A total of 40 pairs of primary tumor and para-cancerous tissues were obtained from patients with GC (28 males and 12 females; average age, 58.93±9.17 years) undergoing surgical resection at the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). The study was approved by the Ethics Committee of Chongqing Medical University. Informed consent was obtained from each patient prior to surgery. The clinicopathological characteristics of the patient samples were confirmed by two pathologists according to the diagnostic criteria of World Health Organization (2010-2012). Metastasis was found in 28 cases, and 25 patients were diagnosed with stage I or II gastric cancer. All samples were immediately stored in liquid nitrogen until further use.

AGS (cat. no. CRL-1739) and normal gastric mucous epithelium cells (GES-1; cat. no. 28200) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). BGC-823 (cat. no. TCHu 11), MKN-45 (cat. no. TCHu130) and SGC-7901 (cat. no. TCHu 46) were purchased from Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Science, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Science) and incubated at 37°C in a humidified atmosphere of 5% CO₂. Short hairpin RNA (shRNA) sequences targeting H19 (sh-H19) and Snail1 (sh-Snail1), as well as the negative control (sh-NC) were obtained from Genepharm Co. Ltd. (Shanghai, China). Following annealing, shRNAs were integrated into the lentiviral pU6-Luc-Puro vector (Genepharm Co. Ltd.). To generate the H19 overexpression model, the H19 fragment was amplified using PCR and then subcloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). pcDNA3.1 vectors containing wild-type (WT) H19 or Snail1, mutant (MUT) H19 or Snail1 were obtained from RioBio (Guangzhou, China). miR-22-3p mimics, inhibitors and negative control (miR-NC) were synthesized by Genepharma Co. Ltd. (Shanghai, China). All transfections were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. The cells were harvested at 48 h post-transfection for use in further experiments.

Targetscan (www.targetscan.org) and LncBase Predicted v.2 (http://www.microrna.gr/LncBase) were employed to predict the potential targets of miR-22-3p or H19. Wild-type segments of the 3′UTR of H19/Snail1 containing potential binding sites of miR-22-3p were cloned into the pMIR-REPORT firefly luciferase vector (Applied Biosystems, Foster City, CA, USA). The mutant fragment of H19/Snail1 was used as a control. 293 cells (ATCC) were co-transfected with the luciferase reporter vector and miR-22-3p mimics or miRNA negative control (miR-NC) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, the luciferase activity was evaluated using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s protocols. The activity was normalized to Renilla luciferase.

Transfected AGS and SGC-7901 cells were seeded at a density of 1×10⁴/well in 96-well plates. Proliferation rates were determined by Cell Counting kit-8 assay (CCK-8 Assay; Beyotime, Shanghai, China) at days 1, 2, 3 and 4 post-transfection. The absorbance at 450 nm was measured using a microplate reader (9200, Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.

The migration and invasion of the cells were evaluated by a Transwell assay. The AGS and SGC-7901 cells were harvested at 48 h post-transfection. For the migration assay, 5×10⁴ cells in serum-free medium were inoculated onto the upper chamber (BD Biosciences, Franklin Lakes, NJ, USA) with an 8-µm membrane. For the invasion assay, 2×10⁵ cells in serum-free medium were inoculated onto the upper chamber pre-coated with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). Culture medium containing 10% FBS was added to the lower chamber. Non-migratory cells were removed using a cotton swab following overnight incubation at 37°C, while the migrated or invaded cells in the lower chamber were fixed in 4% paraformaldehyde for 10 min at room temperature, and stained using methanol and 0.1% crystal violet. Finally, the number of migratory or invasive cells were observed and counted using an inverted light microscope (magnification, ×100; Olympus Corp., Tokyo, Japan).

