CircPTPRA promotes the progression of pancreatic ductal adenocarcinoma via the miR-140-5p/LMNB1 axis

doi:10.21203/rs.3.rs-1917234/v1

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Research Article

CircPTPRA promotes the progression of pancreatic ductal adenocarcinoma via the miR-140-5p/LMNB1 axis

https://doi.org/10.21203/rs.3.rs-1917234/v1

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Background

Growing evidence suggests that circular RNAs (circRNAs) are important factors in cancer progression. Nevertheless, the role of circRNAs in the progression of pancreatic ductal adenocarcinoma (PDAC) remains unclear.

Methods

CircPTPRA was identified based on our previous circRNA array data analysis. Wound healing, transwell and EdU assays were performed to investigate the effect of circPTPRA on the migration, invasion and proliferation of PDAC cells in vitro. RNA pull-down, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were conducted to verify the binding of circPTPRA with miR-140-5p. Subcutaneous xenograft models were constructed for in vivo experiments.

Results

CircPTPRA was significantly upregulated in PDAC tissues and cells compared to normal controls. Moreover, circPTPRA overexpression was positively correlated with lymph node invasion and worse prognosis in PDAC patients. In addition, overexpression of circPTPRA promoted PDAC migration, invasion, proliferation and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, circPTPRA upregulates LaminB1 (LMNB1) expression by sponging miR-140-5p and ultimately promots the progression of PDAC.

Conclusions

This study revealed that circPTPRA plays an important role in the progression of PDAC by sponging miR-140-5p. It can be explored as a potential prognostic marker and therapeutic target for PDAC.

PDAC is a malignant tumor with a high mortality rate. The increasing morbidity and mortality rates of PDAC present a serious health burden worldwide[1, 2]. According to the latest research from the USA, PDAC ranks 10th (males) or 8th (females) among cancers in terms of incidence, but ranks 4th in terms of mortality in both males and females[3]. A study from China came to the same conclusion: The mortality rate of PDAC ranked higher than the morbidity rate[4]. The early symptoms of PDAC are ambiguous, and local invasion or distant metastasis are prone to occur[5]. Most patients miss the opportunity for surgery because they are already in the advanced stage of the disease when they are diagnosed[6]. Compared with other malignant tumors, PDAC is not effectively treated with combined therapy, including immunotherapy and targeted therapy, and the 5-year survival rate is still less than 10%[2, 3]. Therefore, it is necessary to further explore the molecular network of PDAC to find more reliable biomarkers and potential therapeutic targets.

CircRNAs, as a special type of RNA, were first found in plant viruses[7]. CircRNAs belong to the long-chain noncoding RNA family and have a covalent closed-loop structure produced by the back-splicing of pre-mRNA transcripts[8]. Unlike linear RNAs, circRNAs lack 5' caps and 3' poly(A) tails, so they are resistant to exonuclease and are stably present in eukaryotic cells[9]. The cellular roles of circRNAs include modulating transcription, regulating splicing, sponging microRNAs (miRNAs), forming circular RNA‒protein complexes, and being translated into proteins or peptides[9]. In recent years, the roles of circRNAs in tumors have received much attention[10], especially certain circRNAs that play a key role in the process of PDAC[11]. For instance, the circEYA3/miR-1294/c-Myc axis promotes the progression of PDAC by inducing energy production[12], and the circNEIL3 regulatory loop promotes PDAC progression via miRNA sponging and A-to-I RNA editing[13].

miRNAs are approximately 18–22 nucleotides long and have binding sequences that are partially complementary to the target mRNA transcripts, known as miRNA response elements (MREs)[14]. As endogenous noncoding RNAs, miRNAs directly bind to the 3'UTR of mRNAs through MREs to form RNA-induced silencing complexes (RISCs), which regulate the expression of target genes at the posttranscriptional level[15]. However, in addition to the traditional microRNA/RNA function, there is an opposite RNA/microRNA function known as the competing endogenous RNA (ceRNA) mechanism, which was first reported in 2011. CircRNAs, lncRNAs and pseudogenes block the inhibitory effect of miRNAs on mRNAs by competitively binding to MREs of miRNAs[16]. Here is a classic example, circular RNA-ciRS-7 contains more than 70 selectively retained miRNA target sites and inhibits miR-7 activity by binding to miR-7 and Argonaute (AGO) protein, resulting in increased levels of miR-7 targets[17]. Therefore, the ceRNA mechanism regulated by circRNAs enriches the RNA interaction network and is also a hot topic in the field of cancer research[18]. Interestingly, it will be meaningful to explore the role of certain circRNAs in PDAC. Although few studies have reported that dysregulated circRNAs affect PDAC progression, more work is needed to uncover the underlying mechanisms of unknown circRNAs in PDAC.

In this study, we identified a novel circRNA (circPTPRA, also known as hsa_circ_0006117) by Arraystar Human CircRNA Array Analysis and quantitative real-time PCR (qRT‒PCR). Using the circRNA database for sequence alignment, we found that circPTPRA was generated from exon 8 and exon 9 of the PTPRA gene. Functionally, circPTPRA promotes PDAC migration, invasion and proliferation in vitro and in vivo. Mechanistically, circPTPRA sponges miR-140-5p to upregulate LMNB1 expression and promotes PDAC progression. Clinically, circPTPRA is highly expressed in PDAC and is significantly associated with poor prognosis and regional lymph node invasion. In summary, our study demonstrated that the circPTPRA/miR-140-5p/LMNB1 axis plays a key role in PDAC progression, and it is worth mentioning that circPTPRA may serve as a potential biomarker and therapeutic target in PDAC.

