Patient information and tissue specimens
Ninety six pairs of pancreatic cancer tissues and normal tissues from gemcitabine-treated PADC patients, which were histopathologically and clinically diagnosed, were obtained from Fujian Provincial Hospital. All experiments involving human samples and clinical data were approved by the Accreditation Committee of Fujian Provincial Hospital.
For Immunohistochemistry staining, slides were incubated with primary antibodies targeting RPN6 (Cat. No. ER64693, HUABIO) overnight at 4 °C, followed by incubation with secondary antibodies at room temperature for 30 min. Next, sections were stained with DAB solution for 10 min. The staining intensities of RPN6 were assigned as follows: 0, negative; 1, weak; 2, medium; and 3, strong. The positive rate of tumor cells was scored as follows: 1, 0–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The IHC scores were calculated by multiplying the staining intensity and positive rate scores. The sections were reviewed by two pathologists.
Establishment of cell lines and transfection
Human PDAC cells (PATU8988T, AsPC-1, BxPC-3, PANC-1, and SW1990) and human pancreatic ductal epithelial (HPDE) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 2 mM L-glutamine. BxPC-3 cells were maintained in RPMI 1640 medium supplemented with 2.5 g/L glucose, 1 mM sodium pyruvate, and 10% fetal bovine serum. Cells were cultured in a 37 °C, 5% CO2 incubator with routine mycoplasma check once every month. Gemcitabine-resistant PANC-1 (PANC-1-GR) cells and gemcitabine-resistant BxPC-3 (BxPC-3-GR) cells were established by our laboratory. Briefly, PANC-1 cells were treated with gemcitabine by a stepwise increase in drug concentration from 1 to 80 μM (1, 5, 10, 20, 40, and 80) every 2 weeks, until cells became resistant to 80 μM of gemcitabine. In contrast, BxPC-3 cells were exposed to gemcitabine ranging from 0.5 to 40 μM (0.5, 2, 5, 10, 20, and 40) every 2 weeks until cells became resistant to 40 μM of gemcitabine. The miR-615-3p mimic, inhibitor, and corresponding control oligonucleotides were purchased from Shangya (Fuzhou, China). The transfection of miR-615-3p mimics, inhibitor, or corresponding NCs into cells was performed using Lipofectamine 3000 (Invitrogen). Lentivirus shRNAs were constructed by Genechem (Shanghai, China). The cells were transfected with lentivirus according to the manufacturer’s instructions. The sequences of shRNA against specific targets are available in Table S1.
The total RNA of the cell samples was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The circRNA sequencing analysis was performed at Genechem (Shanghai, China) following the standard experimental procedures below.
RNA quantification and qualification
RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit® 2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Library preparation for circRNA sequencing
A total of 5 μg RNA per sample was used as input material for the RNA sample preparations. Firstly, ribosomal RNA was removed by Epicentre Ribozero™ rRNA Removal Kit (Epicentre, USA), and the rRNA-free residue was cleaned up by ethanol precipitation. Subsequently, the linear RNA was digested with 3 U of RNase R (Epicentre, USA) per μg of RNA. Following the manufacturer’s recommendations, the sequencing libraries were generated by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). Briefly, fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. In the reaction buffer, dNTPs with dTTP were replaced by dUTP. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferentially 150 ~ 200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Clustering and sequencing
According to the manufacturer’s instructions, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the libraries were sequenced on an Illumina Hiseq 4000 platform, and 150 bp paired-end reads were generated.
CircRNA sequencing-data analysis
Raw data (raw reads) of fastq format were firstly processed through in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, ploy-N, and low-quality reads from raw data. At the same time, the clean data’s Q20, Q30, and GC content were calculated. All the downstream analyses were based on clean data with high quality. The genome and gene model annotation files were downloaded directly from the genome website. Index of the reference genome was built using Bowtie2 v2.2.8, and paired-end clean reads were aligned to the reference genome using Bowtie. The circRNAs were detected and identified using find_circ and CIRI2. Prior to differential gene expression analysis, the read counts were adjusted by edgeR program package for each sequenced library through one scaling normalized factor. Differential expression analysis was performed using the edgeR R package. Fold change > 2 or < 0.5 and P-value < 0.05 was set as the threshold for significantly differential expression.
