Long noncoding RNA 00976 promotes pancreatic cancer progression through OTUD7B by sponging miR-137 involving EGFR/MAPK pathway
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Accumulation evidence indicates the vital role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including pancreatic cancer (PC). However, the role and the molecular mechanism of long non-coding RNA 00976 is unclear in pancreatic cancer.
In situ hybridization (ISH) and qRT-PCR was performed to investigate the association between linc00976 expression and the clinicopathological characteristics and prognosis of patients with PC. Subsequently, linc00976 over-expression vector and shRNAs were transfected into PC cells to up-regulate or down-regulate linc00976 expression. Loss- and gain-of function assays were performed to investigate the role of linc00976 in proliferation and metastasis in vitro and vivo. ITRAQ, bioinformatic analysis and rescue assay were used to illustrate the ceRNA mechanism network of linc00976/miR-137/OTUD7B and its downstream EGFR/MAPK signaling pathway.
linc00976 expression was overexpressed in PC tissues and cell lines and was positively associated with poorer survival in patients with PC. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, migration and invasion in vivo and in vitro, whereas its overexpression reversed these effects. Based on Itraq results and online database prediction, Ovarian tumor proteases OTUD7B was found as a downstream gene of linc00976, which deubiquitinated EGFR mediates MAPK signaling activation. Furthermore, Bioinformatics analysis and luciferase assays and rescue experiments revealed that linc00976/miR137/OTUD7B established the ceRNA network modulating PC cell proliferation and tumor growth.
The present study demonstrates that linc00976 enhances the proliferation and invasion ability of PC cells by upregulating OTUD7B expression, which was a target of miR-137. Ultimately, OTUD7B mediates EGFR and MAPK signaling pathway, suggesting that linc00976/miR-137/OTUD7B/EGFR axis may act as a potential biomarker and therapeutic target for PC.
KeywordsLinc00976 Pancreatic cancer miR-137 OTUD7B EGFR
Cell counting kit
Long non-coding RNA
Ovarian tumor proteases 7
Real-time quantitative reverse transcription PCR
Pancreatic cancer is currently one of the most severe gastrointestinal malignancies. The symptoms are atypical, advanced disease progression is rapid, and there are no sensitive early diagnostic indicators or effective treatment initiation points in the clinic . The development of pancreatic cancer is a biological process involving multiple genes and multiple steps . Although studies have made great progress on many levels and in many fields, including genes, proteins and cells, much of the malignant biological mechanism of pancreatic cancer remains to be elucidated [3, 4]. Therefore, further study about the progress of pancreatic cancer, especially the search of effect therapy target, is of great significance for improving the curative effect and prognosis of pancreatic cancer. Epigenetic regulation, including noncoding RNAs (which can be divided into long and short noncoding RNAs, according to their length), plays an important role in the complex regulatory network in the process of stem cell differentiation and tumor development, providing new ideas and research directions for the pathogenesis and treatment of tumors . Studies have shown that there are approximately 10,000–20,000 human lncRNAs that contain poorly conserved sequence, are unstable , and occur at low copy numbers. However, they can regulate gene expression at multiple levels of epigenetic regulation, by affecting DNA methylation, histone modification, random chromosome inactivation, coding and other noncoding RNAs, and small peptides, without changing the nuclear acid sequence [7, 8]. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) constitute the majority of regulatory noncoding RNAs [9, 10]. miRNAs are function as vital regulator in multiple Physiological and pathological processes and play a critical role in mRNA stability and translation by post-transcriptionally regulation at 3′ untranslated regions (3′-UTRs) of mRNAs .
Recently, there has gradually been discovered in the study of deubiquitination enzymes in cancer and other disease. Deubiquitination enzyme OTUD7B belongs to the ovarian tumor family of deubiquitination enzymes, and is similar to that of the cysteine proteases, but it has different functions [12, 13, 14, 15]. The mechanism of the association between OTUD7B and the occurrence and development of tumor cells is unclear, and the specific function of its action on cells has not been fully clarified. Studies have shown that OTUD7B can act on EGFR to affect the activation of the EGFR signaling pathway [16, 17].