PDAC tissue specimens

Tissue samples from PDAC patients were provided by Southwest Hospital, Army Medical University [93 PDAC and 31 normal adjacent tissue (NAT) samples], Wuhan Tongji Hospital (34 PDAC and 27 NAT samples) and Soochow University (61 PDAC samples). Tissue microarrays (TMAs) were made by Shanghai Outdo Biotech Company (Shanghai, China). These PDAC patients were evaluated for clinical staging according to the American Joint Commission on Cancer (AJCC) 8th edition guidelines. None of the PDAC patients received chemotherapy or/and radiotherapy before surgery. All patients were followed up regularly until August 2021. Informed consent was obtained from all patients prior to obtaining tissue samples, and all procedures were approved by the designated Hospital Ethics Committee.

CircRNA array data

To determine the abnormal expression of circRNAs in PDAC, we previously used Arraystar Human CircRNA Array Analysis to measure circRNA expression in 20 PDAC tissues and paired NATs. These data were published in the NCBI GEO datasets on 07 Oct 2016 (GSE79634)[19]. At the same time, we analyzed the GEO dataset (GSE69362) from other units[20]. The GSE69362 dataset contains information for 6 PDAC tissues and paired NATs. Fold change ≥ 2 and p values ≤ 0.05 were set as thresholds to identify the significant differentially expressed circRNAs (DEcircRNAs).

Cell lines and cell culture

The human normal pancreatic ductal epithelial cell line (HPDE6-C7) was purchased from Genio Biotechnology Co., Ltd. (Guangzhou, China). The human PDAC cell lines AsPC-1, BxPC-3, CFPAC-1, PANC-1 and SW1990 were purchased from Genechem Co., Ltd. (Shanghai, China). These cells were cultured in complete growth medium (Gibco, USA) containing 10% fetal bovine serum (HyClone, USA) under humidified conditions with 95% air, 5% CO₂ at 37°C according to the manufacturer's instructions.

Plasmid and siRNA transfection and lentiviral infection

To construct the overexpression plasmids of circPTPRA, LMNB1, EIF4A3 and FUS, synthetic human circPTPRA cDNA was cloned into the vector pLC5-ciR by Geneseed Biotech Co., Ltd. (Guangzhou, China), synthetic human LMNB1, EIF4A3 and FUS cDNAs were cloned into the vector pEX-3 by GenePharma (Shanghai, China), and the empty plasmid was used as a control. The siRNA sequences of circPTPRA, LMNB1, EIF4A3 and FUS, as well as mimics and inhibitor of miR-140-5p, were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China). Lipofectamine™ 3000 (Invitrogen, USA) and Opti-MEM™ (Gibco, USA) were used as transient transfection reagents according to the manufacturer's instructions. Total RNA was extracted 48 h after transfection. The sequences of siRNAs, mimics and inhibitor are shown in Additional file 1 Table S1.

The overexpression and knockdown lentiviruses of circPTPRA and LMNB1 were constructed by Genechem Co., Ltd. (Shanghai, China). After sequencing to verify the correctness of the sequence, lentiviral infection was performed according to the manufacturer's instructions. Stable cells were selected by culturing in medium containing 5 µg/ml puromycin (Beyotime, China), and the expression of circPTPRA and LMNB1 was confirmed by qRT‒PCR.

RNA extraction and qRT‒PCR analysis

According to the manufacturer's instructions, total RNA was extracted from PDAC cells by using the Ultrapure RNA Kit (CWBio, China), and then the RNA concentration was measured by a NanoPhotometer-N50 (Implen, Germany). One microgram of total RNA in a final volume of 20 µl was used for reverse transcription (RT) with PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan) and T100™ Thermal cycler (Bio-Rad, USA). qRT‒PCR was performed on a CFX96 PCR system (Bio-Rad, USA) using TB Green Premix Ex Taq II (Takara, Japan) to detect the expression of the relevant genes following the manufacturer's protocol. The human GAPDH gene was used as a control, and the expression of related genes was calculated by the 2^−△△CTmethod. Three replicate wells were set up for each qRT‒PCR reaction, and three independent replicate experiments were performed. All primer sequences used for the qRT‒PCR assay are summarized in Additional file 1 Table S1.

RNase R and actinomycin D treatment

Total RNA was treated with Ribonuclease R (Geneseed, China). Ten micrograms of total RNA was obtained and divided into two parts (5 µg/group); the samples were incubated at 37°C for 30 min with or without Ribonuclease R (3 U/µg), and then the expression of circPTPRA and PTPRA mRNA was measured by qRT‒PCR.

Actinomycin D (2 µg/ml, CST, USA) was added to the complete medium and incubated with the PDAC cells. Then, total RNA was extracted after 0, 3, 6, 12, and 24 h, respectively. Finally, the expression of circPTPRA and PTPRA mRNA was measured by qRT‒PCR.

Western blotting

Proteins were extracted from PDAC cells with RIPA buffer mixed with protease and phosphatase inhibitors (Beyotime, China), and the protein concentration was determined with a BCA protein concentration assay kit (Beyotime, China). Appropriate amounts of protein were electrophoresed on a SurePAGE™, Bis-Tris, 10×8, 4–20% gel (GenScript, China).Then, the protein was transferred to a PVDF membrane (Millipore, USA), and after blocking in 5% skim milk for 1 h, the PVDF membrane was soaked in primary antibody at 4°C overnight. Primary antibodies against LMNB1 (1/1000, ab229025, Abcam, UK), EIF4A3 (1/1000, ab180573, Abcam, UK), FUS (1/1000, ab243880, Abcam, UK), GAPDH (1/1000, #5174, CST, USA), E-Cadherin (1/1000, #14472, CST, USA), N-Cadherin (1/1000, #13116, CST, USA), Snail (1/1000, #3879, CST, USA), β-Catenin (1/1000, #8480, CST, USA), Vimentin (1/1000, #5741, CST, USA). The next day, the PVDF membrane was washed with TBST (1/1000 Tween-20, Solarbio, China) buffer 3 times for 10 min each time. Next, the PVDF membrane was incubated with the specific secondary antibody for 1 h at room temperature, and then the PVDF membrane was washed with TBST buffer 3 times for 10 min each time. Finally, protein bands were visually detected by a ChemiDoc™ XRS+ (Bio-Rad, USA) and ECL detection system (Bioground, China), and the GAPDH band was used as a control.

