Patient samples.

Three tissue microarrays (TMAs) of 5 µm formalin-fixed and paraffin embedded (FFPE) adenocarcinomas were used in this study. One contained one core each representing breast, lung, prostate, pancreas and colon adenocarcinomas, while the remaining two TMAs contained cores originating from breast, lung, prostate, pancreatic, colon, bile duct, endometrial, and ovarian cancer, and three cores originating from stomach adenocarcinomas. Additionally, for stomach, bile duct, endometrial, and ovarian cancer, 5 µm thick FFPE full tissue sections were used. The total number of patient samples for each adenocarcinoma type was five for breast, lung, prostate, pancreas, colon, bile duct, endometrium, and ovary, and four for stomach. The tissue sections were cut at the pathology department at Aarhus University Hospital.

Cell cultures.

Cell lines (Caco-2, HCT116, TFK-1, EGI-1, SKOV-3, CAOV3, Hec-1-a, KLE, SNU-1, 23132/87, A459, H1650, H1975, LNCaP, DU145, MDA-MB-231, and PaTu-8898T) representing nine different adenocarcinomas (colon, bile duct, ovarian, endometrial, stomach, lung, prostate, breast, and pancreatic) were grown under standard conditions, in accordance with the providers’ recommendations at and 5% CO₂. The only exception was MDA-MB-231, which was cultured in 100% air. The normal colon fibroblast cell line, CCD18-Co, was cultivated in MEME (Sigma-Aldrich) containing 10% FBS (Sigma-Aldrich), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich), and 1% non-essential amino acids (Sigma-Aldrich). Cell viability and cell counts were monitored using the Luna automated cell counter (Logos biosystems, Gyeonggi-do, South Korea)

Human cell lines were acquired from the American Tissue Culture Collection (ATCC, Manassas, VA, USA), Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Leibniz, Germany), or kindly donated by Karina Dalsgaard Sørensen and Lene Nejsum from Aarhus University Hospital (Aarhus, Denmark) or David Olagnier, Martin Roelsgaard Jakobsen, and Naiara Santana Codina (Department of Biomedicine, Aarhus University). All cell lines were tested negative for mycoplasma and all donated cell lines were authenticated by Eurofins Genomics Germany Gmbh (Ebersberg, Germany)

DNA and RNA purification.

Total DNA and RNA were isolated from the cell lines using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen), and total RNA was isolated using the QIAwave RNA Mini kit (Qiagen), both according to the manufacturer’s protocol. DNA and RNA extraction from FFPE patient samples were performed using the Allprep DNA/RNA FFPE kit (Qiagen) according to manufacturer’s protocol. The concentrations of total DNA and RNA were measured by spectrophotometry using a NanoDrop One spectrophotometer (Thermo Fisher).

ciRS-7 chromogenic and fluorescent in situ hybridization.

The localization of ciRS-7 within primary FFPE tissue samples was investigated by single molecule chromogenic and fluorescent in situ hybridization using a slightly modified version of the BaseScope Detection Reagent Kit v2- RED protocol (Advanced Cell Diagnostics [ACD], MN, USA), where the nuclei of the cells in the tissues were stained with DAPI prior to mounting. The BaseScope method allows the detection of ciRS-7 using a BSJ-specific probe (BA-Hs-CDR1as-circRNA-Junc, ACD). In brief, the FFPE tissue samples were deparaffinized using Neoclear (Sigma-Aldrich) and treated with pretreatment buffers, allowing the probe to hybridize to ciRS-7 during the hybridization step. Eight amplification steps allowed the signal to be detected using FAST-Red, creating both a chromogenic and fluorescent signal. Counterstaining was done using 50% hematoxylin (Sigma-Aldrich) and 1 µg/ml DAPI (Thermo Fisher Scientific) dissolved in PBS. Data were collected using a Slide Scanner (Upright Widefield Fluorescence, Olympus VS120, Olympus Life Science, Japan) and analyzed using OliVIA (Ver. 2.9.1, Olympus Life Science) and QuPath (Ver. 0.3.1.) [47].

RT-qPCR analyses of ciRS-7 and the LINC00632 transcripts T1, T2, and T3.

