LINC01711 facilitated gastric carcinoma progression by sponging miR-140-3p

Study subjects

This study was performed in full compliance with the ethical guidelines established by the Declaration of Helsinki. Approval was granted by the Ethics Committee of the author’s institution before the study began (No. 2019162, dated 2019.03.05). A cohort of 150 GC patients was recruited from the author’s institution between 2019 and 2020. The enrollment criteria were defined as follows:

1. Patients histopathologically confirmed to have GC were enrolled.

2. Patients or their legal guardians who were fully informed about research procedures and signed the informed consent were enrolled.

3. Patients who had received no prior antitumor therapy were enrolled.

4. Patients with complete follow-up data and comprehensive medical records were enrolled.

5. Patients without serious complications were enrolled.

6. Patients who presented with no acute or chronic comorbidities that might confound the study outcomes were enrolled.

7. Female patients who were not pregnant or breastfeeding were enrolled.

Follow-up survey

Postoperative follow-up was conducted for all enrolled patients for a period of 3–60 months to evaluate both functional recovery and long-term survival outcomes. The study endpoints included disease recurrence, metastasis, and mortality attributable to GC.

Sample collection

The tumor and normal gastric tissues were obtained during the surgical resection. Collected tissues were histologically evaluated and confirmed by at least two pathologists to ensure diagnostic consensus. Following pathological verification, the tissues were immediately frozen in liquid nitrogen and subsequently stored at −80 °C.

Cell culture and transfection

In this study, human gastric epithelial cells (GES-1, SNL-304, SUNNCELL, China) and GC cells (AGS, SNL-103, SUNNCELL, China; MKN45, SNL-173, SUNNCELL, China; MKN74, SNL-176, SUNNCELL, China; HGC-27, SNL-104, SUNNCELL, China; and SNU-16, SNL-538, SUNNCELL, China) were chosen for testing LINC01711 expression characteristics in GC cells. Cells were cultured using Roswell Park Memorial Institute Medium (RPMI 1640, SNM-001B, SUNNCELL, China) containing 10 % fetal bovine serum (FBS, 164210, Pricella, China) and 1 % penicillin-streptomycin solution (PB180120, Pricella, China) at 37 °C in a 5 % CO₂ incubator.

HGC-27 and SNU-16 cell lines were selected for further cell experiments because LINC01711 was the most up-regulated in the two types of cells. After seeding in 24-well plates, cells were cultured overnight in complete growth medium (RPMI 1640 supplemented with 10 % FBS). Following medium removal, the cells were transfected in Opti-MEM I medium (31985070, Biolead, China) using Lipofectamine 3000 (L3000001, Invitrogen, USA) for 6 h with the following constructs: hsa-miR-140-3p mimic (B01001, GenePharma, China), LINC01711-targeting small interfering RNA (si-LINC01711; A01001, GenePharma, China), hsa-miR-140-3p inhibitor (B03001, GenePharma, China), mimic NC (B04001, GenePharma, China), si-NC (A06001, GenePharma, China), and inhibitor NC (B04003, GenePharma, China). The transfection efficiency was detected via qRT-PCR.

Prediction and validation of LINC01711-hsa-miR-140-3p interaction

The potential miRNAs that interact with LINC01711 in GC were screened across multiple databases using the LncBook (https://ngdc.cncb.ac.cn/lncbook/), miRanda (http://www.bioinformatics.com.cn/local_miranda_miRNA_target_prediction_120; score ≥160, ΔG ≤ −25 kcal/mol), and starBase (http://starbase.sysu.edu.cn/index.php) databases. The DIANA bioinformatics suite was employed to investigate putative binding sites between LINC01711 and hsa-miR-140-3p.

To experimentally validate this interaction, wild-type (WT-LINC01711) and mutant (MUT-LINC01711) sequences of LINC01711 were cloned into the luciferase reporter plasmid.

