The present study followed the Declaration of Helsinki and was endorsed by the Ethics Committee of the People's Hospital of Ningxia Hui Autonomous Region (approval no. 2021-10-003). The human tissue samples were obtained from the Human Tissue Bank of People's Hospital of Ningxia Hui Autonomous Region. All patients signed the informed consent before surgery. Tumor tissues and para-cancerous tissues (≥3 cm away from the tumor margin) of 30 patients with pancreatic carcinoma (age range, 53–68 years; 14 male and 16 female) between October2021 and November 2012 were collected during surgery from the People's Hospital of Ningxia Hui Autonomous Region and immediately stored in liquid nitrogen at −196°C. None of the subjects had undergone chemotherapy or radiotherapy prior to the surgery.

Normal human pancreatic duct epithelial cell line (HPDE6-C7), human pancreatic carcinoma cell lines (Hs766T, SW1990, CAPAN-1) and 293T were obtained from the American Type Culture Collection; the JF305 pancreatic carcinoma cell line was purchased from Yuchi (Shanghai) Biological Technology Co., Ltd. (cat. no. SCO423). All the cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) in 5% CO₂ at 37°C.

Hs766T and JF305 cells were inoculated into 6-well plates at a density of 2×10⁵ cells/well. Small interfering (si)RNA oligonucleotides targeting CTBP1-AS2 (si-CTBP1-AS2-1, 5′-GAGATCTAAGAAAAAATTCCAGA-3′; si-CTBP1-AS2-2, 5′-GCGCGTTATCATGACTTCTATTT-3′), scrambled siRNA [negative control siRNA (si-NC); 5′-TTCTCCGAACGTGTCACGTTT-3′], miR-141-3p mimic (5′-UAACACUGUCUGGUAAAGAUGG-3′), miR-141-3p inhibitor (5′-CCAUCUUUACCAGACAGUGUUA-3′), mimics NC (5′-UCACAACCUCCUAGAAAGAGUAGA-3′), inhibitor NC (5′-CAGUACUUUUGUGUAGUACAAA-3′), pcDNA3.1 vector overexpressing ubiquitin-specific protease 22 (USP22) and empty plasmids were provided by Guangzhou RiboBio Co., Ltd. The transfection was performed by Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction at 37°C and 5% CO₂ for 6 h, with final concentrations of 50 nM siRNAs, miRNA mimics and inhibitor, and 2 µg overexpression plasmid. Finally, cells were incubated at 37°C for 48 h and harvested for the following study.

TRIzol^® (Thermo Fisher Scientific, Inc.) was used to extract lncRNA or mRNA from cells (5×10⁵) according to the manufacturer's instructions. A mirVana miRNA isolation kit (Ambion; Thermo Fisher Scientific, Inc.) was used to extract miRNA from tissues and cell lines according to the manufacturer's instructions. cDNA synthesis was conducted using the Mir-X miRNA First-Strand Synthesis kit (Takara Biotechnology Co., Ltd.) for miR-141-3p and a PrimeScript RT reagent kit (Promega Corporation) was adopted for CTBP1-AS2 and USP22 according to the manufacturer's instructions. An ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for performing qPCR with a SYBR Green Premix Ex Taq kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. The reaction conditions were as follows: 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. GAPDH and U6 served as internal references, and the 2^−ΔΔCq method (11) was used for calculating the data. Primer sequences are listed in Table I.

Pancreatic carcinoma cells were seeded into 96-well plates (2×10³ cells/well) and cultured for 0, 24, 36 and 72 h. At each time point 10 µl of CCK-8 solution (5 mg/ml; Beyotime Institute of Biotechnology) was added, and the cells were incubated for an additional 4 h. A microplate reader (Thermo-Fisher Scientific, Inc.) was used to measure the absorbance at 450 nm, which indicated the viability of the cells.

An EdU Labeling/Detection kit (Guangzhou RiboBio Co., Ltd.) was used. The cells were inoculated into 96-well plates (5×10³ cells/well) for 24 h and then incubated with 50 mmol/l EdU solution in 5% CO₂ at 37°C for 2 h. The cells were then fixed for 30 min at room temperature with 4% paraformaldehyde and incubated with glycine for 10 min at room temperature. Next, 100 µl of PBS containing 0.5% TritonX-100 was added into each well and the cells were incubated for 10 min at room temperature. Then the cells were washed twice with PBS and stained with 1X Apollo fluorochrome at room temperature for 30 min in the dark. Next, the cell nuclei were stained by 1X DAPI staining solution for 15 min at room temperature. A Nikon Eclipse Ti fluorescence microscope (Nikon Corporation) was used to observe the cells and to determine the percentage of EdU-positive cells, which indicated the proliferative capacity.