In this research, Linc00976 was significantly overexpressed in PC and correlative with clinical pathology features in PC patients. Function assays eluted that Linc00976 promoted proliferation and metastasis of PC cells both in vitro and in vivo via up-regulating its host gene OTUD7B involving EGFR deubiquitination and downstream pathway MAPK activation. Further mechanistic studies indicated that Linc00976 exerts oncogene function by competitively interacting with miR-137 and acting as a microRNA sponge, thus inhibiting miR-137 and upregulating OTUD7B expression.
Materials and methods
The cell lines we used in the study were purchased from American Type Culture Collection (ATCC). CFPAC-1, MIA-PaCa-2 and PANC-1 cells were cultured in high-glucose DMEM (Gibco, NY, USA), while BxPC-3, CFPAC-1, ASPC-1, Panc03.27, and Capan-2 cells were cultured in RPMI-1640 (Gibco, NY, USA). The cells used in the research were MIA-PaCa-2 and PANC-1. All the cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
qRT-PCR, western blotting analysis
The characteristics of the primers used for real-time PCR and plasmid sequence
Sequence (5′ - > 3′)
RNA fluorescence in situ hybridization analysis (FISH)
The different groups cells were seeded in the confocal dish at a density of 2 × 105.The paraformaldehyde (4%) was used to fixed the cell at 37 °C for 30 min. According to the manual, the probe was added into the dish in the dark at indicated temperature and time.
After twice washing with PBST, the cells were stained with DAPI and incubated for 5 min at 37 °C. After twice washing with PBS containing Tween-20 (PBST), the cells were blocked using glycerin. Images were captured using a microscopy (ZEISS) at 400× magnification. The regents used in the FISH assay was purchased from Ribobio (Guangzhou, China).
Cell viability analysis
For the Cell Counting Kit-8(CCK-8) assay, according to the instruction (Dojindo, Japan), the different group cells were plated in 96-well plates with a density of 4000 cells. After culturing 0, 24, 48, 72 and 96 h, 200 μl medium contained of CCK-8 regent was fixed and then added into the wells. After incubating for the indicated time, the absorbance value was detected at 450 nm with a microplate reader.
Colony formation assay
Pancreatic cancer cells were inoculated in culture dishes at 1000 cells/plate. They were placed at 37 °C and 5% CO2 for 10 days, and then the medium was discarded. The paraformaldehyde (4%) was used to fixed the cells 15 min, and then crystal violet was used to stain the cells 30 min. The clones were counted after imaging, and statistical analysis was performed on the results of the count.
Cell cycle assay
According to the manufacturer’s protocol of BD (Becton, Dickinson and Company, USA) cell cycle regent. Pancreatic cancer cells were collected after centrifugation in the PBS. After fixing in 75% ethanol for 24 h, the mixture was centrifuged, and the supernatant was discarded. After washing away the residual ethanol with PBS, the cells were stained with 200 μl PBS contained PI (propidium iodide) 10 μ incubated for 5 min at 37 °C. At last, the cell suspension was detected and analyzed in the flow cytometer.
The different groups cells were seeded in the confocal dish at a density of 5 × 105.The paraformaldehyde (4%) was used to fixed the cell 10 min. After washing with PBS, the triton (1%) was used to transparent the cell for 5 min. According to the manual, the cell was incubated with dyeing agent for 30 min in the dark and stained with 1 × Hochest33342 and incubated for 5 min at 37 °C. After twice washing with PBS containing Tween-20 (PBST) the images were captured using a microscopy (ZEISS) at 400× magnification. The regents used in the EDU assay was purchased from Ribobio (Guangzhou, China).