The M-MLV Reverse Transcriptase kit (Invitrogen, Waltham, MA, USA) was used to reverse-transcribe 500 ng of RNA according to the manufacturer’s protocol and diluted 1:10 in nuclease free water (VWR, Avantor, PA, USA). RT-qPCR was performed using 4 µL of diluted cDNA and 6 µL of qPCR master mix consisting of 5 µL SYBR green PCR Master mix (Applied biosystems, Thermo Fisher Scientific, MA, USA) and 5 µM of each primer (S1 Table). The PCR amplification was performed using a LightCycler 480 instrument (Roche Life Science, Mannheim, Germany) with the following cycling conditions: one cycle of 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds. The commonly used reference genes GUSB, UBC, and PUM1 [48–50] were used to normalize the data using geometric means and relative gene expression was calculated as [51]. Laser-capture microdissected samples were normalized to UBC. The experiments were done as technical triplicates at the qPCR level.

NanoString nCounter analysis of ciRS-7 across cell lines.

The NanoString nCounter SPRINT instrument (NanoString Technologies, USA) was used to analyze 100 ng of total RNA using a custom panel (S2 Table) including ciRS-7, the T1, T2, and T3 transcripts according to the manufacturer’s instructions with a 20-hour hybridization time. The data were processed with the nSOLVER 4.0 software (NanoString), and normalization was performed relative to the positive controls included in the panel, as well as to the geometric mean of the reference genes ACTB, MRPL19, PUM1, SF3A1, and GUSB [48]. The data were exported to Excel (Microsoft Corporation, USA) and the background threshold was defined as the mean of the negative controls plus two times the standard deviation.

Bisulfite treatment and sequencing of the LINC00632 T3 promoter CpG island.

Bisulfite-treatment of 500 ng of genomic DNA for each sample was performed using the EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. A PCR to amplify the T3 promoter was subsequently performed using Taq recombinant polymerase (Thermo Fisher) according to the manufacturer’s recommendations with 10 mM dNTPs (Invitrogen, USA) and 10 µM forward and reverse primers from a previously published assay [16]. The PCR was run with the following cycling conditions: 94 °C for three minutes, 45 cycles of 94 °C for 20 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds followed by one cycle of 72 °C for 10 minutes. Subsequently, the amplicons were Sanger sequenced as previously described [16]. Sanger sequencing chromatograms can be found in S2 File. The methylation percentage of individual CpG sites was quantified by assessing the peak intensities of thymine and cytosine using SnapGene Viewer (v. 6.1.2, GSL Biotech, CA, USA). Subsequently, the ratio between the bases was calculated, considering intensities under 100 as background [52].

Laser-capture microdissections.

Five µm thick FFPE colon adenocarcinoma tissue sections from five colon cancer patients were mounted on membrane slides (Arcturus PEN Membrane Glass Slides, Applied Biosystems, USA) and stained with Mayers hematoxylin at the pathology department at Aarhus University Hospital (Aarhus, Denmark) and at Aarhus University. Laser-capture microdissection was performed on a Leica LMD 7 Laser Microdissection Microscope, where the cancer cell and stromal cell fractions were collected in 0.2 ml tubes respectively. Samples were stored at -18°C until they were processed as described above in the DNA and RNA extraction section.

Sensitive Melting Analysis after Real-Time Methylation-Specific PCR (SMART-MSP).

SMART-MSP [53] was performed on bisulfite treated cell line DNA as described above. A previously published assay, which is less susceptible to normalization errors caused by copy number changes and aneuploidy, was used for normalization [54,55], while previously published primers were used to specifically target several CpG sites in the T3 promoter [16]. Bisulfite-converted fully methylated and fully unmethylated DNA (Epitect Control DNA, Qiagen) were used as positive and negative controls, respectively. The negative control was considered negative when amplification occurred after >35 PCR cycles. qPCR was performed using a 96-well plate with 2 µL of bisulfite-treated DNA and 8 µL of LightCycler 480 High-Resolution Melting Master (Roche Life Science) including primers, in each well as technical triplicates. The PCR amplification was carried out on a LightCycler 480 instrument II (Roche Life Science) with the following cycling conditions: one cycle of 95 °C for 10 min, followed by 45 cycles of 95 °C for 10 seconds, 60 °C for 20 seconds and 72 °C for 20 seconds. The melting program was carried out using the following conditions: 95 °C for 15 seconds, 40 °C for 1 minute, and 20 acquisitions/°C from 40 °C to 95 °C. The experiments were conducted using technical replicates at the level of qPCR on three biological replicates. Methylation levels were calculated using the method [51] when comparing the samples to a fully methylated genomic control.