Transwell chambers (24-well; pore size of 8 µm; Corning, Inc.) were used to evaluate cell invasion and migration. Briefly, for the migration assay, pancreatic carcinoma cells in 200 µl FBS-free RPMI-1640 medium were transferred into the upper compartment (1×10⁵ cells/well) and the lower compartment contained 600 µl of RPMI-1640 medium containing 10% FBS. After 24 h, the cells in the upper compartment were removed, the cells on the lower surface of the filter were stained with 0.1% crystal violet for 30 min at room temperature and the number of migrated cells was counted under a microscope (Olympus Corporation). The cell invasion assay was carried out following the same procedures after the filter was precoated with 60 µl of Matrigel at 37°C for 2 h (BD Biosciences).

Apoptosis was examined using an Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology, Inc.). Briefly, 48 h after transfection, cells were harvested, resuspended in binding buffer and incubated with 5 µl of Annexin V-FITC and 10 µl of PI in the dark at room temperature for 15 min. The apoptotic rate (early + late apoptotic cells) were analyzed by flow cytometry using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.) and FlowJo software (version 10.0; BD Biosciences) (12).

Wild-type (WT) and mutant-type (MUT) CTBP1-AS2 luciferase reporter plasmids (CTBP1-AS2-WT and CTBP1-AS2-MUT) and USP22 3′UTR luciferase reporter plasmids (USP22-3′UTR-WT and USP22-3′UTR MUT) were obtained from Shanghai GenePharma Co., Ltd. Subsequently, these luciferase reporters were co-transfected with either miR-141-3p mimics or mimics NC into 293T cell line (1×10⁵ cells/well) by using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. After 48 h, the luciferase activity of the cells was detected by the Dual-Luciferase Reporter Assay System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Proteins were extracted from pancreatic carcinoma tissues and cell lines using RIPA lysis buffer (Sigma-Aldrich; Merck KGaA), and a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) was used for protein quantification. Proteins (30 µg/lane) were separated using 10% SDS-PAGE (Sigma-Aldrich; Merck KGaA) and then transferred to polyvinylidene difluoride membranes (MilliporeSigma). After being blocked with 5% skimmed milk at 4°C for 2 h, the membranes were incubated with primary antibodies (anti-USP22; 1:2,000; cat. no. LS-C102769; LifeSpan BioSciences, Inc.; anti-GAPDH; 1:1,000; ab9385; Abcam) at 4°C overnight and then incubated with a HRP-conjugated secondary antibody (1:10,000; ab6721; Abcam) for 1 h at 37°C. Amersham ECL Select Western Blotting Detection Reagent (Cytiva) was used to visualize the protein bands. ImageJ software 1.8.0 (National Institutes of Health) was used to quantify the protein bands.

The GEPIA database (http://gepia.cancer-pku.cn) is an interactive web server for tumor gene expression analysis by using data from The Cancer Genome Atlas Pan-Cancer project and Genotype-Tissue Expression database. The GEPIA database was searched to analyze the expression of CTBP1-AS2 in pancreatic carcinoma tissues and normal pancreatic tissues. StarBase database (http://starbase.sysu.edu.cn) was used to search for the miRNAs with potential binding sites for CTBP1-AS2 and mRNAs that bind to miR-141-3p.

Each experiment was conducted at least three times, and all data are expressed as mean ± standard deviation or median ± interquartile range. SPSS 19.0 software (IBM Corp.) was used for statistical analysis. Data were analyzed using unpaired or paired Student's t-test, one-way ANOVA with Tukey's post hoc test, Pearson's correlation analysis and χ² test. P<0.05 was considered to indicate a statistically significant difference.

By searching the GEPIA database it was found that CTBP1-AS2 expression in patients with pancreatic carcinoma tumor tissues was significantly higher compared with that in normal pancreatic tissues (Fig. 1A). Similarly, RT-qPCR showed that CTBP1-AS2 expression was significantly higher in patient tumor tissues compared with para-carcinoma tissues (Fig. 1B). Additionally, compared with HPDE6-C7 normal pancreatic duct epithelial cells, CTBP1-AS2 expression was significantly higher in pancreatic carcinoma cell lines, Hs766T, SW1990, CAPAN-1 and JF305 (Fig. 1C). Since CTBP1-AS2 exhibited the highest expression level in Hs766T and JF305 cells, these two cell lines were selected to perform the subsequent loss-of-function experiments.

To analyze the clinical significance of high CTBP1-AS2 expression in pancreatic carcinoma, the relationship between patient clinicopathological characteristics and CTBP1-AS2 expression levels were evaluated (Table II). Based on the median value of CTBP1-AS2 expression level, 30 patients were divided into high and low CTBP1-AS2 expression groups (n=15 patients/group). The results showed that high CTBP1-AS2 expression was associated with lymph node metastasis and advanced clinical stage of patients with pancreatic carcinoma. Together, these data indicated that CTBP1-AS2 may be a crucial regulatory factor for pancreatic carcinoma development and may serve a cancer-promoting role.