Wound healing assay
The different groups cells were plated in six-well plates and allowed to grow to confluence. The cell wound was scratched a 200 μl pipette tip. The indicated cells were deprived of serum for 48 h, treated with mitomycin-C. Photos of migration were captured at 0 and 48 h after scraping.
The indicated cells were deprived of serum were seeded in the upper Transwell chamber, and the complete medium contained 10% FBS was added into the bottom chamber. After cultured in the normal condition for 24 h, the cell in the upper chamber would wipe out by cotton swab, and the migrated cells were fixed with paraformaldehyde and stained with crystal violet. The transwell invasion experiment required matrigel to be coated in the upper chamber before cell seeding.
In vivo assay
For the proliferation assays, PANC-1 cells of linc00976 knockdown, linc00976 overexpressed and negative control were subcutaneously injected into BALB/c nude mice. The mice were weighed every week and euthanized in 7 weeks after injection. The tumors were dissected and weighed (4–6 weeks old, female, n = 6 per group).
For the metastasis assays, spleen capsule injected liver metastasis model was used to illustrate the different groups PC cells metastasis ability. A total of 1× 106 cells in 150 μL PBS were injected into the spleen. of nude mice. The mice were weighed every week and euthanized 12 weeks after injected. The liver tissues were harbested and photo. IHC was used to detected the metastasis loci in the liver.
All conditions and procedures for the animal experiments were approved by the Animal Care Committee of WuHan University.
ISH (in situ hybridization)
Experiments were performed with the RNA ISH Kit (BersinBi, Beijing, China) according to the manufacturer’s instructions. Briefly, PC cells were fixed in 4% paraformaldehyde for 20 min, washed with distilled water, treated with pepsin (1% in 10 mM HCl), and incubated with 20 nM ISH probe in hybridization buffer (100 mg/mL dextran sulfate, 10% formamide in 2X SSC) at 90 °C for 3 min. Hybridization was performed at 37 °C for 18 h, followed by a wash step and an incubation with digoxin antibodies for 1 h. Finally, DAB was applied to the samples to detect the signals. The ISH images were captured using an Aperio ImageScope system. Each sample was examined by two pathology specialists who were blinded to the diagnoses and outcomes. Staining intensities and percentages of positive cells were recorded. The relative expression of each sample was calculated as the product of the expression intensity and percentage of positivity.
iTRAQ (isobaric tags for relative and absolute quantitation)
Lysis buffer containing 8 M carbamide, 30 mM HEPES, 1 mM PMSF, 2 mM EDTA, and 10 mM DTTwas added to cells and centrifuged at 20,000×g and 4 °C for 30 min to collect the supernatant. Protein concentrations were determined using the Bradford method. For digestion, each sample was reduced with 10 mM dithiothreitol for 60 min at 56 °C and alkylated using 55 mM iodoacetamide for 60 min at room temperature in the dark. The proteins were digested with 1 μg/μl trypsin at a weight ratio of 1:30 (trypsin:protein) overnight at 37 °C. Tryptic peptides were lyophilized and resuspended in 0.5 M Triethylammonium bicarbonate. Following trypsin digestion, each iTRAQ reagent was dissolved in isopropanol and added to the appropriate peptide mixture. A total of 3 biological replicates of the knockdown of Linc00976 group were labelled with iTRAQ tags 113, 114 and 115, respectively. Similarly, 3 biological replicates of the negative control group were labelled with iTRAQ tags 118, 117 and 116, respectively. The labelled peptide mixtures were incubated at room temperature for 2 h and obtained by vacuum-drying. Then, peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, CA, USA), and analyzed using a mass spectrometer (TripleTOF 6600; SCIEX, Framingham, MA, USA). The instrument parameters were as follows: Mass range, 350–2000 m/z for time-of-flight mass spectrometry (TOF MS) and 100–1500 m/z for TOF MS/MS; dynamic exclusion, 12.0 s. Mass spectra raw data were analyzed with ProteinPilotä software (version 5.0; SCIEX). Peptides were identified using a false discovery rate of < 1%. Proteins were considered differentially expressed if they differed in at least 2 of the 3 biological replicates. The criteria of P < 0.05 and fold change (ET/LPS) > 1.2 were selected to identify up- and down-regulated proteins.
