Clinical tissue samples

A total of 95 pairs of PDAC tissues and adjacent tumor tissues were collected from Xuzhou Medical University Affiliated Hospital. Among them, 50 patients had not received radiotherapy or chemotherapy while 45 patients received GEM neoadjuvant therapy. This study was approved by Institutional Ethics Committee of Xuzhou Medical University Affiliated Hospital and informed consent were signed by all patients.

Cell culture and transfection

The normal human pancreatic duct cell line hTERT-HPNE and PDAC cell lines PANC-1, CFPAC-1, BxPC-3 and MIA-PaCa2 were purchased from Chinese Academy of Science (Shanghai, China) cultured RPMI 1640 medium (Hyclone, USA) containing 10% Fetal Bovine Serum (FBS) (Gibco,USA), 100u/ml penicillin and 100 µg/ml streptomycin (Beyotime, China) in a cell incubator at 37℃ with 5% CO2. For the construction of GEM-resistant PDAC cell lines, PANC-1 and CFPAC-1 cells were cultured with GEM (MCE, USA) at increasing concentration gradients. For 5-AzaC treatment, CRC cells were treated with 5µM of 5-AzaC (MCE, USA) for 72 h. All small interfering RNA (siRNA) and full-length plasmid of hsa_circ_0007919, LIG1, FOXA1, TET1 and negative control were purchased from GenePharma (Suzhou, China) and transfected into cells using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s protocol. All sequences of siRNAs are shown as follows:

si-hsa_circ_0007919#1: 5’-GACAGAUCCAGGUGGAAGCTT-3’;

si-hsa_circ_0007919#2: 5’-ACAGAUCCAGGUGGAAGCATT-3’;

si-LIG1#1: 5’- AGAAGAUAGACAUCAUCAAAG-3’;

si-LIG1#2: 5’- CGUCAUUUCUUUCAAUAAAUA-3’;

si-FOXA1: 5’- GGAUGUUAGGAACUGUGAAGA-3’;

si-TET1: 5’- CGAAGCUACUGCAAAUCAACA-3’;

si-Ctrl: 5’- UUCUCCGAACGUGUCACGUTT-3’.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from tissues and cells by RNA isolater Total RNA Extraction Kit (Vazyme, China), cDNA was synthesized by HiScript II Q RT SuperMix for qPCR (Vazyme, China) and the expression was detected by ChamQ SYBR qPCR Master Mix (Vazyme, China). All the data were normalized to GAPDH/U6 and the data from tissues were quantified by 2−ΔCt method while others were quantified by 2−ΔΔCt method. All the primers were synthesized by GENEray (Shanghai, China) and the sequences are shown as follows:

hsa_circ_0007919-F: 5’-AGGTGGAAGCAGGGAAAG-3’;

hsa_circ_0007919-R: 5’-TCATGGGCAGCAACAGG-3’;

ABR-F: 5’-GGTGGATTCCTTCGGCTAT-3’;

ABR-R: 5’-CACTTGGGCTCCGCTGT-3’;

LIG1-F: 5’-GCCCTGCTAAAGGCCAGAAG-3’;

LIG1-R: 5’-CATGGGAGAGGTGTCAGAGAG-3’;

FOXA1-F: GCAATACTCGCCTTACGGCT-3’;

FOXA1-R: TACACACCTTGGTAGTACGCC-3’;

TET1-F: CATCAGTCAAGACTTTAAGCCCT-3’;

TET1-R: CGGGTGGTTTAGGTTCTGTTT-3’;

LIG1 P1-F: GCTAAAACCTCCTCCCC-3’;

LIG1 P1-R: CATGAAGCATGTGACCG-3’;

GAPDH-F: 5’-GGAGCGAGATCCCTCCAAAAT-3’;

GAPDH-R: 5’-GGCTGTTGTCATACTTCTCATGG-3’;

U6-F: 5’-CTCGCTTCGGCAGCACA-3’;

U6-R: 5’-AACGCTTCACGAATTTGCGT-3’.

Identification of hsa_circ_0007919

qPCR product amplified by hsa_circ_0007919 primer was validated by Sanger-seq (Sangon, China). Total gDNA was extracted by FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, China) and qPCR products amplified from cDNA and gDNA were separated in 1% agarose gel. Total RNA was treated with RNase R (Epicentre, USA) at 37℃ for 30 min and were detected by qRT-PCR just as described above.

Half maximal inhibitory concentration (IC50) detection assay

A total of 12 groups of 4 × 103 PDAC cells were placed into 96-well plate separately, then cells were treated with GEM at concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2 and 102.4µM for 48 h and then detected as CCK-8 assay described.

CCK-8 assay

Cells after transfection or GEM treatment were collected and counted, then 4 × 103 cells were placed into 96-well plate and cultured in incubator at 37℃. 24, 48, 72 and 96 h after, cells were incubated with 100 µl serum-free medium and 10 µl CCK-8 solution (Glpbio, USA) at 37℃ for 2 h and measured at 450 nm wavelength (SPARK, Switzerland).