RNase R treatment
RNase R (Geneseed, Guangzhou, China) was applied to digest linear RNA. RNAs extracted from PANC-1-GR and BxPC-3-GR cells were divided into two groups for RNase R treatment and control. The sample was incubated for 30 min at 37 °C with 3 U/μg of RNase R. For analysis, qRT-PCR was used to detect the expression of MTHFD1L and circ-MTHFD1L. GAPDH in the control group was used as the internal reference. Three independent experiments were applied in triplicate.
Actinomycin D (ActD) treatment
1 × 105 cells per well of PANC-1-GR and BxPC-3-GR cells were seeded in a 6-well plate overnight, and 2 mg/L actinomycin D (Sigma, USA) was added into the well for 4, 8, 12, and 24 h. The cells were harvested according to the time of treatment. Then qPCR was performed to analyze the stability of MTHFD1L mRNA and circ-MTHFD1L. Three independent experiments were applied in triplicate.
RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
According to the manufacturer’s protocol, total RNA from PDAC cells, tissues, and matched non-cancerous tissues was isolated using the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Dalian, China). Bulge-loop™ miRNA RT-qPCR Primers were applied to determine the level of miRNAs. The real-time PCR reactions were performed using StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, MA, US). The program settings on temperature cycling were followed as instructed by the manufacturer. The relative circRNA/mRNA and miRNA expression levels were normalized to GAPDH and U6, respectively, using the 2−△△CT method. The sequences of primers are listed in Table S1.
Western blot analysis
In brief, proteins were isolated from PDAC cells and tumor tissues using RIPA buffer (Solarbio, Beijing, China) supplemented with proteinase inhibitors, and the protein concentration was determined with BCA reagent (Beyotime, Beijing, China). Cell lysates were separated on SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes. (Millipore), After the membranes were blocked in 5% skim powdered milk for 2 h, they were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in this study included: RPN6 (Cat. No. ER64693, HUABIO), RAD50 (Cat. No. sc-56,209, Santa Cruz), RAD51 (Cat. No. sc-398,587, Santa Cruz), BRCA1 (Cat. No. sc-6954, Santa Cruz), BRCA2 (Cat. No. sc-518,154, Santa Cruz), PCNA (Cat. No. EM111201, HUABIO), P53 (Cat. No. ET1605–16, HUABIO), P21 (Cat. No. 10355–1-AP, Proteintech), and GAPDH (Cat. No. ET1601–4, HUABIO). Next, the membranes were incubated with secondary antibodies (HUABIO, Hangzhou, China) at room temperature for 1 h. After washing three times, the targeted proteins were visualized using enhanced chemiluminescence (ECL) reagent (Millipore, MA, USA). GAPDH was used as the loading control in this study.
Colony formation assay
Cells were seeded into 6-well plates at an initial density of 800 cells/well and grew for 24 h, then treated with gemcitabine at half the IC50 value of the respective cells for 48 h, after which the culture was continued for 10 days. The colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA). The visible colonies were counted using a light microscope. The numbers of colonies in triplicate wells were measured for each treatment group.
Cell proliferation assay
Cell proliferation was assessed by a Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan). Cells (5 × 103cells per well) were cultured in 96-well-plates for 0, 24, 48, 72, and 96 h, and treated with gemcitabine at the IC50 value of the respective cells, after which CCK-8 solution (10 μl) was added to each well, and the plates were further incubated for 2 h. The number of cells was quantified by measuring the absorbance at 450 nm on a microplate reader. Three independent experiments were performed. The sensitivity to gemcitabine of pancreatic cancer cells was determined using the CCK-8 assay.