FISH and miRNA scope assay in PDAC cells and TMAs

The Cy3-labeled circPTPRA probe and FAM-labeled miR-140-5p probe were designed and synthesized by GenePharma (Shanghai, China). On the first day, the PDAC cells were cultured in 15mm confocal glass-bottom dishes (NEST, China). On the second day, the FISH experiment was carried out according to the instructions of the RNA FISH kit (GenePharma, China), and the probes and PDAC cells were coincubated at 37℃ overnight. On the third day, photographs were taken by confocal microscopy (Leica Microsystems, Germany). All probe sequences used for the FISH assay are summarized in Additional file 1 Table S1.

The expression of circPTPRA was detected by FISH experiments with TMAs. The average density (average density = cumulative optical density IOD value/positive pixel area) was used to evaluate the expression of circPTPRA. The higher the average density was, the higher the expression of circPTPRA. Next, according to the median value of the mean density, the samples were divided into a circPTPRA low expression group and a circPTPRA high expression group.

Moreover, we used a miR-140-5p special probe (ACD, USA) to carry out miRNA scope experiments. The expression of miR-140-5p in the TMAs was detected according to the instructions of the miRNA scope™ HD detection kit (ACD, USA). The mean density (mean density = cumulative optical density IOD value/positive pixel area) was used to evaluate the expression of miR-140-5p. The higher the mean density was, the higher the expression of miR-140-5p. Finally, according to the median value of the mean density, the samples were divided into a miR-140-5p low expression group and a miR-140-5p high expression group.

RIP

We performed RIP experiments according to the protocol of the Magna RIP kit (Millipore, USA). On the first day, the PDAC cells were lysed with RIP lysis buffer. Then, the samples were divided into two groups, and 50 µL of magnetic beads was added to each group, followed by incubation with 5 µg of anti-Ago2 (ab186733, Abcam, UK) and anti-IgG (Millipore, USA), respectively, at 4°C overnight. The next day, the magnetic bead-bound complexes were immobilized on a magnetic stand, and unbound material was washed away, followed by precipitation of RNA with absolute ethanol at -80°C overnight. On the third day, RNA was extracted and assessed by qRT‒PCR.

CircRNA pull-down assay

A biotin-labeled circPTPRA probe was designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China), and cell lysates were prepared with IP Lysis Buffer (Beyotime, China). The RNA pull-down assay was performed according to the instructions of the Pierce^™ Magnetic RNA‒Protein Pull-Down Kit (Thermo Scientific, USA). The 50 pmol biotin-labeled circPTPRA probe was gently mixed with 50 µL magnetic beads with a pipette; then, the probe and beads were coincubated for 15–30 min at room temperature with agitation. Cell lysates were added to the circPTPRA probe-bound magnetic beads and mixed by pipetting or gentle vortexing, and then the mixtures were coincubated at 4°C for 30–60 min with agitation or rotation. Finally, the magnetic bead-bound complexes were immobilized on a magnetic holder, the unbound material was washed away, and the RNA was extracted and assessed by qRT‒PCR. The probe sequences used for the RNA pull-down assay are summarized in Additional file 1 Table S1.

Dual-luciferase reporter assay

The binding sites of circPTPRA, LMNB1 and miR-140-5p were predicted, and wild-type and mutant-type circPTPRA and LMNB1 fragments were constructed and cloned into the dual-luciferase reporter vector by GenePharma (Shanghai, China). Dual-luciferase reporter plasmids (WT or Mut-circPTPRA/LMNB1) were cotransfected into PDAC cells with miR-140-5p mimics or miR-140-5p mimics NC, respectively. After 48 h, Firefly and Renilla luciferase activities were assayed according to the Dual-Glo® Luciferase Assay System (Promega, USA).

Nude mouse subcutaneous tumor model

The animal experimental model was approved by the Animal Experiment Ethics Committee of Chongqing Medical University, and all procedures were in compliance with agency regulations. Five nude mice (4–5 weeks, Ensiweier, China) in each group were used to establish a subcutaneous tumorigenesis model. The PDAC cell suspensions (0.1 ml) contained 5 × 10⁶ cells, and the cell suspensions and a high concentration of Matrigel (Corning, USA) were mixed 1:1. Next, the cell suspensions and the Matrigel mixture were randomly injected into the axilla of the right upper limb of nude mice. When subcutaneous tumors formed, they were measured weekly with calipers. The tumor volume = (length × width²)/2. Nude mice were sacrificed 30 days after PDAC cells were inoculated, and then subcutaneous tumor tissues were obtained. The volume and weight of the tumors were measured, and the tumors were fixed with 4% paraformaldehyde (Biosharp, China) for immunohistochemistry (IHC) to determine the H-score as follows: H-score = ∑(PI×I) = (percentage of weakly stained cells ×1) + (percentage of moderately stained cells ×2) + (percentage of strongly stained cells ×3). In the formula, I represents the classification of positive cells: no staining, 0 points; weak positive (light yellow), 1 point; medium positive (tan), 2 points; and strong positive (tan), 3 points. PI indicates the percentage of positive cells. The higher the H-score value is, the stronger the comprehensive positive intensity.

Statistical analysis

GraphPad Prism version 7.0 (GraphPad Software, USA) and SPSS version 23.0 (SPSS, USA) were used for the statistical analysis. In this study, the quantitative data were expressed as the mean ± standard deviation. For univariate analysis: if the data followed a normal distribution, the differences between the two samples were analyzed by Student’s t test, while one-way analysis of variance (ANOVA) was used for multiple groups; otherwise, the nonparametric Mann‒Whitney test was used. Two-way ANOVA was used for multivariate analysis. The Kaplan‒Meier method was used for survival analysis, and the log-rank test and the Gehan-Breslow-Wilcoxon test were used for the comparison of survival curves. According to test sensitivity, the log-rank test was applied to assess the long-term survival effect, and the Gehan-Breslow-Wilcoxon test was was applied to assess the short-term survival effect. We utilized Pearson's correlation analysis to examine the correlation between two variables. We used p < 0.05 as an indicator of statistical significance.