Apoptosis detection assay

Cells after transfection or GEM treatment were collected by EDTA-free trypsin solution (Beyotime, China), then cells were washed by PBS and incubate with Annexin V and PI solution for 10 min and detected by the flow cytometer (BD, USA) according to the manufacturer’s protocol of Cell Apoptosis Detection Kit (Biosharp, China).

Western blot assay

Total protein was extracted from cells by RIPA lysis solution (Beyotime, China) and quantified by BCA Protein Assay Kit (Beyotime, China), protein was separated in SDS-PAGE and transferred to PVDF membrane (Millipore, Germany). After blocking with 5% skim milk, the membrane was incubated with primary antibodies and secondary antibodies and detected with Super ECL Detection Reagent (Yeasen, China) using a luminescent imaging system (Tanon, China). All used antibodies are shown as follows: anti-caspase3 (19677-1-AP, Proteintech, USA), anti-caspase9 (10380-1-AP, Proteintech, USA), anti-BCL2 (68103-1-Ig, Proteintech, USA), anti-GAPDH (60004-1-Ig, Proteintech, USA), HRP-goat anti-rabbit IgG (H + L) (BF03008, Biodragon, China), HRP-goat anti-mouse IgG (H + L) (BF03001, Biodragon, China), anti-γ-H2AX (AP0687, Abclonal, China), CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) (SA00013-4, Proteintech, USA), anti-LIG1 (18051-1-AP,, Proteintech, USA), anti-Ki67 (GB111499, Servicebio, China), anti-FOXA1 (GTX100308, GeneTex, USA), anti-TET1 (AB_2793752, Active Motif, USA), anti-QKI (13169-1-AP, Proteintech, USA).

Single cell gel electrophoresis

0.8% normal melting point agarose (Vicmed, China) was placed on glass slide, then 5 × 103 cells in 0.6% low melting point agarose (Biosharp, China) was place above and electrophoreted in a horizontal electrophoresis tank after lysis, at last cells were stained with PI solution (Biosharp, China) and the picture was photographed by inverted fluorescence microscope (Olympus, Japan).

DNA ladder assay

Total DNA of cells after transfection was extracted by FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, China). Briefly, cells were collected and treated by RNase Solution and Proteinase K at room temperature. Then cells were mixed with buffer GB and anhydrous ethanol, after abstersion with washing buffer, DNA was dissolved into elution buffer. At last, DNA was separated in 1% agarose gel and photographed using luminescent imaging system (Tanon, China).

Immunofluorescence (IF)

Cells were fixed by 4% paraformaldehyde (Vicmed, China) and blocked by 5% Bovine Serum Albumin (BSA) (Solarbio, China), then cells were incubated with primary antibody, fluorescent secondary antibody and DAPI (Bioss, China) and photographed by confocal laser microscope (ZEISS, Germany).

Stable inhibition cell lines construction and xenograft model

PANC-1 and CFPAC-1 GEM-resistant cells were infected by hsa_circ_0007919 inhibition lentivirus (GenePharma, China) or negative control lentivirus and selected by puromycin (Solarbio, China) for over 2 weeks, the efficiency of lentivirus was detected by qPCR. 5 × 106 lentivirus-infected cells were injected into blank region of nude mice (Gempharmatech, China) and were treated with GEM (50 mg/kg, i.p.) every 4 days. After measuring volumes of tumors every 5 days, the mice were sacrificed and the tumors were harvested 25 days after injection. All sequences of shRNAs are shown as follows:

sh-hsa_circ_0007919: 5’-CACCGAGGTGGAAGCAGGGAAAGTTCGA AAAAATTGATCAATGCCGAGGA-3’;

sh-Ctrl: 5’-CACCGTTCTCCGAACGTGTCACGTTTCGAAAAACGTG ACACGTTCGGAGAA-3’.

Immunohistochemical (IHC)

Tumors were fixed by 4% paraformaldehyde, paraffin embed and sliced into sections, sections were hydrated by xylene and gradient alcohol (Sinoreagent, China). Antigen of sections were repaired by citrate solution and blocked by goat serum, then sections incubated with primary and secondary antibodies according to the manufacturer’s protocol of SP Kit (ZSGB-BIO, China) and stained by DAB Staining Kit (ZSGB-BIO, China) and hematoxylin solutions (Sinoreagent, China). The pictures of sections were photographed by inverted microscope (Olympus, Japan). Relative staining score was calculated using an IHC score analysis method according to the proportion of positively stained cells and the intensity of staining. The proportion of positive cells was scored as follows: 0 (0–5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), 4 (> 75%) and the intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong).

TUNEL assay

The TUNEL assay was performed according to the manufacturer’s protocol of TUNEL Apoptosis Detection Kit (Color Development) (Beyotime, China). Tissue sections was hydrated as described in IHC, after treated with Proteinase K at 37℃, the tissues were incubated with 3% hydrogen peroxide solution and biotin labeling solution containing TdT enzyme and biotein-dUDP away from light at 37℃, then the tissues were stained using Streptavidin-HRP solution and DAB staining solution and photographed by inverted microscope (Olympus, Japan).