RT-qPCR was used to determine the expression levels of H19, miR-22-3p, Snail1, E-cadherin (E-Cad), α-smooth muscle actin (α-SMA), vimentin (VI) and fibronectin (FN). Total RNA was extracted from the tissues or cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. miRNA was extracted using the miRNeasy Mini kit (Qiagen, Shenzhen, China) according to the manufacturer’s instructions. RNA was reverse transcribed using a PrimeScript RT kit (Takara, Dalian, China) according to the manufacturer’s instructions. For the assessment of miR-22-3p, a TaqMan MicroRNA Assay kit (Applied Biosystems) was used and qPCR was performed using the Applied Biosystem 7500 Real-Time PCR System; U6 was used for normalization. For H19, Snail1, E-Cad, α-SMA, VI and FN, qPCR was performed using SYBR-Green PCR Master Mix (Takara). Relative expression was calculated and normalized to endogenous GAPDH. The forward and reverse primer sequences were as follows: H19 forward, 5′-ATCGGTGC CTCAGCGTTCGG-3′ and reverse, 5′-CTGTCCTCG CCGTC ACACCG-3′; Snail1 forward, 5′-GAAGATGCACATCCGA AGC-3′ and reverse, 5′-AGTGGGAGCGGAGAAAGG-3′; E-Cad forward, 5′-CAATGGTGTCCATGTGAACA-3′ and reverse, 5′-CCTCCTACCCTCCTGTTCG-3′; α-SMA forward, 5′-TCCCTTGAGAAGAGTTACGAGTTG-3′ and reverse, 5′-ATGATGCTGTTGTAGGTGGTTTC-3′; VI forward, 5′-CGCTTCGCCAACTACAT-3′ and reverse, 5′-AGGGCA TCCACTTCACAG-3′; FN forward, 5′-CCAAACCTCAAGC TCCCGTCA-3′ and reverse, 5′-GAGATTCTGCACATCAC GGTCA-3′; and GAPDH forward, 5′-GCAAGAGCACAAG AGGAAGA-3′ and reverse, 5′-ACTGTGAGGAGGGGAG ATTC-3′. The PCR program used for the thermocycler was as follows: 95°C for 5 min, followed by 45 cycles of 95°C for 15 sec, 60°C for 20 sec and 72°C for 10 sec. Relative expression levels were evaluated via the 2^−∆∆Cq method (25). The results of significant difference tests between the relative and raw numerical value are the same.

Total protein was isolated from the tissues or cells using radioimmunoprecipitation assay buffer (Beyotime). Protein concentrations were evaluated by a BCA Protein Assay kit (Beyotime). Equal amounts (40 µg) of extracted protein were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% (w/v) non-fat milk in TBST buffer (Beyotime) for 1 h at 37°C. The membranes were then incubated with anti-E-Cad (1:5,000; cat. no. ab40772), anti-α-SMA (1:2,000; cat. no. ab124964), anti-VI (1:1,000; cat. no. ab20346), anti-FN (1:1,000; cat. no. ab18265), anti-Snail1 (1:500; cat. no. ab82846) and anti-GAPDH (1:1000; cat. no. ab8245) (all from Abcam, Cambridge, MA, USA) at 4°C overnight. Following 3 washes in TBST for 10 min, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1,000; cat. no. 7076) or goat anti-rabbit IgG (1:1,000; cat. no. 7074) (both from Cell Signaling Technology, Danvers, MA, USA) for 1 h at 37°C. The protein bands were visualized by an enhanced chemiluminescence kit and quantified by densitometric analysis using ImageJ software (NIH, Bethesda, MD, USA).

All animal experiments were ethically approved by the Research Ethics Committee of Chongqing Medical University (Chongqing, China). A total of 10 female BALB/C nude mice (5-6 weeks old) with a weight of 17-22 g were obtained from the Experimental Animal Centre of the Third Military Medical University (Chongqing, China). The mice were routinely housed in a humidity- (80%); and temperature-controlled (22±2°C) environment for at least 3 days prior to the experiments. A total of 2×10⁶ SGC-7901 cells transfected with sh-NC or sh-H19 were suspended in 200 µl phosphate-buffered saline and injected subcutaneously into right side of the armpit regions of mice (5 mice/group. At 6 weeks post-injection, the mice were sacrificed, and the tumors were isolated and measured. Tumor volume was calculated using the following formula: V (mm³) = 0.5 × length × width². The tumor tissues were snap-frozen in liquid nitrogen until further use.