Identification and characterization of the oncogene circPTPRA

Exploring the expression profile of circRNAs in PDAC can provide more reliable biomarkers and new therapeutic targets. Our team previously used Arraystar Human CircRNA Array Analysis to measure circRNA expression in 20 PDAC tissues and paired NATs (Fig. 1A). These data were deposited in the NCBI GEO on 07 Oct 2016 (GSE79634)[19]. With the cutoff criteria of fold change ≥ 2 and p values ≤ 0.05, we found 289 DEcircRNAs, of which 128 circRNAs were significantly upregulated and 161 circRNAs were significantly downregulated in PDAC tissues versus NATs. (Fig. 1B). Similar data were reported by Shibin Qu et al, named GSE69362[20]. The GSE69362 samples consisted of 6 PDAC tissues and paired NATs. We downloaded and analyzed the data of GSE69362 from the NCBI GEO (https://www.ncbi.nlm.nih.gov/). With the cutoff criteria of fold change ≥ 2 and p values ≤ 0.05, there were 111 significantly upregulated circRNAs in GSE69362. Finally, combined with the data of GSE79634 and GSE69362, we identified 20 highly expressed candidate circRNAs (Fig. 1C). Among them, circPTPRA was highly expressed in PDAC cells compared with HPDE6-C7 cells (Fig. 1D). Therefore, circPTPRA was chosen for further study. Through the annotations of circBase (http://www.circbase.org/)[21] and circBank (http://www.circbank.cn/)[22], we know that circPTPRA is also called hsa_circPTPRA_009 or hsa_circ_0006117. CircPTPRA is formed by the circularization of exons 8 and 9 of the PTPRA gene (chr20: 2944917–2945848 strand: +) and has a length of 421 bp. Sanger sequencing verified the backsplicing site of circPTPRA (Fig. 1E). Actinomycin D and RNase R treatment showed that compared with PTPRA mRNA, circPTPRA was more stable and had a longer half-life (Fig. 1F-H). Moreover, the existence of circPTPRA was also confirmed by nucleic acid electrophoresis experiments, and circPTPRA could be amplified by divergent primers from cDNA but not from gDNA in AsPC-1 and PANC-1 cells, respectively (Fig. 1I). In addition, FISH experiments showed that Cy3-labeled circPTPRA was mainly enriched in the cytoplasm of AsPC-1 and PANC-1 cells (Fig. 1J). Taken together, these results confirmed the stability of circPTPRA, which is upregulated and prominently localized in the cytoplasm of PDAC cells and may contribute to disease progression.

CircPTPRA promotes the progression of PDAC in vitro and in vivo

To verify the function of circPTPRA, we designed two specific short interfering RNAs (siRNAs) targeting the back-splice site of circPTPRA, named si-circPTPRA#1 and si-circPTPRA#2. We also constructed a circPTPRA overexpression plasmid (OE-circPTPRA). The expression of PTPRA mRNA was not affected after transfection with si-circPTPRA or OE-circPTPRA in AsPC-1 and PANC-1 cells, respectively. Furthermore, qRT‒PCR was used to verify the efficiency of circPTPRA interference and overexpression in AsPC-1 and PANC-1 cells (Fig. 2A-C). The effects of circPTPRA on cell migration and invasion were verified by wound healing and transwell assays. The results showed that downregulation of circPTPRA in AsPC-1 cells could significantly inhibit the migration and invasion ability of cells, whereas overexpression of circPTPRA in PANC-1 cells could significantly promote the migration and invasion ability of cells (Fig. 2D-G). EdU assays showed that the downregulation of circPTPRA in AsPC-1 cells inhibited proliferation, while overexpression of circPTPRA in PANC-1 cells promoted proliferation (Fig. 2H-I). Additionally, the sequences of si-circPTPRA#1 and OE-circPTPRA were cloned into lentiviruses, and stable cells were constructed (PANC-1-sh-circPTPRA#1, PANC-1-Vector, PANC-1-OE-circPTPRA). In nude mice, overexpression of circPTPRA promoted the growth of subcutaneous tumors (Fig. 2J-L). IHC results showed that overexpression of circPTPRA induced the expression of Ki-67 (Fig. 2M). In summary, these results confirmed the role of circPTPRA in promoting PDAC progression.

CircPTPRA sponges miR-140-5p in PDAC cells

It has been reported that the regulation of downstream gene expression by sponging miRNAs is one of the main functions of circRNAs, which is known as the ceRNA mechanism[16]. Previous results showed that circPTPRA was mainly enriched in the cytoplasm (Fig. 1J), which suggested that circPTPRA may have a ceRNA mechanism. The results of the RIP assay showed that circPTPRA could be significantly enriched by anti-AGO2, which indirectly confirmed our speculation (Fig. 3A). The circRNA-microRNA interaction was predicted with Arraystar's home-made miRNA target prediction software based on TargetScan (https://www.targetscan.org/vert_80/) and miRanda (http://www.microrna.org/microrna/home.do)[23, 24]. We found five miRNAs (miR-140-5p, miR-152-5p, miR-582-3p, miR-26b-3p, miR-96-5p) that may directly bind to circPTPRA (Fig. 3B). To verify the binding of the above five miRNAs and circPTPRA, we designed a biotin-labeled circPTPRA probe for the RNA pull-down assay. First, to verify the specificity of the probe, the expression level of circPTPRA in the RNA pull-down product was detected by qRT‒PCR. The results showed that, compared with the random sequence NC probe, circPTPRA was mainly captured by the biotin probe in AsPC-1 and PANC-1 cells (Fig. 3C). Moreover, among the five potential target miRNAs, only miR-140-5p was significantly enriched by the biotin-labeled circPTPRA probe in both AsPC-1 and PANC-1 cells (Fig. 3D-E). The RNAhybrid database (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/)[25] was used to predict the binding sequences between circPTPRA and miR-140-5p. The 3D structure showed that there are two binding sites between circPTPRA and miR-140-5p (Fig. 3F). The prediction results showed that miR-140-5p may bind to the “AACCACT” base sequence on circPTPRA. Then, we mutated the predicted binding sites of circPTPRA, and the wild-type and mutant-type circPTPRA fragments were constructed and cloned into the dual-luciferase reporter vector, respectively (Fig. 3G). In AsPC-1 and PANC-1 cells, the relative intensity of firefly luciferase was significantly decreased after cotransfection with wild-type circPTPRA vector and miR-140-5p mimics compared to miR-140-5p NC. However, when cotransfected with mutant-type circPTPRA vector, miR-140-5p mimics or miR-140-5p NC did not affect the relative intensity of firefly luciferase (Fig. 3H-I). The results indicate that circPTPRA can specifically bind miR-140-5p in the "seed" region. FISH experiments showed that Cy3-labeled circPTPRA and FAM-labeled miR-140-5p were colocalized in the cytoplasm (Fig. 3J), which also verified that circPTPRA and miR-140-5p can bind to each other. Collectively, these data demonstrated that circPTPRA directly sponge miR-140-5p.

