Tissue specimens
Breast cancer tissues and adjacent normal tissues of 189 breast cancer patients who underwent surgical treatment at Qilu Hospital of Shandong University were collected from May 2017 to July 2021. Tissue specimens were frozen in liquid nitrogen immediately after surgery until further use. All patients signed informed consents.
RNA extraction and quantitative real-time PCR (qRT-PCR)
RNA-easyTM Isolation Reagent (Vazyme, Nanjing, China) was used to extract total RNA from tissues. The quality and concentration of RNA were measured on a Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies). For circ_0075796 and mRNAs, cDNA was synthesized using ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan). SYBR® Green Realtime PCR Master Mix (TOYOBO, Osaka, Japan) was used for qRT-PCR on a LightCycler480 machine (ROCHE, Basel, Switzerland). For miRNAs, reverse transcription was performed using SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). QRT-PCR was conducted using qPCR SYBR Green Master Mix (CloudSeq, Shanghai, China). GAPDH was used as an internal standard control for circ_0075796 and mRNAs, while U6 was used for miR-452-3p. Related primers were detailed in Supplementary Table S1. The relative expression of RNAs was calculated using the 2−△△Ct method.
Cell culture
Breast cancer cell line MDA-MB-231 and BT474 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 were cultured in DMEM medium (FBS, Gibreast cancero, USA) containing 10% fetal bovine serum (FBS, Gibreast cancero, USA). BT474 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibreast cancero, USA). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
Plasmid construction and cell transfection
For the overexpression of circ_0075796, 902bp cDNA fragment was cloned into pLC5-ciR vector, which was then verified by PCR product sequencing analysis (Supplementary Fig. S1A). MDA-MB-231 cells and BT474 were seeded in 6-well culture plates and cultured to 70-90% confluence before transfection, respectively. According to the manufacturer's instructions, circ_0075796 overexpression plasmid and negative control (Geneseed Biotech Co., Ltd., Guangzhou, China) were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The transfection efficiency was detected by qRT-PCR analysis 48 hours after transfection.
Methyl thiazolyl tetrazolium (MTT) assay
MDA-MB-231 cells were seeded in 96-well plates at a density of 5000 cells per well after transfection. MTT solution (20µL) (Sigma, Saint Louis, MO, USA) was added to each well, and the OD value was measured after incubation for 4 hours. The OD values at 0, 24, 48, 72 and 96h after transfection were recorded for statistical analysis.
Cell counting kit-8 (CCK-8) assay
BT474 cells were seeded in 96-well plates at a density of 5000 cells per well after transfection. CCK-8 reagent (10µL) (Bestbio, Shanghai, China) was added to each well, and the OD value was measured after incubation for 2 hours. The OD values at 24, 48 and 72h after transfection were recorded for statistical analysis.
Colony formation assay
The transfected MDA-MB-231 and BT474 cells (700 cells/well) were plated into 6-well plates. The plate was placed in a 37°C incubator containing 5% CO2 to induce colony growth. After cultivation for 10 days, cell surface was rinsed using cooled PBS. Cell colonies were fixed with 4% paraformaldehyde, stained with crystal violet and finally observed under a microscope.
Transwell assay
Transwell inserts precoated with or without Matrigel (BD, Biosciences, USA) were used for the determination of cell invasion and migration, respectively. Transfected cells (1×105) were suspended in 100µL serum-free medium and inoculated into upper chamber of the transwell. For migration assays, the Matrigel (BD, Biosciences, USA) was not coated on the membrane of the upper chamber, while the Matrigel (BD, Biosciences, USA) was coated for invasion assays. Medium containing 10% FBS was placed in the bottom chamber as a chemoattractant. Transfected cells were incubated at 37°C with 5% CO2 for 24h, then the cells on the lower surface were fixed with methanol, stained with crystal violet, and finally observed under a microscope.
Wound healing assay
The transfected MDA-MB-231 cells and BT474 cells were cultured in 6-well plates. The monolayer of cells was created with a scratch using a 200-µL pipette tip and then washed with PBS. Images of cell migration were captured at 0 and 24 h for MDA-MB-231 cells and BT474 cells, respectively.
Cell cycle and apoptosis assays
For cell cycle assays, cells were stained with propidium iodide using Cell Cycle Detection Kit (Bestbio, Shanghai, China) and detected by flow cytometry. The ratios of cells in the G1, S, G2 phases were counted and compared. To detect cell apoptosis, cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit (Bestbio, Shanghai, China) and analyzed by flow cytometry. The ratio of normal cells, early apoptotic cells, late apoptotic cells and total apoptotic cells was compared with the value of the control group in each experiment.
Fluorescence in situ hybridization (FISH)
circ_0075796 specific probe was used for in situ hybridization. Nuclei were counter by staining with 4,6-diamidino-2-phenylindole (DAPI). All the procedures were conducted according to the manufactory’s instruction (Geneseed, Guangzhou, China).
The final specimen was analyzed on a laser confocal microscope, TCS SP2 AOBS (Leica, Wetzlar, Germany). The probe was labeled with digoxin and the sequence is 5'-ggatacattccttgcaaatc-3'.
Bioinformatics analysis
Starbase [30] was applied to predict the potential binding sites between QKI and circ_0075796. Miranda [31] and TargetScan [32] databases were used to predict the relationship between 836 disease-related miRNAs (from the Human microRNA Disease Database, version 2.0) and circ_0075796. The target genes of the candidate miRNAs and the potential binding sites between them were further predicted based on these two databases.
Methylated RNA immunoprecipitation (MeRIP)
In brief, total RNA was extracted from three pairs of breast cancer tissues and adjacent normal tissues using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) and then fragmented using 2µL fragmentation buffer later. The fragmented RNA was then incubated with anti-m6A onoclonal antibody and A/G magnetic beads in IP buffer at 4°C for 2 hours for immunoprecipitation. Next, the bound RNA was eluted from the beads in IP buffer. SuperScriptTM III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) was applicated for reverse transcription of the eluted RNA after purification. QRT-PCR was conducted using qPCR SYBR Green Master Mix (CloudSeq, Shanghai, China) in QuantStudio 5 real-time PCR System (Thermo Fisher, Waltham, MA, USA). The Ct difference between input and the immunoprecipitated RNA was identified, and the relative enrichment was calculated using the 2−△△Ct method.
Statistical analysis
All statistical analyses were performed using SPSS 20.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 5.0 (SPSS, Chicago, IL, USA). Data differences between two groups were analyzed using the Student’s t test. One way analysis of variance (ANOVA) was used to analyze data differences between three or four groups. Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic value of circ_0075796 for breast cancer. The correlations between circ_0075796 expression level and the clinicopathological parameters of breast cancer patients were analyzed using the chi-squared test. P < 0.05 was considered to be statistically significant.