpressing circHSDL2 or the control were made for LC-MS as required by Biotree. In brief, samples were prepared by protein extraction, protein digestion and peptide desalting. Then total peptides of the samples were isolated and tested with nano-UPLC (EASY-nLC1200) connected to a Q-Exactive HFX Orbitrap device (Thermo Fisher Scientific) with nano-electrospray. A reversed-phase column (100 μm ID × 15 cm, ReprosilPur 120 C18AQ, 1.9 μm, Dr. Maisch, Germany) was used in the separation. All procedures were finished in Biotree.

Western blotting

Breast cancer cells were ice-lysed with an RIPA buffer. Proteins were extracted after centrifugation and quantified with a BCA kit (Biosharp, China). Same quantities of proteins (30 µg) were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After blocking for 2 h with 5% skim milk, the membranes were immunoblotted with primary antibodies at 4 ℃ overnight, including anti-ZNF704 (Santa Cruz, #SC-514,109, USA), anti-MST2/Krs-1 (Santa Cruz, #SC-130,405), anti-YAP1 (Abcam, #ab52771, UK), anti-YAP1 (phosphor S127) (Abcam, #ab76252), anti-LATS1/2 (Affinity, #DF7517, USA), anti-phospho-LATS1/2 (Ser909/Ser872) (Affinity, #AF8163), anti-TAZ (Cell Signaling Technology, #72,804, USA) and anti-GAPDH (Proteintech, USA). The membranes were cultured with a secondary antibody (Beyotime) for 1 h after washing with TBST and visualized using an enhanced chemiluminescence (ECL) plus kit (Millipore) and an integrated chemiluminescence Datar (CLINX, ChemiScope 5300 Pro, China). The Datas were tested on Data J.

Animal experiments

Approval was offered by the Animal Care and Use Committee of Nanjing Medical University. Athymic nude mice (female BALB/c, 4 to 6 weeks old) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. The MDA-MB-231 cells stably expressing circHSDL2 or the control (5 × 106 cells/mouse) were injected subcutaneously into the right flank (n = 5, each group) to build a subcutaneous implantation model. Tumor length and width were detected every 2 days. Tumor volumes were estimated as volume (mm3) = 0.5 * length * witdth2. The tumors were weighed at the end point of the assay. For lung metastasis assay, MDA-MB-231 cells stably expressing circHSDL2 or the control were luciferase-labeled, and injected into the caudal vein. Lung metastasis was detected using a visible light 3D imaging system for animals (PerkinElmer, IVIS Spectrum, USA).

Immunohistochemistry

Fresh samples of xenograft tumor tissues were fixed with formalin, dehydrated with gradient aqueous alcohol, embedded with paraffin and cut into 4-µm sections. Then the sections were grown with anti-ZNF704, anti-TAZ and anti-MST2/STK3 (Abcam, ab52641). Pictures were taken under the inverted microscope.

Statistical analysis

Statistical analysis was finished on GraphPad Prism 7 (GraphPad, CA, USA) and SPSS 20.0 (IBM, SPSS, IL, USA). Groups were compared via Student’s t test. Data were acquired from three independent assays and expressed as mean ± standard deviation (SD). P < 0.05 implied significance.