Fresh gastric cancer tissue samples were obtained from patients diagnosed with gastric cancer at the Affiliated Hospital of Yan’an University. Immediately following surgical resection, pathological samples were collected and promptly preserved in liquid nitrogen. Additionally, paraffin-embedded tissue samples from gastric cancer patients with confirmed pathological diagnoses were also acquired from the same hospital.

Human gastric cancer cell lines HGC 27,MKN 28, and AGS, as well as the human normal gastric mucosal cell line GES-1, were provided by the Medical Translational Center of Yan’an University School of Medicine. HCG27 and MKN28 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). AGS and GES cells were cultured in DMEM medium supplemented with 10% FBS. All cells were maintained in a cell culture incubator at 37°C with 5% CO2.

Total RNA was extracted from gastric cancer cell lines and normal/tumor paired tissues using TRIzol reagent (AG) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA using the RT-PCR reverse transcription kit (Aikerui Biosciences, China, AG11711). qRT-PCR was performed using SYBR Green Pro Taq HS premix qPCR kit (Aikerui Biosciences, China, AG11701) on a Roche Cobas Z480, with U6 and GAPDH as internal references. Relative expression levels were calculated using the 2^-ΔΔCT method. Primer sequences are listed in Supplementary Table 1 .

MiR-582-5p mimic, mimic control (mimic-NC), MiR-582-5p inhibitor,(inhibitor-NC) small interfering RNA targeting ATG7 (si-ATG7), and its negative control (si-NC) were purchased from Aikerui Biosciences. Transfection was performed using jetPRIME^® transfection reagent (Polyplus) following the manufacturer’s protocol. Cells were collected for subsequent studies 48 hours post-transfection. Sequences for small interfering RNA, MiR-582-5p mimic, MiR-582-5p, and mimic control (miR-NC) are detailed in Supplementary Table 1 .

Cells (2 × 103) were seeded in 96-well plates and cultured until specific time points. Each well was treated with 10 μL CCK-8 reagent and incubated for 2 hours in the culture incubator. Optical density (OD) at 450 nm was measured using a microplate reader.

Cells in logarithmic growth phase were evenly seeded in 6-well plates (approximately 4 × 10⁵ cells per well) and cultured in the incubator. After 24 hours, cells were transfected according to the Polyplus transfection reagent instructions, with culture medium without antibiotics added. After 6 hours of transfection, scratches were made using a 100 µl pipette tip. Images were taken under a microscope as 0-hour controls, and wound healing was observed and quantified at 24 and 48 hours post-scratch. Cell migration rate was calculated using the formula: Cell migration rate = [(initial blank area - blank area at specific time point)/initial blank area] × 100%.

Transwell chambers were used to evaluate the migration and invasion capabilities of cancer cells. Transfected gastric cancer cells were seeded in the chambers. For invasion assays, matrix gel was coated on the upper chamber, while for migration assays, cells were directly seeded on the upper chamber. The lower chamber was filled with medium containing 20% FBS. After 48 hours of incubation, cells were fixed with 4% paraformaldehyde for 30 minutes and stained with 0.25% crystal violet.

The wild-type (WT) fragment of ATG7 3′-UTR containing the binding sequence of miR-582-5p was cloned into pmirGLO vector to construct ATG7-WT. Mutant (MUT) fragment (ATG7-MUT) with mutated binding sequence was also cloned into pmirGLO vector. Gastric cancer cells were co-transfected with ATG7-WT or ATG7-MUT and miR-582-5p mimic or miR-NC using Polyplus reagent. After 48 hours, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter gene assay system.

Total protein from cells and tissues was extracted using RIPA buffer. Protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk at room temperature for 2 hours, followed by overnight incubation with primary antibodies (GAPDH, P62, ATG7, Beclin1) at 4°C. After washing, membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit IgG) at room temperature for 2 hours. Protein bands were visualized using ECL exposure strips.

All experiments were repeated three times, and data are presented as mean ± SD (standard deviation). Student’s t-test was used for statistical analysis. P< 0.05 was assumed to be statistically significant.

To investigate the expression of miR-582-5p in gastric cancer and its relationship with clinical pathological parameters, we first identified low expression of miR-582-5p in gastric cancer using the UALCAN database (https://ualcan.path.uab.edu/) ( Figure 1A ). We further examined miR-582-5p expression in 41 pairs of gastric cancer and normal tissue samples by qPCR, confirming low expression in gastric cancer tissues ( Figure 1B ). Additionally, we assessed miR-582-5p expression in gastric cancer cell lines and normal gastric epithelial cells, demonstrating consistently low expression in gastric cancer cell lines ( Figure 1C ). Analysis of the correlation between miR-582-5p expression and clinical pathological parameters in patients indicated a significant association with T stage, while no correlation was observed with gender, age, tumor size, differentiation grade, or N stage ( Table 1 ).