Briefly, Cells (5 × 103cells per well) were seeded onto 96-well plates and incubated at 37 °C overnight. Cells were then treated with different concentrations of gemcitabine. After incubation for 48 h, CCK-8 solution (10 μl) was added to each well, and the plates were further incubated for 2 h. The number of cells was quantified by measuring the absorbance at 450 nm on a microplate reader. Dose-response curves were plotted on a semilog scale as the percentage of the control cell number, which was obtained from the sample with no drug exposure. The IC50 was determined by the intersection of the gemcitabine concentration and the midpoint of the 450 nm reading.
Cell cycle assay
A cell cycle assay kit (Cat. no. C1052) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The drug-resistant cells were treated with gemcitabine at half the respective IC50 concentrations for 24 h, and then cells were fixed with 70% ethanol at 4 °C for 24 h and then resuspended in ice-cold PBS before they were resuspended with a staining solution containing RNase A and propidium iodide (PI). The DNA content was assessed using a flow cytometer, and the number of cells in the different cell cycle phases was counted using ModFit version 5.0 (Verity Software House, Topsham, ME, USA). Three independent experiments were performed.
Fluorescence in situ hybridization (FISH)
The specific fluorescently labeled circ-MTHFD1L and miR-615-3p FISH probes were designed and synthesized by Servicebio (Wuhan, China). The FISH experiment was performed according to the manufacturer’s instructions. All images were acquired on Nikon A1Si Laser Scanning confocal microscope (Nikon Instruments Inc., Japan). The sequences of probes are listed in Table S1.
Dual-luciferase reporter assay
The full-length wild-type (WT) sequence of circ-MTHFD1L, the RPN6 3′ untranslated region (UTR), and the indicated mutant circ-MTHFD1L and RPN6 3’UTR (Mut) containing the predicted miR-615-3p binding sites were separately synthesized and cloned into the dual-luciferase reporter vector PGL3 (Genechem, Shanghai, China). The resulting dual-luciferase reporter plasmids (WT or Mut) were co-transfected with the miR-615-3p mimic or inhibitor into PANC-1-GR or BxPC-3-GR cells, respectively, using Lipofectamine 3000. After 48 h of incubation, the relative firefly luciferase activities concerning the corresponding Renilla luciferase activities were measured and analyzed using a Dual-Luciferase Assay System (Promega, Fitchburg, WI, USA) following the manufacturer’s protocol.
RNA pull-down assay
A biotin-labeled probe targeting circ-MTHFD1L and a random oligo probe (RiboBio, Guangzhou, China) were incubated with M280 streptavidin dynabeads (Invitrogen, USA) at 25 °C for 2 h to generate probe-coated beads. The cell lysates were incubated with the probe-coated beads mixture at 4 °C overnight. Subsequently, the circ-MTHFD1L/miRNA/bead complexes were washed three times and eluted from the beads. Then, the enrichment of circ-MTHFD1L and related miRNAs in the precipitated complexes was evaluated by qRT-PCR. The probe sequence of RNA pull-down is shown in Table S1.
RNA immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) assay was performed using Magna RIP™ RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) following the manufacturer’s protocol. Transfected cells were lysed in complete RNA immunoprecipitation lysis buffer after transfected with miR-615-3p mimics or negative control. Then, the cell extract was incubated with magnetic beads conjugated with anti-Argonaute 2 (AGO2) or anti-IgG antibody (Millipore, Billerica, MA, USA) for 6 h at 4 °C. The beads were washed and incubated with Proteinase K to remove proteins. Finally, isolated RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), then the purified RNA was subjected to agarose gel electrophoresis and qRT-PCR analysis.
Neutral comet assay
Neutral comet assays were performed using an OxiSelect Comet Assay Kit (Cell Biolabs, USA) per the manufacturer’s protocol. Briefly, cells were dissociated with Accutase and washed with PBS, and replicates were suspended in OxiSelect comet agarose (Cell Biolabs, USA). Neutral electrophoresis was conducted at 30 V for 30 min. Data were collected with a fluorescent microscope with a FITC filter and analyzed using the OpenComet software . All steps after agarose treatment were conducted in the dark to prevent additional DNA damage.