HGC-27 and SNU-16 cells, cultured in RPMI 1640 medium with 10 % FBS, were plated in 48-well culture plates upon reaching 80 % confluence. The cells were then co-transfected with either WT-LINC01711 or MUT-LINC01711 constructs, along with a Renilla luciferase plasmid (for normalization; 11402ES60, YEASEN, China). At 24 h post-transfection, luciferase activity was measured using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA) following the addition of a luciferase substrate. Firefly luciferase signals were normalized to Renilla luciferase fluorescence intensity to account for transfection efficiency.

Cell proliferation assay

HGC-27 and SNU-16 cell proliferation was tested using the CCK-8 assay. Following plating in 96-well culture plates, cell proliferation was monitored at 24 h intervals (0–72 h) by measuring the optical density at 450 nm (OD₄₅₀). At each time point, cells were incubated with the CCK-8 reagent (C0038, Beyotime, China) for 2 h at 37 °C, followed by absorbance measurement at 450 nm.

Cell migration and invasion assay

Cell migration ability was examined using a Transwell chamber assay. The permeable membrane (FTW064-12Ins, Beyotime, China) was rehydrated with the serum-free medium at 37 °C for 30 min. Cells were serum-starved for 12 h, trypsinized, washed twice with PBS, and resuspended in serum-free medium. Then, the cell suspension was added to the upper chamber, while the lower chamber was filled with complete medium (RPMI 1640 medium containing 10 % FBS). Following a 24 h incubation, the upper membrane surface was washed with calcium-free PBS to remove non-migrated cells. Migrated cells were fixed with 4 % paraformaldehyde for 30 min, stained with 0.1 % crystal violet (20 min), and washed with PBS. Cells were counted under an inverted light microscope (Olympus, USA).

The invasive potential of cells was determined using the Transwell assay with membranes that were pre-coated with Matrigel. Matrigel (50 mg/L, 1:8 dilution; C0372-1 ml, Beyotime, China) was used to coat the upper chamber membrane. The subsequent steps were performed identically to the migration assay.

Total RNA extraction

RNA extraction was performed using TRIzol reagent (15596026CN, Invitrogen, USA).

Tissue RNA Extraction: Fresh tissue samples were minced into 1–3 mm fragments and homogenized in 1 mL of TRIzol reagent using a sterile pestle. Chloroform (C0761520275, Nanjing Reagent, China) was added to promote the isolation of RNA from the samples. The mixture was centrifuged, and the upper aqueous phase was mixed with isopropanol (C0690546005, Nanjing Reagent, China) to pellet RNA. The pellet was washed with 75 % ethanol (C0691560024, Nanjing Reagent, China), and the RNA was air-dried and resuspended in RNase-free water.

Cell RNA Extraction: The procedure for cell RNA extraction was identical to tissue RNA extraction, but omitted the step of grinding cells. Only RNA samples with OD_260/280 ratio of 1.8–2.0 and concentration greater than 40 μg/mL were used for further study.

Real-time quantitative PCR

PrimeScript RT Master Mix kit (RR037Q, Takara, Japan) was used for the cDNA synthesis with 2 μg of the obtained RNA. MX3000P system (Stratagene, Germany) performed quantitative real-time PCR (qRT-PCR) with the following reaction parameters: initial denaturation: 94 °C for 3 min; denaturation: 94 °C for 20 s; annealing/extension: 62 °C for 40 s; amplification cycles: 40. Gene-specific primers for LINC01711 (forward 5′-GAT​CTG​CTC​CAT​GTC​GTG​TGA​TG-3′, reverse 5′-ACT​GGC​TGA​GAA​CCA​CAG​GTC​T-3’; QH51305S, Beyotime, China) and hsa-miR-140-3p (forward 5′-GCG​CGT​ACC​ACA​GGG​TAG​AA-3′, reverse 5′-AGT​GCA​GGG​TCC​GAG​GTA​TT-3’; QH77781S, Beyotime, China), as well as 0.3 μL of LINC01711/hsa-miR-140-3p cDNA were included in qRT-PCR. All samples were run in triplicate, and mean Ct values were determined for each experimental group. Relative expression levels were calculated using the 2^−ΔΔCt method with appropriate normalization controls.