Gene set enrichment analysis (GSEA)

GSEA was performed on the normalized data using the GSEA v2.0 tool (http://www.broad.mit.edu/gsea/). We compared the expression of genes in PANC-1 GEM-resistant cells transfected with hsa_circ_0007919 siRNA or negative control siRNA. Three gene sets were used for analysis (KEGG_BASE_EXCISION_REPAIR, KEGG_MISMATCH_REPAIR, KEGG_NUCLEOTIDE_EXCISION_REPAIR), and the detailed genes in the gene sets can be found in MSigDB (http://software.broadinstitute.org/gsea/msigdb/genesets.jsp). The P values of the differences between the two gene sets were analyzed with the Kolmogorov–Smirnov test.

Fluorescence in situ hybridization (FISH)

Cells were fixed by 4% paraformaldehyde and incubated with hybridization solution containing probes at 37℃ overnight, then cells were washed by SSC solution and stained with DAPI according to the manufacturer’s protocol of FISH Kit (RIBOBIO, China). The images were photographed by confocal laser microscope (ZEISS, Germany).

Nuclear-cytoplasmic fractionation assay

Nuclear and cytoplasmic RNA was extracted by Cytoplasmic & Nuclear RNA Purification Kit (Norgen Biotek, Canada) according to the manufacturer’s protocol, then the expression of hsa_circ_0007919 in nucleus and cytoplasm was detected by qPCR.

Chromatin isolation by RNA purification (ChIRP)

ChIRP was used to detect the protein binding with hsa_circ_0007919 and performed according to the manufacturer’s protocol of ChIRP kit (Bersinbio, China). Briefly, cells were cross-linked with paraformaldehyde and lysed through sonication, and then the lysis solution was incubated with biotin-labeled hsa_circ_0007919 probes (RIBOBIO, China) and magnetic beads, ultimately the protein was extracted and detected by WB.

Co-immunoprecipitation (Co-IP)

Total protein extracted by cell lysis buffer for IP (Beyotime, China) was incubated with antibodies and magnetic beads, binding proteins were extracted by 2×SDS-PAGE Sample Loading Buffer (Beyotime, China) and detected by WB.

RNA binding protein immunoprecipitation (RIP)

RIP assay was conducted according to the manufacturer’s protocol of RNA Immunoprecipitation Kit (GENESEED, China), mixture of RNA and protein was collected and incubated with antibodies and magnetic beads, RNA was extracted by adsorption column and detected by qPCR.

Chromatin immunoprecipitation (ChIP)

ChIP assay was conducted according to the manufacturer’s protocol of Simple ChIP Enzymatic Chromatin IP Kit (CST, USA). Cells were mixed by 1% paraformaldehyde and chromatin was digested into fragments by enzymes. Then the solution was incubated with antibodies and magnetic beads and DNA was extracted from beads by purified centrifugal column, the binding DNA fragments were detected by qPCR.

Methylation-specific PCR (MS-PCR)

Total DNA was extracted by FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, China), then DNA was denaturated and bisulfite convered by EZ DNA Methylation-Gold Kit (ZYMO RESEARCH, USA) and amplificated by Methylation Specific PCR Kit (TIANGEN, China) according to the manufacturer’s protocol, the DNA was ultimately separated in 1% agarose gel and photographed by luminescent imaging system. Methylated primer and unmethylated primer located in LIG1 promoter were generated by MethPrimer 2.0 (http://www.urogene.org/methprimer2/) and the sequences are shown as follows:

LIG1 M-F: 5’-GAGAAGAAGGTTCGTTTTCGTAG-3’;

LIG1 M-R: 5’-ATAAAATAAATAAAATACCCCGAAT-3’;

LIG1 U-F: 5’-GAGAAGAAGGTTTGTTTTTG-3’;

LIG1 U-R: 5’-AAAATAAAATAAATAAAATACCCCAAAT-3’.

Luciferase reporter assay

Luciferase reporter assay was conducted according to the manufacturer’s protocol of Dual Luciferase Reporter Gene Assay Kit (Beyotime, China). Cells transfected with pGL3-basic plasmid containing LIG1 promoter (Genecreate, China) and pRL-TK control plasmid with or without hsa_circ_0007919 inhibition were collected for lysis, then the firefly luciferase detection reagent and renilla luciferase detection reagent was added into the solution and measured by Multifunctional microplate reader (SPARK, Switzerland) separately.

Statistical analysis

All values are expressed as mean ± standard deviation (SD). The significance of the differences was measured by Student’s t-test or one-way ANOVA. Kaplan–Meier analysis was used for survival analysis, and the differences of survival probabilities were measured by the log-rank test. The correlations between the expression of hsa_circ_0007919 and various clinicopathological variables were analyzed by Chi-Squared test. p < 0.05 was considered significant. Statistical analyses were performed using SPSS version 25.0 (SPSS, Inc., USA).