4–6 week-old male athymic BALB/c nude mice (Slac Laboratory Animal Co. Ltd., Shanghai, China) were housed and fed in standard pathogen-free conditions. For the subcutaneous cell line-derived xenograft (CDX) model, Gemcitabine-resistant PANC-1 cells (PANC-1-GR) were prepared as stable gene expression cell lines after transduction with sh-NC or sh-circ-MTHFD1L, vector or circ-MTHFD1L. Subcutaneous injections of 5 × 106 cells in 100 μL serum-free DMEM and Matrigel (1:1) were performed on each nude mouse (n = 6 in each group). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Gemcitabine was suspended in an oral vehicle containing Cremophor (Sigma-Aldrich), 95% ethanol, and water in a ratio of 1:1:6. Four weeks later, the mice were treated with gemcitabine (50 mg/kg/mouse twice a week by tail vein injection). The mice were sacrificed after 4 weeks of treatment. For two generations of the subcutaneous gemcitabine-resistant CDX model, PANC-1-GR cells were transplanted subcutaneously into the axilla of 4–6 week-old male BALB/c nude mice (Slac Laboratory Animal Co. Ltd., Shanghai, China). After 4 weeks, all mice were treated with gemcitabine (50 mg/kg/mouse twice a week by tail vein injection). 10 weeks later, the most resistant xenograft was isolated and mechanically disaggregated into approximately 1–2 mm3 tissue blocks to implanted subcutaneously into the axilla of 4–6 week-old male BALB/c nude mice for the second CDX generation. Two weeks after implantation, all mice were treated with sh-circ-MTHFD1L lentivirus (twice a week) and randomized into four groups (n = 6 mice per group). The four groups were treated as follows: gemcitabine alone, olaparib alone, gemcitabine combined with olaparib, and negative control. The mice were euthanized in the sixth week, and tumors were isolated for further studies. The subcutaneous tumor size was measured and recorded every 2 days using the Vernier caliper: tumor volume (mm3) = (L × W2)/2, where L is the long axis, and W is the short axis. All the animal experiments were approved by the Animal Welfare Committee of Fujian Medical University (Fuzhou, China).
The public datasets GSE79634, GSE110580, GSE112264, and GSE113486, were retrieved from the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. Differential expression analysis was performed using the limma R package with Fold change > 2 or < 0.5 and P-value < 0.05 as the screening criterion. The downstream miRNAs of circ-MTHFD1L were predicted through ENCORI (ClipExpNum ≥1, https://starbase.sysu.edu.cn/), CircInteractome (https://circinteractome.irp.nia.nih.gov/), Circbank (MiRanda binding site ≥1 & targetscan binding site ≥2, http://www.circbank.cn/) and CircAtlas (http://circatlas.biols.ac.cn/), and then intersected miRNAs served as a candidate miRNA. The downstream target genes of miR-615-3p were predicted through ENCORI (ClipExpNum ≥1, https://starbase.sysu.edu.cn/), miRDB (Target Score ≥ 50, http://mirdb.org/), mirDIP (Integrated score > 0.266, http://ophid.utoronto.ca/mirDIP/), miRTarBase (Sum ≥1, https://miRTarBase.cuhk.edu.cn/) and TargetScan (Conserved sites ≥1, http://www.targetscan.org/), and then intersected with the downstream target genes of miR-615-3p to identify the candidate gene. The expressions of MTHFD1L and RPN6 in pancreatic cancer and normal pancreatic tissue from TCGA and GTEx databases and the survival curves of MTHFD1L and RPN6 in TCGA database were obtained by GEPIA2 (http://gepia2.cancer-pku.cn/).
GraphPad Prism 8.0 and SPSS 23.0 were applied for statistical analysis. Differences between the indicated groups were compared using the Student’s t-test and one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) test. Correlations were evaluated by Pearson correlation analysis. The cumulative overall survival (OS) and progression-free survival (PFS) rates were calculated using the Kaplan-Meier method, and significance was evaluated with the log-rank test. A P-value < 0.05 was considered to indicate a statistically significant result.