To explore the biological function of MiR-582-5p in gastric cancer cells, we constructed a MiR-582-5p overexpression gastric cancer cell line. We verified the transfection efficiency using q-PCR ( Figure 2A ). Next, we assessed the effect of MiR-582-5p overexpression on the proliferation ability of gastric cancer cells using the CCK8 assay, which showed that overexpression of MiR-582-5p significantly inhibited the proliferation of gastric cancer cells ( Figure 2B ). Meanwhile, wound healing assays demonstrated that overexpression of MiR-582-5p suppressed the invasion and migration capabilities of gastric cancer cell lines ( Figure 2C ). These experimental results confirm that MiR-582-5p acts as a tumor suppressor gene, effectively inhibiting the malignant biological functions of gastric cancer cells. Subsequently, we investigated whether MiR-582-5p affects the sensitivity of gastric cancer cells to oxaliplatin. We demonstrated through CCK8 and transwell assays that MiR-582-5p can enhance the sensitivity of gastric cancer cells to oxaliplatin ( Figures 2D, E ).

To further confirm the biological function of MiR-582-5p in gastric cancer cells, we constructed a MiR-582-5p knockdown gastric cancer cell line. The transfection efficiency was verified by q-PCR ( Figure 3A ). The CCK8 assay assessed the effect of MiR-582-5p knockdown on the proliferation ability of gastric cancer cells, revealing that knockdown of MiR-582-5p significantly promoted the proliferation of gastric cancer cells ( Figure 3B ). Meanwhile, wound healing assays demonstrated that knockdown of MiR-582-5p enhanced the invasion and migration capabilities of gastric cancer cells ( Figure 3C ). Next, we investigated the effect of MiR-582-5p knockdown on the sensitivity of gastric cancer cells to oxaliplatin. CCK8 and transwell assays confirmed that knockdown of MiR-582-5p can reduce the sensitivity of gastric cancer cells to oxaliplatin ( Figures 3D, E ).

To further investigate the mechanism by which MiR-582-5p affects gastric cancer cell biological functions and sensitivity to oxaliplatin, we assessed the expression of ATG7 at both the RNA and protein levels following MiR-582-5p overexpression and knockdown using q-PCR and western blotting. The results showed that overexpression of MiR-582-5p led to low expression of ATG7, while knockdown of MiR-582-5p resulted in high expression of ATG7 ( Figures 4A, B ). This suggests that ATG7 might be a potential downstream target of MiR-582-5p and that MiR-582-5p could exert its effects by targeting ATG7. To confirm this, we performed a dual-luciferase reporter assay to verify the binding sites. The results indicated that MiR-582-5p has binding sites in the 3′-UTR of ATG7 mRNA ( Figure 4C ).

Next, we further explored the impact of ATG7 on the biological functions of gastric cancer cell lines. First, using the GEPIA database, we found that ATG7 is highly expressed in gastric cancer ( Figure 5A ), and that patients with high ATG7 expression have a lower overall survival (OS) compared to those with low expression ( Figure 5B ). We used the normal gastric mucosal epithelial cell line GES-1 as the control group and selected three gastric cancer cell lines (HGC27, MKN28, AGS) as the experimental group. Total RNA and protein were extracted from each group of cells, and the expression levels of ATG7 were measured using RT-qPCR and western blotting. The results showed that ATG7 expression levels were significantly upregulated in the three gastric cancer cell lines (HGC27, MKN28, AGS) compared to the normal gastric mucosal epithelial cell line GES-1 ( Figures 5C , D ) (*p<0.05, **p<0.01, ***p<0.001).

By examining ATG7 expression in gastric cancer cells, we observed high levels of ATG7 expression. To investigate whether its overexpression affects the biological functions of gastric cancer, we designed two siRNAs targeting ATG7 and established transiently transfected gastric cancer cell lines with reduced ATG7 expression. We validated the knockdown efficiency through RT-qPCR and Western blotting ( Figures 6A , B ). Functional assays were performed on HGC27, MKN28, and AGS cells with transient ATG7 knockdown. The results showed that compared to the negative control (NC) group, proliferation capacity was decreased in HGC27 and AGS cells in the CCK8 proliferation assay ( Figure 6C ). Scratch and Transwell assays demonstrated reduced migration and invasion capabilities of cells following ATG7 knockdown ( Figures 6D , E ). Additionally, using Transwell and CCK8 assays, we evaluated the impact of ATG7 knockdown on gastric cancer cell resistance to oxaliplatin, revealing suppressed proliferation, invasion, migration, and oxaliplatin resistance after ATG7 knockdown ( Figures 6F, G ) (*P < 0.05, **P < 0.01, ***P < 0.001).

To further demonstrate that MiR-582-5p exerts its effects by targeting ATG7, we conducted rescue experiments by co-overexpressing ATG7 and MiR-582-5p in gastric cancer cell lines. Western blotting confirmed that overexpression of MiR-582-5p reduced ATG7 expression, which was reversed upon simultaneous overexpression with ATG7 ( Figure 7A ). Subsequent functional assays and assessments of oxaliplatin sensitivity showed that co-overexpression of ATG7 reversed MiR-582-5p-induced inhibition of cell proliferation, as demonstrated by CCK8 assays ( Figure 7B ). Transwell assays further validated that co-overexpression of ATG7 reversed the phenotypic changes induced by MiR-582-5p overexpression in gastric cancer cells, including their sensitivity to oxaliplatin ( Figures 7C–E ).