Methylation-mediated LINC00261 suppresses pancreatic cancer progression by epigenetically inhibiting c-Myc transcription

Background: Due to the limitations of strategies for its early diagnosis and treatment, pancreatic cancer (PC) remains a substantial human health threat. We previously discovered a methylation-mediated lncRNA, LINC00261, which is downregulated in PC tissues. However, the underlying role of LINC00261 in PC remains largely unknown. Methods: Quantitative real-time PCR and in situ hybridization were performed to evaluate the expression levels of LINC00261 in PC, adjacent nontumor and normal pancreas tissues. The clinical significance of LINC00261 was assessed in multicenter PC samples. The functions of LINC00261 in PC were investigated by gain- and loss-of-function assays in vitro and in vivo. Potential downstream pathways and mechanisms were explored via RNA sequencing and bioinformatic analyses. RNA immunoprecipitation and chromatin immunoprecipitation assays were used to validate the underlying mechanisms. Pyrosequencing and targeted demethylation of the LINC00261 promoter were performed to explore the upstream epigenetic mechanisms and therapeutic potential. Results: LINC00261 was significantly downregulated in PC tissues, and its expression was positively associated with the prognosis of PC patients. Phenotypic studies indicated that LINC00261 overexpression significantly suppressed PC cell proliferation, migration and metastasis in vitro and in vivo. c-Myc was identified as a downstream target of LINC00261. LINC00261 repressed c-Myc transcription by physically interacting and binding with the bromo domain of p300/CBP, preventing the recruitment of p300/CBP to the promoter region of c-Myc and decreasing the H3K27Ac level. Moreover, the methylation level of the LINC00261 promoter was high in PC tissues and was correlated with poor prognosis. Targeted demethylation of the LINC00261 promoter inhibited PC progression both in vitro and in vivo. Conclusions: Our findings indicate that methylation-mediated LINC00261 suppresses PC progression by epigenetically repressing c-Myc expression. These findings expand the therapeutic potential of LINC00261, possibly providing evidence to support the development of epigenetic drugs or therapeutic strategies. This research adds further insights into the etiology of PC and indicates that LINC00261 may be a prognostic and therapeutic target in PC.


Figure S8. (A)
The mRNA levels of p300/CBP was assessed by qRT-PCR, in the NC WT and mut-p300/CBP groups. (B)The WB levels of p300/CBP was assessed by WB analysis, in the WT and mut-p300/CBP groups. All plasmids were labeled with 3-flag.

Figure S2
Up-regulated Gene Down-regulated Gene

Figure S11
Table S1 Information of the primer.

Microarray analysis
Two independent datasets, GSE15471 (including 39 NP and 39 PC tissues) and

GSE16515 (including 16 NP and 36 PC tissues), were downloaded from the Gene Expression
Omnibus (GEO). The original log 2 transformed normalized signaling data were processed, and differential analyses were performed using the R package limma (downloaded from www.bioconductor.org). The differentially expressed lncRNAs were visualized using the R package pheatmap. All bioinformatic analyses were performed in R (version 3.6.1, www.r-project.org). The intersection of the differentially expressed lncRNAs in both datasets was visualized via a Venn diagram generated using an online tool (bioinformatics.psb.ugent.be/webtools/Venn/). In addition, the Gene Expression Profiling Interactive Analysis (GEPIA, gepia.cancer-pku.cn) online database was used to estimate the associations between lncRNA expression levels and survival.

RNA sequencing analysis
PANC-1 cells were transfected with si-LINC00261 and control siRNA. Biological triplicates were established for each group. Total RNA was extracted from the samples using the TRIzol method. The quality and purity of total RNA extracted from the samples were analyzed by NanoDrop 2000 measurement and denaturing gel electrophoresis. RNA-seq was performed by SHBIO (Shanghai, China) with the Illumina HiSeq 2500 platform. Differential analyses between the si-LINC00261 group and the control group were performed using the R package limma. Detailed information about the RNA-seq protocol, including the sample preparation methods, study design and raw sequencing data, has been uploaded to the GEO database (GSE152012). All the original data would be scheduled to be released on Jun 8,

DNA methylation analysis
Genomic DNA was extracted from cells and tissues (OMEGA, USA  Table S1. Methylation data are presented as the percentage of average methylation in all observed CpG sites. DNA methylation screen and pyrosequencing analysis were performed by SHBIO (Shanghai, China).