Statistical analysis

Multivariate Cox proportional hazards regression was used to assess the independent prognostic value of clinical characteristics (performed in SPSS). The Kaplan–Meier Plotter database, combined with the Kaplan–Meier survival curve analysis (in SPSS), was further used to explore the predictive value of LINC01711 for GC patient survival probability. Data are presented as mean ± standard deviation (SD). All datasets underwent normality testing using the Shapiro–Wilk test (in SPSS) before statistical analysis. The independent-sample t-test (two groups, normally distributed data), Wilcoxon rank-sum test (two groups, non-normally distributed data), one-way ANOVA with Tukey’s post-hoc test (more than two groups, normally distributed data), and Kruskal–Wallis test with Dunnett’s test (more than two groups, non-normally distributed data) were performed in SPSS to test the differences of data between groups. The association between the expression of LINC01711 and hsa-miR-140-3p was assessed using the Pearson correlation coefficient analysis. The figures in this study were drawn using GraphPad Prism.

The expression of LINC01711 in GC and its impact on GC patients

Compared with normal tissues, the expression of LINC01711 was upregulated in GC tissues (Figure 1A). A high LINC01711 expression level (≥1.93) was associated with advanced tumor-node-metastasis (TNM) stage (p = 0.016, Table 1), larger tumor size (p = 0.049, Table 1), and positive lymph node metastasis (p = 0.026, Table 1). Furthermore, compared with the low LINC01711 expression level, both the Kaplan–Meier Plotter database (Figure 1B, hazard ratio (HR): 1.52, 95 % confidence interval (CI): 1.05–2.20, p = 0.024) and Kaplan–Meier survival curve (Figure 1C, p = 0.025) showed a significant correlation between the high LINC01711 expression and a lower survival probability in GC patients.

Figure 1:

The significance of LINC01711 expression for the survival probability of gastric cancer (GC) patients. A, the bar chart showed the expression levels of LINC01711 in normal and GC tumor tissues. B, the Kaplan–Meier plotter database demonstrated the association between LINC01711 expression levels and GC patient survival probability. C, the Kaplan–Meier survival curve showed the association between LINC01711 expression levels and GC patient survival probability in this study.

The Cox proportional hazards model further confirmed high LINC01711 expression as an independent risk factor for the low survival probability of GC patients (Table 2, HR: 2.446, 95 % CI: 1.108–5.399, p = 0.027). In addition, the TNM stage (Table 2, HR: 2.227, 95 % CI: 1.071–4.632, p = 0.032) and lymph node metastasis status (Table 2, HR: 2.064, 95 % CI: 1.036–4.111, p = 0.039) were also independent risk factors for the low survival probability of GC patients.

The miRNA that interacts with LINC01711 in GC

Compared with gastric epithelial cells, there was an upregulation of LINC01711 in GC cells (Figure 2A). The HGC-27 and SNU-16 cell lines were chosen for further cellular experiments as they showed the highest LINC01711 expression levels among all GC cell lines analyzed in this study (Figure 2A). To explore the miRNAs that interacted with LINC01711, the LncBook, miRanda, and starBase databases revealed 64, 96, and 79 potential miRNA interactions, respectively (Figure 2B). Intersection analysis of these datasets yielded seven common miRNAs, including hsa-miR-1914-3p, hsa-miR-5194, hsa-miR-665, hsa-miR-140-3p, hsa-miR-6799-3p, hsa-miR-3944-3p, and hsa-miR-2467-3p (Figure 2B and C). The characteristics of these seven miRNAs expression in GC cells showed that the expression of hsa-miR-140-3p was significantly downregulated in both HGC-27 and SNU-16 cells than in GES-1 cells (Figure 2C). Hsa-miR-140-3p has been proven to be an inhibitor of the progression of GC [12]. Therefore, this study further explored the association between LINC01711 and hsa-miR-140-3p, as well as the effect of the LINC01711/hsa-miR-140-3p on GC cell activities.