CircPTPRA promotes PDAC progression by sponging miR-140-5p

According to the literature, miR-140-5p acts as a tumor suppressor gene to inhibit PDAC progression[26]. In this article, we found that the expression of miR-140-5p was decreased in PDAC cells (Additional file 2 Fig. S1 A). Furthermore, we used the RNA scope assay to detect the expression of miR-140-5p in PDAC tissues. The results showed that miR-140-5p was significantly decreased in PDAC, which was positively correlated with the prognosis of patients (Additional file 2 Fig. S1 B-C). To demonstrate whether circPTPRA promotes PDAC progression by sponging miR-140-5p, we performed rescue experiments in vitro. First, we used qRT‒PCR to measure the efficiency of miR-140-5p mimics and inhibitor in AsPC-1 and PANC-1 cells, respectively (Fig. 4A). The wound healing, transwell and EdU experimental results showed that interfering with miR-140-5p in AsPC-1 cells significantly promoted cell migration, invasion, proliferation and reversed the tumor suppressor effect of si-circPTPRA#1 (Fig. 4B-C, F-G, J-K). On the other hand, upregulation of miR-140-5p significantly inhibited cell migration, invasion, proliferation and reversed the oncogenic effect of OE-circPTPRA in PANC-1 cells (Fig. 4D-E, H-I, L-M). Overall, these data suggested that miR-140-5p can act as a downstream target gene for circPTPRA and exert anticancer effects on PDAC cells.

CircPTPRA promotes LMNB1 expression by competitively sponging miR-140-5p

To search for potential downstream target genes of circPTPRA, we used stable sh-circPTPRA#1-transfected cells for whole transcriptome sequencing. In PANC-1 cells, the interference efficiency of sh-circPTPRA#1 was verified by qRT‒PCR (Fig. 5A). With the cutoff criteria of fold change ≥ 2 and Q-value ≤ 0.05, 332 genes were significantly downregulated, while 173 genes were significantly upregulated in the experimental group compared with the sh-NC group (Fig. 5B). Based on the data of TargetScan and starBase v2.0 (https://starbase.sysu.edu.cn/index.php)[24, 27], we searched for mRNAs that may be regulated by miR-140-5p. miRNAs usually interact with the 3'UTR of target genes and downregulate their expression. We combined data on downregulated genes from RNA-seq with data from TargetScan and starBase v2.0[24, 27] to screen out 11 candidate genes that may be potential downstream targets of circPTPRA and miR-140-5p (Fig. 5C). Subsequently, the above 11 candidate genes were analyzed by the GEPIA (http://gepia.cancer-pku.cn/) and OncoLnc (http://www.oncolnc.org/) databases. The results showed that only LMNB1 was abnormally highly expressed in PDAC and associated with a poor prognosis in patients (Additional file 3 Fig. S2 A-C). Then, the expression of LMNB1 was detected in our clinical specimens, and the same results were obtained (Fig. 5D-F). In addition, the expression of LMNB1 was positively correlated with the expression of circPTPRA, and the expression of both LMNB1 and circPTPRA was negatively correlated with the expression of miR-140-5p in PDAC (Additional file 3 Fig. S2 D-F). The results from the RNAhybrid database[25] showed that LMNB1 shares the same sequence (the “AACCACT” base sequence) and binds the "seed" region of miR-140-5p, and then we constructed wild-type and mutant-type dual luciferase reporter plasmids targeting the 3'UTR of LMNB1 (Fig. 5G). In AsPC-1 and PANC-1 cells, miR-140-5p mimics significantly reduced the relative activity of firefly luciferase in the wild-type LMNB1 group, but the relative activity of firefly luciferase did not change significantly after transfection with miR-140-5p mimics in the mutant-type LMNB1 group (Fig. 5H-I). Mechanistically, miR-140-5p was negatively correlated with the expression of LMNB1. The Western blotting results showed that miR-140-5p mimics could significantly inhibit LMNB1 expression, while the miR-140-5p inhibitor could enhance LMNB1 expression (Fig. 5J). On the other hand, the expression of LMNB1 was positively regulated by circPTPRA, but miR-140-5p mimics blocked the effect of OE-circPTPRA on promoting LMNB1 expression, while the miR-140-5p inhibitor reversed the inhibition of LMNB1 expression induced by si-circPTPRA#1 (Fig. 5K-L). In short, these results suggested that circPTPRA can competitively sponge miR-140-5p to promote LMNB1 expression.

