The starBase v3.0 database [13] was utilized to predict the binding sites between lncRNA OIP5-AS1 (ENSG00000247556) and hsa-miR-145-5p, with the following search parameters: species set to Homo sapiens (hg38), CLIP-Data threshold ≥ 1, Degradome-Data ≥ 0, and Pan-Cancer ≥ 0. The predicted interaction (8mer site, chr15:41301483-41301503[+]) was supported by Ago CLIP-seq data with a TDMD Score of 0.9194 and phyloP conservation score of 0.509.

The TargetScan Human database [14] was used to predict the targeted binding sites of hsa-miR-145-5p within the 3’UTR of DNAJA3 (ENST0000262375.6, 1243 bp). The search was restricted to broadly conserved sites among vertebrates, identifying a 7mer-A1 binding site (position 25 - 31) with a Context++ score of −0.03 (percentile 63) and a weighted context++ score of −0.03.

The human immortalized gastric mucosal epithelial cell line (GES-1) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin. Cells were incubated in a humidified incubator at 37˚C with 5% CO₂.

The H. pylori standard virulent strain (CagA+/VacA+, ATCC 43504) was inoculated onto Columbia agar plates containing 5% defibrinated sheep blood and cultured for 72 hours in a microaerophilic environment (85% N₂, 10% CO₂, 5% O₂) before harvesting.

GES-1 cells were seeded into 6-well plates and cultured to 70% - 80% confluence. Prior to infection, the culture medium containing penicillin/streptomycin was removed, and cells were gently washed three times with sterile PBS to eliminate residual antibiotics, followed by replacement with antibiotic-free infection medium. The H. pylori suspension was then added to the cells at a multiplicity of infection (MOI) of 100:1. Cell samples were collected at 0 h (Control), 6 h, 12 h, and 24 h post-co-culture for RNA extraction. For protein extraction, to coincide with the peak transcriptional changes observed in initial assays, Control and H. pylori-infected cells were harvested specifically at 24 h post-infection.

Total cellular RNA was extracted using TRIzol reagent. For mRNA and lncRNA detection, RNA was reverse transcribed into cDNA using a reverse transcription kit according to the manufacturer’s instructions. For miRNA detection, cDNA was synthesized using the All-in-One^™ miRNA First-Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China) with stem-loop RT primers specific for miR-145-5p and U6 provided in the kit. qPCR reactions were performed using SYBR Green Master Mix. The primer sequences for OIP5-AS1, DNAJA3, and GAPDH (internal control for lncRNA/mRNA) are listed in Table 1. For miR-145-5p, the forward primer sequence is listed in Table 1, and the reverse primer was the universal primer supplied with the All-in-One^™ Kit. U6 snRNA served as the internal control for miRNA quantification. All experiments were performed with three independent biological replicates (n = 3), with each sample run in technical triplicate (three qPCR replicates per sample). Relative expression levels were calculated using the 2-ΔΔCt method.

Table 1. Primer sequence.

Total cellular proteins were extracted using RIPA lysis buffer containing PMSF protease inhibitors. After determining the protein concentration via the BCA method, equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% non-fat milk for 1 hour, the membranes were incubated with specific primary antibodies: Anti-DNAJA3 (1:3000, Proteintech) and Anti-Tubulin (1:5000, Proteintech) overnight at 4˚C. The following day, membranes were incubated with HRP-conjugated secondary antibodies (1:3000, Proteintech). Protein bands were visualized using an ECL chemiluminescence kit, and densitometry analysis was performed using ImageJ software.

All experiments were independently repeated at least three times. Quantitative data were presented as Mean ± SD. Statistical analysis was performed using GraphPad Prism 8.0 software. Comparisons between two groups were conducted using Student’s t-test, and comparisons among multiple groups were performed using One-way ANOVA followed by Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant.

Based on bioinformatics predictions, we identified potential interactions among OIP5-AS1, miR-145-5p, and DNAJA3. Data obtained from the starBase database indicated that the OIP5-AS1 sequence contains an 8mer binding site (TargetSeq 5’ auccuuUUCAUGAGAACUGGAa 3’) highly complementary to the hsa-miR-145-5p sequence (miRseq 3’ ucccuAAGGACCUUUGACCUg 5’) (Figure 1(A)). Furthermore, TargetScan data revealed that the 3’ UTR of DNAJA3 mRNA (Position 25-31) contains a canonical 7mer-A1 site (...CUGGAAA...) that binds to hsa-miR-145-5p (Figure 1(B)). This provides a structural basis for OIP5-AS1 to act as an miRNA sponge for miR-145-5p, thereby potentially regulating DNAJA3.

Figure 1.Bioinformatics prediction of binding sites in the putative OIP5-AS1/miR-145-5p/DNAJA3 regulatory relationship (A) Interaction and predicted binding sequence between hsa-miR-145-5p and lncRNA OIP5-AS1 based on starBase data. (B) Interaction and canonical pairing sequence between hsa-miR-145-5p and the DNAJA3 3’ UTR region based on TargetScan data.

To investigate whether this predicted network is activated during H. pylori infection, we examined the expression dynamics in GES-1 cells across a time gradient (Control, 6 h, 12 h, 24 h). RT-qPCR results demonstrated that H. pylori infection significantly induced OIP5-AS1 expression in a time-dependent manner, peaking at 24 h post-infection (approximately 2.0-fold that of the control group, P < 0.0001) (Figure 2, left).

Conversely, the relative expression of miR-145-5p decreased progressively over time, reaching its lowest point at 24 h (approximately 0.5-fold compared to control, P < 0.0001) (Figure 2, middle). Concurrently, the relative mRNA expression of DNAJA3 exhibited a continuous upward trend, similar to OIP5-AS1, reaching approximately 1.8-fold at 24 h (P < 0.0001) (Figure 2, right). These highly correlated expression trends strongly support the existence of the OIP5-AS1/miR-145-5p ceRNA interaction in vitro.

Figure 2.H. pylori infection alters the expression profiles in GES-1 cells over time. RT-qPCR analysis showing the relative expression levels of lncRNA OIP5-AS1 (left), miR-145-5p (middle), and DNAJA3 mRNA (right) at control, 6 h, 12 h, and 24 h post-infection. Data are presented as Mean ± SD (n = 3 biological replicates). **P < 0.01 compared to the Control group (One-way ANOVA with Tukey’s post hoc test).

To confirm whether the changes at the transcriptional level translated to the protein level, we performed Western blot analysis. Based on the peak mRNA expression at 24 h, we assessed protein levels at the identical time point (24 h post-infection). Results showed that compared to the uninfected control group, the protein level of DNAJA3 (40 kDa) was visibly elevated in the H. pylori infection group, with Tubulin (55 kDa) serving as a stable loading control (Figure 3(A)). Densitometric quantification further confirmed that the relative protein expression of DNAJA3 was significantly upregulated following H. pylori infection (Figure 3(B)). This aligns with the reduction of the inhibitory miR-145-5p and the increase in OIP5-AS1, supporting the preliminary correlation logic of the network.

Figure 3.H. pylori infection induces high protein expression of DNAJA3. (A) Representative Western blot images showing the protein expression of DNAJA3 (40 kDa) and the internal loading control Tubulin (55 kDa) in control and H. pylori-infected groups (harvested at 24 h post-infection). (B) Densitometric quantitative analysis of DNAJA3 relative protein expression. Data are presented as Mean ± SD (n = 3 biological replicates).