5-aza and SAHA treatments.

In 6-well plates, 100,000 cells were seeded 24 hours prior to treatment. 5-aza monotreatment was performed every 24 hours for four days with either 0.5 µM or 2.5 µM 5-aza (STEMCELL technologies, Canada) freshly prepared in low FBS media and harvested 120 hours after seeding. Cells treated with media containing PBS instead of 5-aza served as a negative control. SAHA monotreatment was performed by seeding 100,000 cells in 6-well plates 24 hours prior to treatment, followed by treatment every 24 hours with either 0.5 µM, 1.5 µM, or 4.5 µM SAHA (MedChem Express LLC, USA) dissolved in DMSO for three days before harvesting after 96 hours. The negative control was treated with 0.3% DMSO. For 5-aza and SAHA combination treatment, 100,000 cells were seeded in 6-well plates 24 hours prior to treatment, followed by treatment with 2.5 µM 5-aza every 24 hours freshly prepared in low FBS media for four days. Forty-eight hours after seeding, the cells were treated with either 0.5 µM, 1.5 µM, or 4.5 µM SAHA dissolved in DMSO every 24 hours simultaneously with the 5-aza treatment for three days. The negative control was treated with PBS for the first 24 hours, and with PBS and 0.3% DMSO for the remaining timepoints. The cells were harvested after 120 hours, and RNA and DNA purifications were performed as previously described.

Chromatin Immunoprecipitation (ChIP)-qPCR.

To study the presence of histone modifications, ChIP-qPCR was performed targeting H3K9Ac using anti-H3K9Ac (Thermo Fisher) or H3K27me using anti-H3K27me3 (Thermo Fisher) antibodies. ChIP was performed using the MAGnify Chromatin Immuniprecipitation System (Invitrogen, Waltham, MA, USA) as described by the manufacturer, using mouse IgG as a negative control. The antibodies were conjugated to Dynabeads Protein A/G for 1 hour at 4 °C. In brief, 1,000,000 cells were harvested and fixated with 37% formaldehyde (Sigma-Aldrich) for 10 minutes. The reaction was stopped using 1.25 M glycine. The cells were subsequently washed with cold PBS (VWR), pelleted (13,000 rpm at 4°C for 10 minutes), and lysed using lysis buffer. Ultrasonication was performed at high intensity for 15 minutes on the Bioruptor Sonication system Version 1.1 (Diogenode, USA) to create 200–500 basepairs (bp) long DNA-chromatin fragments, which were validated using gel electrophoresis. The chromatin was diluted 1:10 and conjugated antibodies were added. An input control was reserved for the reverse crosslinking and DNA purification steps. After IP, the DNA-chromatin complexes were reverse crosslinked, and DNA was purified using DNA purification magnetic beads according to the MAGnify protocol. qPCR was performed as previously described using primers targeting the genomic region 1,000 bp upstream from the T3 transcript, whilst UBC and RARβ1 were employed as controls for H3K9Ac and H3K27me3, respectively (S3 Table). The Ct values for the T3 transcript were normalized to the 1% input control, which was adjusted to 100% by subtracting 6.6 cycles from the measured Ct value, while the degree of histone modification (% Input) was calculated as .

Statistical analysis.

To assess potential linear correlations, linear regression with an F-test for significance was employed. Unpaired t-tests were performed to compute the P-values between groups as detailed in the figure legends. Plotting of data and statistical analyses were performed using Prism 10.0.2 (GraphPad, USA). P-values below 0.05 were considered statistically significant.