The present study included 58 patients with PAAD (28 men and 30 women; age range, 23–66 years; mean age, 45±16.1 years) selected from 122 patients with PAAD who were admitted at The Second Affiliated Hospital of Xi'an Jiaotong University between January 2012 and January 2014. All patients were followed up from the date of admission until January 2019 to record their survival conditions. Follow-up was performed every month through outpatient visit and/or telephone calls. All PAAD cases were confirmed by histopathological analysis. The inclusion criteria were as follows: i) Patients newly diagnosed with PAAD; ii) patients with no other clinical disorders; and iii) patients who received no treatment for any disease six months prior to admission. The exclusion criteria were as follows: i) Patients with recurrent PAAD; ii) patients for whom therapies for PAAD were initiated; ii) patients with other severe clinical disorders, and iii) stage IV patients. According to the American Joint Committee on Cancer staging systems (14), there were 20, 23 and 15 cases at stage I, II and III, respectively. This study was approved by The Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University. All patients were informed with the details of this study and signed written informed consent.

The human PAAD cell lines Capan-2 and HPAC (American Type Culture Collection) were used in this study. Cells were cultured in McCoy's 5a Medium (Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.) and placed at 37°C in a humidified incubator containing 5% CO₂. Biopsy was performed on all 58 patients with PAAD to collect PAAD lesion tissue and adjacent non-cancer tissue samples within 3 cm around tumors. All tissue samples were stored in liquid nitrogen before use. Histopathological examination was performed to test all tissue specimens. Non-cancer tissues contained <1% cancer cells and PAAD tissues contained >95% cancer cells.

The pcDNA3.1 vector was used as backbone to construct IUR and CD44 expression vectors by Guangzhou RiboBio Co., Ltd. Negative control (NC) miRNA (5′-UGCGACGUUGGACGUGACGAAU-3′) and miR-34a mimic (5′-UGGCAGUGUCUUAGCUGGUUGU-3′) were synthesized by Sangon Biotech Co., Ltd. Capan-2 and HPAC cells were harvested at 70–80% confluence and 5×10⁵ cells were transfected with 10 nM plasmids (pcDNA3.1-IUR, pcDNA3.1-CD44 vectors or empty pcDNA3.1 vector as NC) or 30 nM miR-34a mimic (or negative control miRNA as NC). All transfections were performed through transient transfections using Lipofectamine^® 2000 (Sangon Biotech Co., Ltd.) for 6 h at 37°C. Cells were collected at 24 h post-transfection for subsequent experiments. Untransfected cells were used as the control (C) group.

The weight of tissues ranged from 0.016 to 0.021 g. Tissue specimens (0.015 g) were ground in liquid nitrogen. Capan-2 cells were harvested and counted, and 1×10⁶ cells were collected. Total RNA and miRNA were extracted from tissues and cells using Trizol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers' instructions. During the precipitation and washing step, 85% ethanol was used to precipitate miRNA.

RNA samples were digested with DNase I to remove genomic DNA. Tetro Reverse Transcriptase (Bioline) was used to perform reverse transcriptions (1 µg RNA per reaction, 25°C for 10 min, 52°C for 30 min and 85°C for 10 min). qPCR reaction mixtures were prepared using BlazeTaq™ SYBR-Green qPCR Mix (GeneCopoeia, Inc.). GAPDH was used as the endogenous control to measure the expression levels of IUR and CD44.

For miRNA qPCR, MystiCq^® microRNA cDNA Synthesis Mix (Sigma-Aldrich; Merck KGaA) was used to perform all reverse transcription. The qPCR reaction mixtures were prepared using miScript SYBR Green PCR Kit (Qiagen China Co., Ltd). U6 was used as the endogenous control to measure the expression level of miR-34a. All qPCR reactions were performed three times.

The primer sequences were as follows: IUR, forward 5′-AGCGGTTTCCTCTTGTTTGTG-3′, reverse 5′-CTTTTGGGTGAGAAAACAAGCC-3′; CD44, forward 5′-ACCTGCCCAATGCCTTTGATGGA-3′, reverse 5′-CAAAGCCAAGGCCAAGAGGGATG-3′; GAPDH, forward 5′-GCTTTCTTTCCTTTCGCGCT-3′, reverse 5′-TTTGCGGTGGAAATGTCCTT-3′; and miR-34a, forward 5′-TGGCAGTGTCTTAGCTGGT-3′. Universal miRNA reverse primer and U6 forward primer were provided in the miScript SYBR-Green PCR Kit. PCR reactions were performed as follows: 95°C for 1 min, and then 95°C for 10 sec and 61°C for 40 sec.