Data are presented as the means ± standard deviation unless otherwise indicated and were analyzed using Graphpad Prism v7.0 software (Graphpad Software Inc., La Jolla, CA, USA). A t-test (two-sided) and one-way analysis of variance (ANOVA) were used for statistical analysis. A student-Newman-Keuls test was performed as a post hoc test following ANOVA. The correlation between the expression levels of Snail1 and miR-22-3p was assessed using Spearman’s correlation analysis, as appropriate. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of H19 and miR-22-3p in 40 paired GC and para-cancerous samples were determined by RT-qPCR. The results revealed that H19 expression was significantly upregulated, whereas the expression level of miR-22-3p was decreased in the GC tissues compared with the para-cancerous tissues (Fig. 1A and D). Furthermore, the effects of H19 and miR-22-3p expression on metastasis and the prognosis of patients with GC were investigated. The results indicated that H19 expression was significantly elevated and that miR-22-3p expression was decreased in patients with GC with lymph node metastasis compared with those without metastasis (Fig. 1B and E). In addition, the expression level of H19 was significantly increased and that of miR-22-3p was decreased in aggressive GC tumors (TNM stage III/IV), compared with stage I/II tumors, suggesting that the upregulation of H19 and the downregulation of miR-22-3p are associated with the development of GC (Fig. 1C and F). Furthermore, the correlation between H19 and miR-22-3p expression was analyzed, and the results indicated that the expression levels of H19 and miR-22-3p inversely correlated in GC tissues (Fig. 1G). Additionally, H19 was significantly upregulated and miR-22-3p was downregulated in GC cells lines compared with GES-1 cells (Fig. 1H and I). These results suggested that the expression of H19 and miR-22-3p is upregulated and downregulated in GC respectively, which is also associated with metastasis in patients with GC. Furthermore, the miR-22-3p level is downregulated by H19 in GC cells, suggesting that H19 may exert its regulatory function in GC via miR-22-3p.

To examine the effects of H19 on the proliferation, invasion and migration of GC cells, the expression of H19 was silenced by sh-H19 in AGS (the lowest expression of H19) and SGC-7901 (the highest expression of H19) cells. The knockdown efficiency of H19 was determined by RT-qPCR (Fig. 2A). The results of CCK-8 assay revealed that the proliferative ability of the AGS and SGC-7901 cells transfected with sh-H19 was decreased compared with the controls (Fig. 2B and C). In addition, Transwell assay indicated that the invasion and migration of the AGS and SGC-7901 cells transfected with sh-H19 were significantly suppressed (Fig. 2D-G). Furthermore, the results of RT-qPCR and western blot analysis revealed that the expression level of E-Cad was notably increased, whereas the expression levels of α-SMA, VI and FN were decreased in the sh-H19-transfected cells compared with the controls (Fig. 2H-J). On the whole, these results suggested that the downregulation of H19 suppressed the growth, metastasis and EMT of GC cells.

To determine whether H19 functions by suppressing its target miRNA in GC, the potential binding sites of miR-22-3p in H19 transcripts were predicted using LncBase Predicted v.2 (Fig. 3A). Luciferase reporter plasmids containing the wild-type H19 (H19-WT) and mutant H19 (H19-MUT) of predicted miR-22-3p binding sites were constructed. The results revealed that transfection with miR-22-3p mimics significantly reduced the luciferase activity in H19-WT, while the luciferase activity in 293 cells transfected with H19-MUT was not affected by miR-22-3p mimics (Fig. 3B). In order to further examine the effects of H19 on the expression level of miR-22-3p, the AGS and SGC-7901 cells were transfected with sh-H19. The cells transfected with sh-H19 exhibited a marked increase in miR-22-3p levels (Fig. 3C). Furthermore, H19 knockdown resulted in a prominent decrease in Snail1 protein expression in the AGS and SGC-7901 cells (Fig. 3D). Additionally, a miR-22-3p overexpression model was established by transfecting the AGS and SGC-7901 cells with miR-22-3p mimics, and the transfection efficiency was confirmed by RT-qPCR (Fig. 3E). Furthermore, the overexpression of miR-22-3p inhibited the expression level of Snail1 in the AGS and SGC-7901 cells (Fig. 3F), suggesting that the miR-22-3p/Snail1 axis may be a potential target of H19 in GC cells.