To examine the biological functions of CTBP1-AS2 in pancreatic carcinoma cells, two CTBP1-AS2 siRNAs (si-CTBP1-AS2-1 and si-CTBP1-AS2-2) were used to knock down CTBP1-AS2 expression in Hs766T and JF305 cell lines. It was demonstrated that CTBP1-AS2 expression levels in both CTBP1-AS2 knockdown groups were significantly reduced compared with the si-NC group (Fig. 2A). CCK-8 and EdU assays confirmed that the cell proliferation was significantly inhibited after CTBP1-AS2 knockdown (Fig. 2B-D). Transwell and Matrigel assays showed that cell migration and invasion, respectively, were notably suppressed after CTBP1-AS2 knockdown (Fig. 2E and F). It was also revealed that Hs766T and JF305 cells transfected with si-CTBP1-AS2 had higher apoptosis rates compared with cells transfected with si-NC (Fig. 2G and H). These results suggested that knocking down CTBP1-AS2 could inhibit the malignant phenotypes of pancreatic carcinoma cells.

LncRNAs can take part in tumor progression as competing endogenous RNAs (ceRNAs), which means that lncRNAs can adsorb miRNAs like molecular sponges to regulate the expression levels of mRNAs (13). To further pinpoint the mechanism by which CTBP1-AS2 serves a role in pancreatic carcinoma, StarBase database was searched and it was found that CTBP1-AS2 likely binds with miR-141-3p (Fig. 3A). RT-qPCR results demonstrated that miR-141-3p expression was significantly reduced in tumor tissues and pancreatic carcinoma cell lines compared with that in the respective para-cancerous tissues and normal human pancreatic duct epithelial cell line (Fig. 3B and C). Furthermore, CTBP1-AS2 expression in pancreatic carcinoma tumor tissues was inversely correlated with miR-141-3p expression (Fig. 3D), and CTBP1-AS2 knockdown markedly promoted miR-141-3p expression in Hs766T and JF305 cell lines compared with si-NC (Fig. 3E). Subsequently, miR-141-3p expression was overexpressed or knocked down in Hs766T and JF305 cells by transfecting with miR-141-3p mimics or miR-141-3p inhibitor, respectively. The results showed that miR-141-3p mimics significantly increased the expression of miR-141-3p compared with mimics NC and miR-141-3p inhibitor decrease the miR-141-3p expression compared with the inhibitor NC (Fig. 3F). No significant difference in miR-141-3p expression was observed between mimic NC and inhibitor NC, so mimic NC was selected as the control of miR-141-3p mimics or miR-141-3p inhibitor in the following study. Dual-luciferase reporter assay results showed that miR-141-3p mimics could significantly reduce CTBP1-AS2-WT luciferase activity but failed to affect the luciferase activity of CTBP1-AS2-MUT, which verified the targeting relationship between CTBP1-AS2 and miR-141-3p (Fig. 3G). To determine whether CTBP1-AS2 could serve a role in pancreatic carcinoma through miR-141-3p, rescue assays were performed. The results showed that the effects of CTBP1-AS2 knockdown on pancreatic carcinoma cell proliferation, migration, invasion and apoptosis could be partially counteracted by inhibiting miR-141-3p (Fig. 3H-M and Fig. S1). These results displayed that CTBP1-AS2 exerted a cancer-promoting effect by repressing miR-141-3p expression in pancreatic carcinoma.

Putative downstream mRNAs interacting with miR-141-3p were also investigated. StarBase database analysis identified a number of potential targets of miR-141-3p, such as E2F transcription factor 3, cyclin G1 and ring finger protein 38. Among them, USP22 was selected in a previous study which demonstrated that USP22 served as a cancer-promoting gene in the progression of pancreatic cancer (14) (Fig. 4A). Results from the dual-luciferase reporter assay showed that miR-141-3p mimics inhibited USP22-WT luciferase activity but did not change USP22-MUT luciferase activity (Fig. 4A). In addition, miR-141-3p inhibitor was transfected into Hs766T and JF305 cell lines and it was found that USP22 mRNA and protein expression levels were significantly decreased following the transfection compared with the control group (Fig. 4B). Notably, USP22 expression levels were significantly enhanced in pancreatic carcinoma cell lines and tumor tissues (Fig. 4C and D, respectively). miR-141-3p expression was negatively correlated with USP22 mRNA expression (Fig. 4E), and CTBP1-AS2 expression was positively correlated with USP22 mRNA expression in pancreatic carcinoma tissues (Fig. 4F). These findings revealed that miR-141-3p targeted and downregulate USP22 expression in pancreatic carcinoma.

Western blot assay results showed that USP22 protein expression was upregulated in Hs766T and JF305 cells transfected with USP22 overexpression plasmid compared with the control (Fig. 5A). In addition, the inhibitory effects of CTBP1-AS2 knockdown on USP22 protein expression could be abolished by co-transfection with miR-141-3p inhibitor (Fig. 5B). In addition, the transfection of USP22 overexpression plasmid could reverse CTBP1-AS2 knockdown-mediated inhibitory effects on the proliferation, migration and invasion of Hs766T and JF305 cells (Fig. 5C-H; Fig. S2). These results suggested that CTBP1-AS2 may serve a cancer-promoting role through regulating the miR-141-3p/USP22 axis.