Reagents and transfections
We purchased siRNAs from RiboBio (Guangzhou, China). We purchased human Linc00976 over-expressing, knocking-down and negative-control lentiviruses from Genechem (Shanghai, China). Linc00976 mutant plasmids were purchased from Genechem (Shanghai, China). All transfections were carried out according to manufacturers’ instructions.
Luciferase reporter assay
PC cells were plated into the 6-well plates at dentist of 5 × 105.Then the corresponding psiCHECK™-2 vector and miR-137 mimics were co-transfected into the indicated well. According to the manufacturer’s protocol (Ribobio, Guangzhou), the lysate was used to measure the firefly and Renilla luciferase activities. Then, the relative luciferase activity was normalized to the firefly luciferase internal control.
PC cells were lysed in lysis buffer to be extracted, and then the BCA method was used to quantity the cell lysates concentration. Cell lysates were incubated in primary antibody at 4 °C overnight and Protein A Sepharose beads was added into lysates for 4 h. The beads were boiled in Loading buffer (2×) after pre-cooled PBS washed. The protein was detected with western blot or Mass spectrometric analysis.
Results were eluted as mean ± SD. Statistical analysis was performed using GraphPad Prism 7.0 (San Diego, CA, USA). Chi-square test was used to analyze the correlation between linc00976 expression levels and clinicopathological features in PC. Kaplan–Meier curve plots method was used to analyze overall free survival rate. Student’s t-test was used for statistical comparison between different groups. *P < 0.05, **P < 0.05.
The expression of Linc00976 and its correlation with the clinical parameters in patients with PC
Association of Linc00976 expression with clinicopathological features from PC patients
Tumor size (cm)
Lymph node metastasis
I and II
III and IV
Blood vessel invasion
Linc00976 promotes the proliferation of pancreatic cancer cells in vitro
Linc00976 promotes the migration and invasion of pancreatic cancer cells in vitro
Linc00976 promotes PC cell proliferation, migration and invasion in vivo
Linc00976 promotes proliferation, migration and invasion by targeting OTUD7B in PC
OTUD7B deubiquitinated EGFR and facilitates downstream pathway MAPK
Linc00976-miR-137/OTUD7B axis on the EGFR downstream MAPK pathway
Recently, lncRNAs have been known as the focus in the field of cancer research involved in the biological and pathological progress of proliferation, metastasis, tumor metabolism and chemoresistance . In order to identify whether Linc00976 was abnormally expressed in PC and its effect on prognosis, ISH and qRT-PCR was used to verify the Linc00976 express level in pancreatic adjacent/tumor tissues and normal/cancer cell lines, and the over survival analysis was used to evaluate prognosis risk. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, cell cycle progression, migration and invasion in vivo and in vitro. These findings illustrated that linc00976 might act as an oncogene and play an important role in the progression of PC.
Furthermore, we used iTRAQ analysis to determine the host gene of linc00976. Function studies revealed that OTUD7B acts as an oncogene promoting proliferation and metastasis in PC cells. OTUD7B is a member of the OTU family of deubiquitinases, which is associated with the tumor rapid progress and deterioration. The main function of ubiquitin proteases in the ubiquitin proteasome system is to remove ubiquitin protein substrates by chemically cleaving either the linkage between ubiquitin molecules in the chain, or between ubiquitin molecules and the substrate. The ubiquitin molecules released from the substrates are involved in the regulation of proteins in the cell, resulting in a feedback loop. In addition, we found that linc00976 overexpress promoted proliferation and metastasis, while the effect was reversed by OTUD7B downregulated. These findings indicated that OTUD7B was the function host gene of linc00976 mediating PC tumorigenesis.
