Two human PAAD cell lines, AsPC-1 and PANC-1, were purchased from the American Type Culture Collection and were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (HyClone; Cytiva) at 37°C in a humidified incubator with 5% CO₂.

Four miRNA-target gene databases were used to predict the miRNAs targeting RMND5A: TargetScan (http://www.targetscan.org/), miRDB (http://www.mirdb.org/), miRcode (http://www.mircode.org/) and TarBase v8 (http://www.microrna.gr/tarbase). In addition, we further evaluated their possible interactions by using starBase (http://starbase.sysu.edu.cn/).

miR-590-5p mimics (5′GAGCUUAUUCAUAAAAGU-GCAG-3′) and negative control (5′CGGUACGAUCGCGGCGGGAUAUC-3′) were synthesized by Guangzhou RiboBio Co., Ltd., and transfected into the PAAD cell lines using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at the concentration of 100 nM. After transfection for 48 h, reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to detect the expression levels of miR-590-5p. The plasmid for overexpressing human RMND5A was constructed using the pHS-AVC (pLV-hef1a-mScarlet-P2A-neo-WPRE-CMV) vector (Beijing Syngentech Co., Ltd.), according to the human RMND5A gene sequence in Genbank (NM_022780.4). HEK293 (CRL-1573, ATCC) cultured in 6-cm dishes were used to pack viral particles for RMND5A overexpression. Briefly, 27 µl FuGENE^® 6 (Promega Corporation), 4 µg of recombinant plasmids or empty vector as control, 3 µg PAX2 (cat. no. 12260; Addgene, Inc.) and 2 µg VSVG (cat. no. 8454; Addgene Inc.) were mixed in 300 ul OPTI-MEM (Gibco; Thermo Fisher Scientific, Inc.), followed by being dropped into 6-cm dish. Then, the cells were placed back in the incubator at 37°C. After 6 h, all the medium was replaced by pre-warmed DMEM medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum. After 48 h, ~4 ml of the lentiviral particles in culture media were collected. For infection, 1 ml of the lentiviral particles and 10 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) were used to infect 0.5 million cells at 37°C for 4 h. After 4 h, more prewarmed medium was added for culturing at 37°C. After 48 h, RT-qPCR analysis was performed to detect the mRNA expression levels of RMND5A.

Total RNA was extracted either using the mirVana miRNA kit (Ambion; Thermo Fisher Scientific, Inc.) or the E.Z.N.A^® Total RNA kit (Omega Bio-Tek, Inc.). For mRNA detection, 1,000 ng RNA was reverse transcribed into cDNA using qScript cDNA Synthesis kit (cat. no. 95047; Quantabio) according to the manufacturer's protocol and quantified using the Fast Start Universal SYBRGreen master mix (cat. no. 4913850001; Merck KGaA). For miRNA detection, 250 ng RNA was reverse-transcribed into cDNA and quantified using the Mir-X™ miRNA qRT-PCR TB Green™ kit (cat. no. 638314; Takara Biotechnology Co., Ltd.). GAPDH and U6 were used as the internal control for mRNA and miRNA, respectively. The QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for qPCR. The thermocycling conditions used were as follows: Pre-denaturation at 95°C for 1 min; 40 cycles of denaturation at 95°C for 15 sec; annealing at 60°C for 15 sec; and extension at 72°C for 30 sec followed by melting curve analysis. For quantification, 2^−ΔΔCq method (17) was used and the primer sequences are listed in Table I.

Total protein was extracted from the AsPC-1 cells using the radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology). The Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) was used to quantify the total extracted protein according to the manufacturer's protocol. A total of 30 µg of proteins were separated using 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore) at room temperature (RT). The membranes were subsequently incubated with rabbit polyclonal anti-RMND5A (1:1,000; cat. no. ab254902; Abcam) and rabbit polyclonal anti-GAPDH antibodies (1:1,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Then, the membranes were incubated with goat polyclonal anti-rabbit IgG secondary antibody (1:1,000; cat. no. ab6721; Abcam) for 1 h at RT. The protein expression levels were measured using an enhanced chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.).

The human PAAD cell lines AsPC-1 and PANC-1 were seeded in a 6-well plate at 1×10⁶ cells/well. When the cells reached 80–90% confluence, the cell monolayer was scraped with a 200-µl pipette tip. After washing with PBS, the cells were cultured for another 24 h. The medium used for this assay was serum-free DMEM. The migrated cells were observed using a phase-contrast light microscope at ×10 objective magnification (Olympus Corporation).

Data are presented as mean ± SD and analysed using GraphPad Prism 8 (GraphPad Software Inc.). A total of 3 biological replicates and 3 technical repeats were performed. For the comparison of two groups, Student's t-test was used. One-way ANOVA followed by Tukey's multiple comparisons test was performed when comparing more than two groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of RMND5A in cancer, the expression levels of RMND5A in 31 types of cancer were analyzed using the Gene Expression Profiling Interactive Analysis platform (http://gepia.cancer-pku.cn). As shown in Fig. 1A and B, the expression levels of RMND5A were significantly higher in PAAD, stomach adenocarcinoma (STAD) and thymoma (THYM) tissues compared with that in normal tissues. Notably, a lower RMND5A expression was associated with better overall survival in patients with PAAD (Fig. 1C). However, in patients with STAD and THYM, RMND5A expression levels were not significantly associated with overall survival (Fig. 1C).

Based on the aforementioned data, the effects of RMND5A overexpression were investigated in PAAD cell lines to determine its function. The transfection efficiency of the RMND5A overexpression plasmid was confirmed using RT-qPCR and western blot analyses (Fig. 2A and B). Notably, it was found that overexpression of RMND5A significantly increased cell migration in both the AsPC-1 and PANC-1 cell lines (Fig. 2C and D).

To predict the potential miRNAs that target RMND5A, in silico analysis was performed by using the TargetScan, miRDB, miRcode and TarBase databases. The overlap analysis identified six potential miRNAs, namely miR-590-5p, miR-30a-5p, miR-27a-3p, miR-148b-3p, miR-30b-5p, and miR-30e-5p (Fig. 3A). In addition, the results of the targeting sites, Cross-linking immunoprecipitation (ClipSeq) PeakCluster and ReadNumber of miRNA-RMND5A interaction in StarBase were checked, which revealed that miR-590-5p was among the top hits. The 3′ untranslated region (UTR) of both miR-590-5p and RMND5A were highly conserved among mammals (Fig. 3B). Finally, the mature miR-590-5p seeding region exhibited a perfect alignment with the RMND5A 3′UTR (Fig. 3C).

To confirm the hypothesis that miR-590-5p targets the RMND5A gene, miR-590-5p mimics were transfected into the PAAD cell lines. The transfection efficiency of miR-590-5p mimics was confirmed using RT-qPCR (Fig. 4A). Following transfection with miR-590-5p mimics, the AsPC-1 and PANC-1 cell lines exhibited significantly decreased RMND5A expression levels (Fig. 4B and C). The wound healing assay demonstrated that miR-590-5p mimics decreased the migration ability of the AsPC-1 and PANC-1 cells (Fig. 4D and 4E). Furthermore, miR-590-5p mimics attenuated the promoting effects of RMND5A overexpression on the migration of the PAAD cells (Fig. 4D and E).