The relative expression levels were normalized to endogenous control and were expressed as 2^−ΔΔCq (15).

Capan-2 cells were harvested and counted. Cells (1×10⁶) were lysed using RIPA solution on ice (Sangon Biotech Co., Ltd.). All protein samples were denatured in boiled water for 5 min. Proteins (20 µg) were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked using 5% skimmed milked in PBS at 23°C for 1 h and were incubated with primary antibodies against CD44 (1:1,000; cat. no. ab157107; Abcam) and GAPDH (1:1,000; cat. no. ab37168; Abcam) at 4°C overnight. Membranes were then incubated with the goat anti-rabbit immunoglobulin G-HRP secondary antibody (1:1,000; cat. no. ab6721; Abcam) at 23°C for 2 h. Signals were developed using RapidStep™ ECL detection reagent (Sigma-Aldrich; Merck KGaA). The data were analyzed via densitometry using ImageJ v1.48 software (National Institutes of Health) and normalized to expression of the internal control GAPDH.

Capan-2 and HPAC cells were harvested and counted. Cells (3×10⁴) were mixed with 1 ml McCoy's 5a Medium containing 1% FBS to prepare cell suspensions. The Transwell upper chamber was filled with 0.1 ml serum-free cell suspension, and the Transwell lower chamber was filled with McCoy's 5a Medium supplemented with 20% FBS. Prior to invasion assay, membranes were coated with Corning^® Matrigel^® matrix (Corning) at 37°C for 6 h. Transwell chambers were incubated at 37°C for 12 h. Subsequently, the membranes were washed with PBS and cells were stained with 1% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. The stained cells were counted under a light microscope.

GraphPad Prism 6 (GraphPad Software, Inc.) was used for data analysis. Western blotting, RT-qPCR, cell invasion and cell migration assays were repeated three times. Data are presented as the mean ± standard deviation. Differences between two groups were compared using paired t-tests. Comparisons between three groups or more were done using one-way ANOVA followed by Tukey's post hoc test. Correlation analysis was performed by linear regression. The 58 patients with PAAD were divided into high- and low-expression groups using the median expression level of IUR in PAAD tissues as cutoff value (cutoff value, 2.11). KM plotter (https://kmplot.com/analysis/) was used for survival analysis, and survival curves were compared using log-rank test. P<0.05 was considered to indicate a statistically significant difference.

The expression of IUR in PAAD and adjacent non-tumor tissues was assessed by RT-qPCR. The results demonstrated that IUR expression was significantly decreased in PAAD tissues compared with non-cancer tissues (Fig. 1A; P<0.05). Survival curves were obtained patients with high and low IUR expression. It was observed that patients in the low-expression group had a significantly lower overall survival rate compared with patients with high IUR expression (Fig. 1B; P<0.05).

The expression of miR-34a and CD44 in PAAD tissue samples was evaluated by RT-qPCR. Correlation analysis demonstrated that the expression of IUR was positively correlated with the expression of miR-34a (Fig. 2A), but negatively correlated with the expression of CD44 (Fig. 2B) in PAAD tissues.

The IUR expression vector, miR-34a mimic and CD44 expression vector were transfected into Capan-2 cells. At 24 h post-transfection, the expression of IUR, miR-34a and CD44 was significantly upregulated compared with the C and NC groups (Fig. 3A; P<0.05). Furthermore, overexpression of IUR resulted in miR-34a upregulation, while overexpression of miR-34a did not affect the expression of IUR (Fig. 3B). In addition, IUR overexpression or miR-34a led to downregulated expression of CD44 in PAAD cells (Fig. 3C; P<0.05). However, overexpression of CD44 did not affect the expression of IUR and miR-34a (Fig. 3D).

Cell invasion and migration assay results demonstrated that, compared with the NC and C groups, overexpression of IUR and miR-34a led to decreased invasive (Fig. 4A) and migratory (Fig. 4B) abilities of Capan-2 cells (P<0.05). Overexpression of CD44 played the opposite role and attenuated the effect of IUR overexpression (P<0.05).

Another PAAD cell line, HPAC, was used to repeat the Transwell assays to further confirm the role of IUR in regulating PAAD cell invasion and migration. Similarly, invasion (Fig. S1A) and migration (Fig. S1B) of HPAC cells were inhibited after the overexpression of IUR and miR-34a (P<0.05). Overexpression of CD44 played the opposite role and attenuated the effects of IUR overexpression (Fig. S1; P<0.05).