Research has repeatedly shown that OTUD7B is overexpressed and hold the vital role in in various types of cancers progress including hepatocellular carcinoma [21, 22] lung adenocarcinoma [23, 24], Breast , squamous cell carcinoma . In addition, OTUD7B as a crucial immune regulator of NF-KB pathway involving Zap70 and TRAF3 deubiquitination and stabilization [27, 28]. However, the mechanism deubiquitinating enzyme OTUD7B promoted PC progress is not clear. Based on the results of Mass spectrometry of Immunoprecipitation proteins interacting with OTUD7B, EGFR may be the substrate in the OTD7B mediated PC tumorigenesis. As the previous report that OTUD7B can act on EGFR deubiquitination and endocytosis to affect the activation of the EGFR signaling pathway. The protein stability and ubiquitination assay eluted OTUD7B may inhibit EGFR ubiquitination, promote the EGFR protein stability. Accumulating evidence indicates that MAPK pathway contributes to the kinds of tumor physiological and biochemical progress, which is the substrate of activated EGFR. Therefore, we hypothesized that OTUD7B may deubiquitinate EGFR and activate downstream pathway MAPK. In addition to defining the regulation ship between OTUD7B and the proliferation capacity of PC, we found that the EGFR/MAPK pathway is involved in this regulation. Preliminary evidence on the role of activated MAPK pathway as mediators of EGFR promoted cancer tumorigenesis. Therefore, we found that Linc00976 promoted proliferation and invasion via MAPK pathway, which is activated by OTUD7B mediated EGFR deubiquitination and stability.
However, the mechanism of linc00976 regulating OTUD7B expression was still not clear. Currently, numerous lncRNAs that serve as endogenous miRNA sponges have been shown to be involved in the initiation and development of PC . Long non-coding RNA XIST exerts oncogenic functions in pancreatic cancer via miR-34a-5p .
The lncRNA UCA1 promotes ovarian cancer cell chemoresistance by sponging miR-143 to activate FOSL2 Signaling Pathway . FISH assay and nuclear RNA extraction was shown that linc00976 mainly locate in the cytoplasm. According to the prediction of bioinformatics software in this study, there were combination sites between linc00976 and miR-137.Our previous research revealed that miR-137 was a tumor pressor in pancreatic cancer growth, invasion and stemness . We also found in this study, there were combination sites between Linc00976 and miR-137. Dual-luciferase reporter gene assay confirmed the regulation of Linc00976 on miR-137 and proved Linc00976 could directly bind to miR-137. At the same time, bioinformatics softwares also predicted that there were combination sites between miR-137 and OTUD7B. Dual-luciferase reporter gene assay proved miR-137 could directly bind to OTUD7B. And we found miR-137 express level was negative correlative with Linc00976 and OTUD7B expression in PC, which was consistent with the ceRNA regulation network in Linc00976/ miR-137/ OTUD7B. Further mechanistic studies indicated that Linc00976 exerts oncogene function by competitively interacting with miR-137 and acting as a microRNA sponge, thus inhibiting miR-137 and upregulating OTUD7B expression.
We acknowledge the generous support of the department of Biliary-Pancreatic Surgery, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
JJ and TC contributed to the experiment design, and data analysis. SL, ZH contributed to the experiment implementation, XG contributed to manuscript draft and data analysis. All authors read and approved the final manuscript.
This study was funded by The National Natural Science Foundation of China (No. 81572429 and No. 81871965 awarded to JJ; No. 81602475 awarded to XG).
Ethics approval and consent to participate
This study was reviewed and approved by the Medical Ethics Committee of Renmin hospital of Wuhan university, Wuhan, China. All animal studies were approved by the Institutional Animal Care and Use Committee of Committee of Renmin hospital of Wuhan university.
Consent for publication
All authors have agreed to publish this manuscript.
The authors declare that they have no competing interests.
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