To further investigate the association between H19 and miR-22-3p, the AGS and SGC-7901 cells were transfected with pc-NC, pc-H19 or co-transfected with pc-H19 and miR-22-3p mimics. The expression of H19 was significantly increased (Fig. 4A) and the level of miR-22-3p decreased (Fig. 4B) in the AGS and SGC-7901 cells transfected with pc-H19. Furthermore, the overexpression of H19 promoted the proliferation (Fig. 4C and D), invasion (Fig. 4E), migration (Fig. 4F) and EMT (Fig. 4G-I) of the AGS and SGC-7901 cells, whereas these effects were substantially abrogated by the upregulation of miR-22-3p. In addition, the overexpression of H19 increased the level of Snail1, which was inhibited by the upregulation of miR-22-3p (Fig. 4J). These results revealed that H19 may promote the proliferation, invasion, migration and EMT of GC cells by downregulating miR-22-3p and upregulating Snail1.

Using the TargetScan database, the binding sites of Snail1 on miR-22-3p was predicted (Fig. 5A). To investigate whether Snail1 is the potential target of miR-22-3p, the WT and MUT fragments of Snail1 were cloned into the downstream of the Firefly luciferase coding domain in pGL-3 vector, and the results revealed that the co-transfection of miR-22-3p and Snail1-WT reduced the relative luciferase activity, whereas the activity remained unaltered in the cells co-transfected with miR-22-3p and Snail1-MUT (Fig. 5B). To further determine whether miR-22-3p can regulate the expression level of Snail1, the AGS and SGC-7901 cells were transfected with miR-22-3p inhibitors. The results confirmed that miR-22-3p was significantly downregulated in the cells transfected with miR-22-3p inhibitors (Fig. 5C), whereas the protein level of Snail1 was elevated (Fig. 5D). Furthermore, Snail1 expression was upregulated in the GC tissues compared with the paired para-cancerous tissues (Fig. 5E), and Snail1 expression inversely correlated with miR-22-3p expression in GC tissues (Fig. 5F), suggesting that Snail1 may be a potential target of miR-22-3p in GC. To further verify the role of Snail1, the AGS and SGC-7901 cells were transfected with sh-Snail1 and Snail1 was significantly downregulated in the cells transfected with sh-Snail1 (Fig. 5G).

To investigate whether the functions of Snail1 as regards the growth, metastasis and EMT of GC cells are regulated by miR-22-3p, the AGS and SGC-7901 cells were transfected with miR-NC, miR-22-3p mimics or co-transfected with miR-22-3p mimics and sh-Snail1, miR-22-3p inhibitors and sh-Snail1, respectively. The results revealed that the overexpression of miR-22-3p suppressed the proliferation (Fig. 6A and B), invasion (Fig. 6E) and migration (Fig. 6F) of the AGS and SGC-7901 cells, while these effects were enhanced by the silencing of Snail1 expression. Conversely, the downregulation of miR-22-3p promoted the proliferation (Fig. 6C and D), invasion (Fig. 6G) and migration (Fig. 6H) of GC cells, whereas these effects were abrogated by the silencing of Snail1 expression. On the whole, the results of the present study suggest that miR-22-3p may inhibit the growth of GC by downregulating Snail1. In summary, H19 may regulate the proliferation, invasion, migration and EMT of GC cells via the miR-22-3p/Snail1 signaling pathway.

To investigate whether sh-H19 suppresses the growth and metastasis of gastric tumors, GC cells transfected with sh-NC or sh-H19 were subcutaneously injected into BALB/C nude mice. At 6 weeks post-injection, the mice were sacrificed and the isolated tumors were measured (Fig. 7A). The mean value of the tumor volume in the sh-H19 group was significantly reduced compared with that in the sh-NC group (Fig. 7B). In addition, the tumor weight in the sh-H19 group was notably decreased compared with that in the sh-NC group (Fig. 7C). Furthermore, the numbers of macroscopic nodules were significantly reduced in the sh-H19 group (Fig. 7D). Additionally, RT-qPCR revealed that the mRNA levels of H19 and Snail1 were downregulated in the sh-H19 group, whereas the expression level of miR-22-3p was elevated (Fig. 7E) in these tumors, suggesting that the downregulation of H19 may suppress the growth of gastric tumors via the miR-22-3p/Snail1 axis in vivo.