The circPTPRA/miR-140-5p/LMNB1 axis promotes PDAC progression in vitro

LMNB1, an oncogene, is also a common downstream target gene of circPTPRA and miR-140-5p, but its role in PDAC has not been reported. To investigate whether circPTPRA promotes PDAC progression via the miR-140-5p/LMNB1 axis in vitro, we designed and synthesized a siRNA and an overexpression plasmid for LMNB1. The efficiency of the siRNA and plasmid was measured by qRT‒PCR in AsPC-1 and PANC-1 cells (Fig. 6A-B). At the protein level, si-circPTPRA#1 inhibited the expression of LMNB1, but the addition of OE-LMNB1 could rescue the inhibitory effect of si-circPTPRA#1 in AsPC-1 cells. Moreover, OE-circPTPRA increased the expression of LMNB1, but the above phenotypes could be rescued after transfection of si-LMNB1 in PANC-1 cells (Fig. 6C). Functionally, increased expression of LMNB1 in AsPC-1 cells promoted cell migration, invasion, proliferation and reversed the tumor suppressor effect of si-circPTPRA#1 (Fig. 6D-E, H-I, L-M). On the other hand, decreased expression of LMNB1 in PANC-1 cells significantly inhibited cell migration, invasion, proliferation and blocked the tumor-promoting effect induced by OE-circPTPRA (Fig. 6F-G, J-K, N-O). Briefly, our results confirmed that LMNB1 promotes PDAC progression in vitro, and it is particularly important that circPTPRA maintains the malignant phenotype of PDAC dependent on the miR-140-5p/LMNB1 axis.

The circPTPRA/miR-140-5p/LMNB1 axis promotes PDAC progression in vivo

To further verify the function of the circPTPRA/miR-140-5p/LMNB1 axis in PDAC progression in vivo, a subcutaneous tumorigenesis model was established with stably transfected PANC-1 cells. Nude mice were sacrificed 30 days after inoculation with PANC-1 cells, and subcutaneous tumor tissues were obtained and photographed (Fig. 7A and Additional file 4 Fig. S3 A). In vivo data confirmed that tumor weight and volume were significantly increased by overexpression of circPTPRA, but sh-LMNB1 partially rescued those phenotypes (Fig. 7B-C). Similarly, tumor weight and volume were significantly reduced by sh-circPTPRA#1, but OE-LMNB1 partially reversed the tumor suppressor effect of sh-circPTPRA#1 (Additional file 4 Fig. S3 B-C). The IHC results also showed that the expression of LMNB1 and Ki-67 in the OE-circPTPRA group was significantly higher than that in the vector group, but sh-LMNB1 partially rescued those phenotypes. In contrast, the expression levels of LMNB1 and ki-67 were significantly decreased in the sh-circPTPRA#1 group, but OE-LMNB1 rescued the above phenotypes (Fig. 7D). We further found that interfering with the expression of circPTPRA in AsPC-1 cells reduced the expression of N-Cadherin, Vimentin and Snail, and increased the expression of E-Cadherin and β-Catenin, but OE-LMNB1 partially rescued the above phenotypes (Fig. 7E). Moreover, overexpression of circPTPRA in PANC-1 cells increased the expression of N-Cadherin, Vimentin, Snail and reduced the expression of E-Cadherin and β-Catenin, but si-LMNB1 partially rescued the above phenotypes (Fig. 7F). Then, specific antibodies and subcutaneous tumor tissues were used for IHC, and the IHC results were consistent with the in vitro results (Fig. 7G). In brief, these data confirmed that the circPTPRA/miR-140-5p/LMNB1 axis could promote PDAC progression in vivo.

Overexpression of circPTPRA is closely associated with a poor prognosis and local lymph node invasion in PDAC patients

To further evaluate the clinical significance of circPTPRA in PDAC, a FISH assay was used to detect the expression of circPTPRA in TMAs. Table S2 shows the detailed clinical data of 130 PDAC patients (Additional file 6 Table S2). The FISH results confirmed that circPTPRA was significantly overexpressed in PDAC tissues compared with paired NATs (Fig. 8A-B). Interestingly, the clinical data of PDAC patients showed that high expression of circPTPRA was closely associated with regional lymph node invasion (Table 1). However, there was no significant correlation between circPTPRA expression and patient sex, age, histological grade, nerve invasion or preoperative serum CA19-9 (Table 1). It is worth noting that compared with patients with low expression of circPTPRA, patients with high expression of circPTPRA had a worse prognosis (Fig. 8C). Briefly, the high expression of circPTPRA in PDAC may be a predictor of a poor prognosis.

Table 1

Correlation of circPTPRA expression with clinical data characteristics of PDAC patients (n = 130)
Clinical data characteristics	Total (n = 130)	circPTPRA expression		P value
Clinical data characteristics	Total (n = 130)	Low (65)	High (65)	P value
Gender
Male	82	42	40
Female	48	23	25	0.5054
Age (years)
< 60	56	29	27
≥ 60	74	36	38	0.6784
Grade
High/ Moderate	99	47	52
Low	31	18	13	0.8705
T stage
T1/T2	73	33	40
T3/4	16	7	9	0.5011
Data missing	41
N stage
N0	89	48	41
N1/2	41	17	24	0.0125*
M stage
M0	125	63	62
M1	5	2	3	0.2639
Nerve invasion
(-)	94	47	47
(+)	36	18	18	0.3823
Preoperative serum CA19-9
≤ 40 (U/ml)	17	9	8
> 40 (U/ml)	36	12	24	0.0715
Data missing	77