Figure 2:

The target miRNA of LINC01711 in GC. A, the bar chart showed the expression levels of LINC01711 in gastric epithelial and GC cells. B, the Venn plot showed the number of LINC01711-targeted miRNAs due to the LncBook, miRanda, and starBase databases. C, the bar chart showed the expression levels of LINC01711-targeted miRNAs in gastric epithelial and GC cells. D, the bar chart showed the expression levels of hsa-miR-140-3p in normal and GC tumor tissues. E, Pearson’s correlation analysis showed a negative correlation between the expression of LINC01711 and hsa-miR-140-3p in GC tumor tissues. F, DIANA tools probed the binding site between LINC01711 and hsa-miR-140-3p. G, the bar chart showed the expression levels of hsa-miR-140-3p in hsa-miR-140-3p mimic or inhibitor transfected GC cells. H–I, dual luciferase assay showed the luciferase activity in WT-LINC01711 and MUT-LINC01711 groups with up- or downregulation of hsa-miR-140-3p in HGC-27 (H) and SNU-16 (I) cells. Different letters (a, b, c) represent significant differences (p < 0.05) from one-way ANOVA with Tukey’s post-hoc test or the kruskal–Wallis test with the Dunnett test.

The expression of hsa-miR-140-3p was downregulated in GC tumor tissue compared with normal tissues (Figure 2D) and was negatively correlated with the expression of LINC01711 (Figure 2E, r = −0.544, p<0.001). The molecular interaction interface of LINC01711 with hsa-miR-140-3p was investigated using the DIANA tools (Figure 2F). The LINC01711-hsa-miR-140-3p interaction was functionally validated by a dual-luciferase reporter assay. The transfection of hsa-miR-140-3p mimic and inhibitor significantly upregulated and downregulated hsa-miR-140-3p expression levels in both HGC-27 and SNU-16 cells, respectively (Figure 2G). In the WT-LINC01711 group, the upregulation of hsa-miR-140-3p led to a significant diminish in luciferase activity, while the downregulation of hsa-miR-140-3p led to a significant increase in luciferase activity (Figures 2H and I). However, in the MUT-LINC01711 group, changes in hsa-miR-140-3p expression did not affect luciferase activity.

Hsa-miR-140-3p mediated the effect of LINC01711 on GC cells

In this study, siRNA-mediated knockdown of LINC01711 resulted in significant downregulation in both HGC-27 and SNU-16 cells (Figure 3A). In contrast, hsa-miR-140-3p inhibition showed no significant effect on LINC01711 expression levels in either cell line (Figure 3A). The downregulation of LINC01711 led to an upregulation of hsa-miR-140-3p (Figure 3B). However, the upregulated hsa-miR-140-3p expression was further suppressed by transfection with the hsa-miR-140-3p inhibitor (Figure 3B).

Figure 3:

The effect of LINC01711/hsa-miR-140-3p axis on GC cell activities. A, the rescue experiments showed that hsa-miR-140-3p could not regulate LINC01711 expression in GC cells. B, the rescue experiments showed that LINC01711 downregulated hsa-miR-140-3p expression in GC cells. C–F, the rescue experiments showed that LINC01711 promoted GC cell proliferation (C, D), migration (E), and invasion (F) by downregulating hsa-miR-140-3p. Different letters (a, b) represent significant differences (p < 0.05) from one-way ANOVA with Tukey’s post-hoc test or the kruskal–Wallis test with the Dunnett test.

For the GC cell activities, the downregulation of LINC01711 inhibited cell activities in both HGC-27 and SNU-16 cells, which included cell proliferation (Figure 3C and D), migration (Figure 3E), and invasion (Figure 3F) abilities. Notably, hsa-miR-140-3p knockdown attenuated the anti-proliferative (Figures 3C and D), anti-migratory (Figure 3E), and anti-invasive (Figure 3F) effects mediated by LINC01711 silencing in GC cells, suggesting hsa-miR-140-3p acts as a crucial downstream effector of LINC01711.