RNA in situ hybridization (ISH)
For the tissue microarray, the LINC00261 probe was labeled with digoxin (Exiqon, Denmark). The reaction NBT/BCIP reaction was incubated for 1 h in a wet box in the dark (diluted NBT/BCIP buffer: 20 µl Roche reagent buffer/1 ml NBT/BCIP diluent). The slide was washed with TBST, and 200 µl of red nuclear fixation dye was added for 1 min. Then, the slide was placed under running water for 10 min and dehydrated with alcohol. The slide was sealed with glycerin buffer. The staining intensity score was defined as 0 (negative), 1 (weak), 2 (moderate), 3 (moderate-strong), and 4 (strong), and the positivity rate score was defined as follows: 0 (negative), 1 (1-25%), 2 (26-50%), 3 (51-75%), and 4 (76-100%). The product of the staining intensity score and positive rate score was considered the total ISH staining score. A total score of ≦ 4 was used for the low LINC00261 expression group, and a total score > 4 was used for the high LINC00261 expression group. 4

Fluorescence in situ hybridization (FISH)
For the PC cell lines, the LINC00261 probe was labeled with cyanine-3 (GenePharma, China). PC cells were fixed in formaldehyde, permeabilized with Triton X-100, and dehydrated with ethanol. The dried cells were mixed with 1 µg/µl of the FISH probe (sequence: see Table S1) in a hybridization buffer and incubated at 37 ℃ overnight. The following day, the slides were washed, dehydrated, and nuclei were finally stained using DAPI. Staining was performed, avoiding light, for 20 min, and cells were observed under a fluorescence microscope.

Immunohistochemistry (IHC) assay
c-Myc, Ki67 was detected in IHC. After de-waxing and hydration, antigen retrieval and blocking, the tissue microarray slide was incubated with specific primary antibodies overnight at 4°C, and followed by the observation using IHC kit purchased from Zsbio (Beijing, China). Primary antibody of c-Myc was described in Table S1.

Western blot assay
Total protein was extracted from cell lysis prepared with cold, freshly RIPA buffer (Sigma, USA) and measured with a BCA Protein Assay kit (Beyotime, China) according to the manufacturer's protocol. The protein samples (30 μg) were separated by 4%-20% gradient SDS-polyacrylamide gel electrophoresis (PAGE, GenScript, USA) and transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes for immunoblotting. The membranes were hybridized with a primary antibody at 4°C overnight with gentle shaking, and followed by incubation with a secondary antibody for 1 h at room temperature.
Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and an ECL chemiluminescence kit (GE, USA) were used to detect bound antibodies. Information of used antibodies were as described in Table S1.

RNA extraction and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues or cells using an Ultrapure RNA kit (Cwbio, China).
The RNA concentration and purity were measured with NanoDrop ND-2000 spectrophotometer (Thermo, USA). Reverse transcription and quantitative real-time PCR were performed using a PrimeScript RT reagent kit and SYBR® Premix Ex Taq™ kit respectively (Takara, Japan) according to the manufacturer's protocol. Each test was repeated in triplicate. Relative mRNA expression levels were calculated using the 2-ΔΔCt method and normalized to GAPDH expression levels. All primer sequences used in the present study are shown in Table S1.

RNA immunoprecipitation
RIP was performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (Merck Millipore, Germany) according to the manufacturer's instructions. PANC-1 and SW1990 cells were lysed in complete RIP lysis buffer, and the cell extracts were incubated with magnetic beads conjugated to specific antibody or control IgG for 12 h at 4℃. Beads were washed and incubated with proteinase K to remove proteins. Finally, purified RNA was subjected to qRT-PCR analysis. The IgG antibody used in RIP (Merck Millipore, Germany).

Chromatin immunoprecipitation
ChIP experiments were performed using a MagnaChIP kit (Millipore) according to the manufacturer's instructions. For the ChIP experiment, PC cells were crosslinked with 1% formaldehyde, and the reaction was quenched by the addition of 125 mM glycine. Then, the cells were washed and lysed with cell lysis buffer, and chromatin was sheared to fragments of 100-500 bp by sonication for seven cycles at high amplitude (cycles of 15 s on followed by 45 s off). Then, 5 µg of specific antibody or IgG control antibody was adsorbed onto protein G magnetic beads and incubated with the chromatin extracts at 4°C overnight. Crosslinking of DNA fragments was reversed by ChIP elution buffer and subsequent incubation at 62°C for 2 h and 95°C for 15 min. Recovered DNA was subjected to RNase treatment and analyzed 6 via qPCR. Sequences of primers used for the ChIP-qPCR quantification are provided in Table   S1.