With the advancement of high-throughput sequencing and bioinformatics technology, an increasing number of new circRNAs with important functions have been discovered. We previously measured circRNA expression in 20 PDAC tissues and paired NATs by Arraystar Human CircRNA Array Analysis[19]. In the present study, we combined our data with the data of GSE69362, and we focused on circRNAs with abnormally high expression in PDAC. We found that circPTPRA was significantly overexpressed in PDAC tissues and cells. The results of gain-and loss-of-function experiments demonstrated that circPTPRA promotes the migration, invasion and proliferation of PDAC cells in vitro and in vivo, suggesting an oncogenic role of circPTPRA in PDAC. However, several studies have reported that circPTPRA upregulates KLF9 by sponging miR-636 or interacting with IGF2BP1 to inhibit the progression of bladder cancer[28, 29]. Thus, circPTPRA can play opposite roles in different tumors, which may be related to tumor heterogeneity. Studies have reported that miR-636 acts as a tumor suppressor[30] or oncogene[29] in other tumors, but its role in PDAC is unclear. IGF2BP1 promotes PDAC cell proliferation by activating AKT, which can be used as a driver and a therapeutic target in PDAC[31]. It is worth considering whether there are synergistic effects and regulatory mechanisms between circPTPRA and IGF2BP1 in PDAC, which would be a meaningful research direction. Interestingly, Tao Liu's team[32] confirmed that the circPTPRA/miR-96-5p axis promotes the development of PDAC, but the shortcoming of their research is the lack of gain-of-function experiments and animal models. Independent research conducted by our team has revealed different mechanisms of circPTPRA in PDAC. More importantly, our results more thoroughly confirmed that circPTPRA promotes PDAC progression through gain-and loss-of-function experiments in vitro and in vivo. The above studies confirmed the key role of circPTPRA in tumors and indicated that circPTPRA has significant research value.

Studies have reported that a few circRNAs can be involved in the translation of proteins or peptides[33], sponge proteins[34], RNA N⁶-methyladenosine (m6A) modification[35], exosome transport[36] and other processes[9], but sponging miRNAs is still one of the important functions of circRNAs, especially in PDAC[11]. For example, circRTN4 promotes PDAC growth and liver metastasis by sponging miR-497-5p[37], and circANAPC7 inhibits PDAC growth and cachexia-induced muscle atrophy by acting as a sponge for miR-373[38]. To explore the possible ceRNA mechanism of circPTPRA in PDAC, we obtained five candidate miRNAs (miR-140-5p, miR-152-5p, miR-582-3p, miR-26b-3p, miR-96-5p) based on the TargetScan and miRanda databases[23, 24]. Although there are potential binding sites between circPTPRA and miR-152-5p, miR-582-3p, and miR-26b-3p, the functions of these three miRNAs in PDAC are still unclear, and whether these three miRNAs can directly bind to circPTPRA requires more experiments to confirm. According to previous studies, circPTPRA inhibits the metastasis of non-small cell lung cancer cells by sponging miR-96-5p[39], and the circPTPRA/miR-96-5p/KRAS/MAPK signaling pathway promotes the development of PDAC[32]. A certain circRNA usually has multiple MREs, so a certain circRNA can bind to different miRNAs in different tumors. Unlike other studies, our results revealed a novel mechanism by which circPTPRA promotes PDAC progression. The results from the dual-luciferase reporter assay confirmed that circPTPRA could specifically bind to miR-140-5p through the “AACCACT” base sequence to form the RISC complex. The binding sequences between circPTPRA and miR-140-5p are different from those between circPTPRA and miR-96-5p. Moreover, the results from rescue experiments showed that circPTPRA could promote the migration, invasion and proliferation of PDAC by sponging miR-140-5p. Furthermore, through the RNA scope assay, we found that miR-140-5p was expressed at low levels in PDAC and positively correlated with the prognosis of PDAC patients.

Studies have revealed the tumor suppressor function of miR-140-5p in digestive system malignancies. Hao Yang et al. reported that miR-140-5p inhibits the growth and metastasis of hepatocellular carcinoma by targeting TGFBR1 and FGF9[40]. Zheng Fang et al. confirmed that miR-140-5p regulates the downstream target gene YES1 to inhibit the proliferation, migration and invasion of gastric cancer[40]. Bo Yuan et al. revealed that the miR-140-5p/CDK8 axis is regulated by lncRNA HCP5 to promote PDAC progression[26]. In our study, LMNB1 was identified as a common downstream target gene of circPTPRA and miR-140-5p by whole-transcriptome sequencing and bioinformatics analysis. Dual-luciferase reporter assay results showed that miR-140-5p directly binds to the “AACCACT” base sequence in the 3’UTR of LMNB1. Although miR-140-5p shares the same binding sequence with TGFBR1, FGF9, YES1, CDK8, and LMNB1, the Western blotting results showed that only LMNB1 was positively regulated by circPTPRA, and miR-140-5p could block the above phenotypes. Moreover, the IHC results showed that LMNB1 was highly expressed in PDAC, positively correlated with the expression of circPTPRA, and negatively correlated with the expression of miR-140-5p. In addition, LMNB1 and circPTPRA synergistically promoted PDAC migration, invasion, proliferation and EMT in vitro and in vivo. Overall, our study confirmes that circPTPRA can also mediate PDAC progression through the circPTPRA/140-5p/LMNB1 signaling axis.

LMNB1 is a member of the lamins family[41], which is necessary for cell survival and plays an important role in the regulation of cell proliferation and senescence, chromosome distribution condensation, and cellular DNA damage repair[42]. In addition, LMNB1 also plays an important role in tumor progression, and the biological function of LMNB1 is closely related to the abnormal proliferation and potential tumorigenicity of tumor cells. For example, LMNB1 and TMED5 are activated by GRSF1 to promote malignant behavior and nuclear autophagy in cervical cancer cells[43]; LMNB1 promotes lung adenocarcinoma cell proliferation through the AKT pathway[44]. Most importantly, LMNB1 is a potential therapeutic target for betulinic acid in PDAC[45], but the molecular mechanism by which LMNB1 promotes PDAC progression is unclear. In this paper, we first confirmed the molecular mechanism of LMNB1 in PDAC, which provides a theoretical basis for LMNB1 as a therapeutic target in PDAC.

CircRNAs are covalently closed endogenous biomolecules without a 5' cap or 3' poly(A) tail, and the regulation of circRNA formation depends on multiple factors[34]. A number of studies have shown that RBPs play the most critical role in the generation of circPTPRA. For instance, EIF4A3 can induce the expression of circMMP9 and circASAP1[46, 47]; similarly, FUS can induce abnormally high expression of circRHOBTB3 and circ_002136 in tumors[48, 49]. Through analysis of the Circular RNA Interactome online database (https://circinteractome.nia.nih.gov/)[50], we found that EIF4A3 and FUS contain binding sites for circPTPRA. To verify whether EIF4A3 and FUS could promote circPTPRA formation, we constructed overexpression plasmids and siRNAs for EIF4A3 and FUS, and Western blotting was used to detect the efficiency of the plasmids and siRNAs (Additional file 5 Fig. S4 A, D). Unfortunately, the qRT‒PCR results showed that neither gain- nor loss-of-function of EIF4A3 or FUS could affect the expression of circPTPRA (Additional file 5 Fig. S4 B-C, E-F). The synthesis of circRNAs is regulated by multiple pathways, which contribute to the diverse expression of circRNAs in different cell types and under different disease conditions. We speculate that circPTPRA is upregulated by other processes in PDAC, including precursor RNA transcription and post-or co-transcriptional processing and turnover. Therefore, more efforts are needed to uncover why circPTPRA is abnormally upregulated in PDAC, which would be a meaningful study.

There are still deficiencies in this study. First, circPTPRA plays an important role in tumor progression. In this study, we only revealed its ceRNA mechanism in PDAC, which does not mean that there are no other important regulatory networks, so mechanisms in other areas need further exploration. Second, LMNB1 is a potential therapeutic target of betulinic acid in PDAC. Our study has not confirmed whether si-circPTPRA can promote the therapeutic effect of betulinic acid on PDAC, which will be our next research plan. Third, the synthesis and degradation of circRNAs are complex processes. In this study, we failed to elucidate the reason for the abnormally high expression of circPTPRA in PDAC, which requires more work to explain. Finally, our results show that circPTPRA can promote the proliferation of PDAC cells in vitro and in vivo, but there was no significant correlation between circPTPRA expression and patient T stage. The reason could be that some patients' data were missing, and a small number of cases are not enough to obtain meaningful analysis results. The results of this study need to be validated in multicenter studies.

In conclusion, our study indicates that circPTPRA plays a key role in PDAC progression and confirms a novel mechanism of circPTPRA in PDAC. We also evaluated the correlation between circPTPRA and clinical data from a large clinical cohort. We confirmed that circPTPRA is abnormally upregulated in PDAC and is closely associated with local lymph node invasion and a poor prognosis. More importantly, the activation of the circPTPRA/miR-140-5p/LMNB1 signaling pathway promoted PDAC migration, invasion, proliferation and EMT in vitro and in vivo (Fig. 8D). However, the mechanism of circPTPRA activation needs further study, and the expression of circPTPRA should be further evaluated in blood, urine and exosomes to improve its clinical application value.

circRNAs

circular RNAs

PDAC

pancreatic ductal adenocarcinoma

FISH

fluorescence in situ hybridization

RIP

RNA immunoprecipitation

EMT

epithelial-mesenchymal transition

LMNB1

LaminB1

miRNAs

microRNAs

MREs

miRNA response elements

RISCs

RNA-induced silencing complexes

ceRNA

competing endogenous RNA

AGO

Argonaute

qRT‒PCR

quantitative real-time PCR

NAT

normal adjacent tissue

TMAs

tissue microarrays

AJCC

American Joint Commission on Cancer

DEcircRNAs

differentially expressed circRNAs

reverse transcription

IHC

immunohistochemistry

ANOVA

analysis of variance

siRNAs

short interfering RNAs

m6A

RNA N⁶-methyladenosine

gDNA

genomic DNA

PAAD

pancreatic adenocarcinoma

Ethical Approval

Informed consent was obtained from all patients prior to obtaining tissue samples, and all procedures were approved by the designated Hospital Ethics Committee. The animal experimental model was approved by the Animal Experiment Ethics Committee of Chongqing Medical University.

Competing interests

The authors declare no competing interests.

Authors' contributions

Wen Fu completed the main experiments, drafted figures and wrote the main manuscript text. Xianxing Wang provided experimental technical support, completed animal experiments and corrected manuscript text. Jifeng Xiang and Shengkai Chen participated in the clinical sample collection, follow up and statistical analysis. Renpei Xia, Fanbo Qin and Zhuo Li were responsible for using the experimental equipments and helping to complete some of the experiments. Huaizhi Wang, Chuanming Xie and Changjiang Liu provided the experimental platform and offered conception, design and critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.

Funding

This study was supported by the Chongqing Science and Technology Commission (No. cstc2021ycjh-bgzxm0022) and the National Natural Science Foundation of China (No. 82072723).

Availability of data and materials

All data generated during this study are included in this article.

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No competing interests reported.

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CircPTPRA promotes the progression of pancreatic ductal adenocarcinoma via the miR-140-5p/LMNB1 axis

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Materials And Methods

PDAC tissue specimens

CircRNA array data

Cell lines and cell culture

Plasmid and siRNA transfection and lentiviral infection

RNA extraction and qRT‒PCR analysis

RNase R and actinomycin D treatment

Western blotting

FISH and miRNA scope assay in PDAC cells and TMAs

RIP

CircRNA pull-down assay

Dual-luciferase reporter assay

Nude mouse subcutaneous tumor model

Statistical analysis

Results

Identification and characterization of the oncogene circPTPRA

CircPTPRA promotes the progression of PDAC in vitro and in vivo

CircPTPRA sponges miR-140-5p in PDAC cells

CircPTPRA promotes PDAC progression by sponging miR-140-5p

CircPTPRA promotes LMNB1 expression by competitively sponging miR-140-5p

The circPTPRA/miR-140-5p/LMNB1 axis promotes PDAC progression in vitro

The circPTPRA/miR-140-5p/LMNB1 axis promotes PDAC progression in vivo

Overexpression of circPTPRA is closely associated with a poor prognosis and local lymph node invasion in PDAC patients

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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