Immunoprecipitation (IP)
After 48 hours of transfection, the PC cells were lysed in an immunoprecipitation lysate buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% NP-40, and a protease inhibitor cocktail tablet) for 15 min on ice. The lysates were centrifuged at 10,000 g for 10 min at 4at 44 4or 10 min at 4min at 44 40 g for 10 mi collected into a new EP tube. The cell lysates were added to 2 µg primary antibody and incubated for 1 hour at 4°C. After incubation, 50 µl Protein A/G PLUS-Agarose (Santa Cruz, CA, USA) was added to the protein-antibody complexes and incubated at 4°C on a rotating device overnight. The immunoprecipitates were washed four times with immunoprecipitation buffer, and a 2x sample loading buffer was added to the beads before boiling for 5 min. The supernatant was collected and used in a Western blot assay.

Cell culture
The human PC cell lines BxPC-3, CFPAC-1, PANC-1 and SW1990 were purchased from American Type Culture Collection (ATCC, USA) and were incubated in complete growth medium with 10% fetal bovine serum (FBS, Gibco, USA), as recommended by the manufacturer. Cultured cells were maintained at 37°C in a humidified incubator with 5% CO2. All cell lines were fingerprinted for authenticity validation.

Cell Transfection and vectors construction
Knockdown siRNA for LINC00261, c-Myc and p300/CBP were purchased from RiboTM promoter.

Cell proliferation and viability assay
For the cell proliferation assay, a total of approximately 25000 PC cells were plated in

Cell cycle assay
For cell cycle analysis, cells were collected and fixed in 70% ethanol at 4°C overnight. Then the cells were incubated with Rnase and propidium iodide (Beyotime, China) according to the manufacturer's protocol. DNA content was measured using a BD Accuri C6 flow cytometer.
All experiments were repeated independently in triplicate. The data were analyzed using the ModFit 3.3 software (BD Bioscience, Sparks, MD, USA).

Migration and invasion assay
For migration assays, 5 × 10 4 cells were suspended in 100 µl of serum-free DMEM and seeded in the top chamber of the transwell (8 μm, 24-well format, Millipore, USA

In vivo animal experiments
Four-to six-week-old female athymic nude mice were purchased from Southwest Hospital (Chongqing, China). All mice were randomized to experimental or control group using a systematic random sampling according the serial number (such as: 1, 3, 5 in experimental group and 2, 4, 6 in control group). All of our animal experimental procedures were carried out in aseptic conditions. To establish the subcutaneously implanted tumor model,

Statistical analysis
Continuous data were tested for normality using the Shapiro-Wilk test at first. Normally distributed continuous data of two groups were expressed as the mean ± standard deviation and compared using a t-test. Nonnormal continuous data or small sample data (n<5) of two groups were expressed as the median (interquartile range) and compared using the Wilcoxon test. Continuous and normal distributed data with three or more groups were tested for normality by evaluating the normality of the residuals using the Shapiro-Wilk test. Variance equality was then tested using Levene's test. If the distribution was normal and variance is equal, data were compared using ANOVA and post-Hoc analyses were performed using Turkey's test. If the distribution was normal and variation is unequal, the ANOVA was corrected using the Welch method and multiple comparisons were performed using the Games-howell's test. If the distribution is nonnormal, Kruskal Wallis test was used as appropriate. Categorical data were expressed as frequencies (percentages) and compared using the Chi-square test. Kaplan-Meier curve, univariate and multivariate Cox regression were performed to analyze survival data. Final screens of covariates and selection of the best-fitted Cox regression model were performed using a stepwise method. Nomogram was developed based on the independent predictors identified by multivariate Cox analysis. For the validation of nomogram, discrimination was evaluated using the C-index (area under curve, AUC) and calibration was evaluated by analyzing observed and expected outcome events followed with Hosmer and Lemeshow Test. P > 0.05 indicated a good performance of discrimination. For in vitro, all experiments were biologically repeated for three times according to the routines of previous studies. All error bar represented standard error of mean.
All tests were two-sided, and P<0.05 was considered statistically significant and expressed as *.