The human GC cell line AGS was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The human GC cell line HGC-27 and the 293T cell line were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). AGS cells were maintained in F-12K medium (ATCC) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). HGC-27 cells were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 1.5 g/l NaHCO₃ (Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA), 2.5 g/l glucose (Sigma-Aldrich; Merck KGaA), 0.11 g/l sodium pyruvate (Sigma-Aldrich; Merck KGaA) and 20% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). 293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.). HGC-27 and AGS cells were continuously exposed to gradually increasing concentrations of CDDP (0.5-10 µg/ml) for >6 months to establish a CDDP-resistant HGC-27 cell line (HGC-27/CDDP) and a CDDP-resistant AGS (AGS/CDDP) cell line.

miR-30b mimic and its scramble control (miR-NC), miR-30b inhibitor and its negative control (anti-miR-30b), small interfering RNA (siRNA) targeting MALAT1 (si-MALAT1) and its negative control (si-NC), were synthesized by GenePharma (Shanghai, China). Full-length sequences of MALAT1 or autophagy-related gene 5 (ATG5) were constructed into a pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.) to generate pcDNA-MALAT1 (MALAT1) or pcDNA-ATG5 (ATG5) overexpression plasmid, respectively. All plasmids or oligonucleotides were transfected into GC cells by Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific Inc.) according to the instructions of the manufacturer. CDDP was purchased from MedChem Express Co., Ltd. (Monmouth Junction, NJ, USA).

Total RNA (mRNAs and microRNAs) was isolated from GC or CDDP-resistant GC cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified by NanoDrop ND 1000 (NanoDrop Technologies, Wilmington, DE, USA). The analysis of miR-30b expression was performed using TaqMan^® MicroRNA Real-Time PCR Assay reagents and primers (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA) with RNU6B (primers also purchased from Applied Biosystems; Thermo Fisher Scientific, Inc.) as an endogenous control. For MALAT1 expression analysis, RNAs were reversely transcribed into first-strand cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and random primers, followed by qPCR detection by SYBR^® Premix Ex Taq™ reagent (Takara, Otsu, Japan) and specific quantitative primers (for MALAT1 or GAPDH). GAPDH was employed to normalize the expression of MALAT1. The primers for MALAT1 or GAPDH were as follows: MALAT1, 5'-CTTAAGCGCAGCGCCATTTT-3' (forward) and 5'-CCTCCAAACCCCAAGACCAA-3' (reverse) ; GAPDH, 5 ' - CAACTCCCTCAAGATTGTCAGCAA-3' (forward) and 5'-GGCATGGACTGTGGTCATGA-3' (reverse).

For the determination of half maximal inhibitory concentration (IC₅₀), cells (10⁴ cells/well) were seeded into 96-well plates and then treated with varying concentrations of CDDP (0-25 µg/ml) for 48 h. For the detection of drug sensitivity, untransfected or transfected cells were treated with CDDP (5 µg/ml) for 48 h. Next, cell viability was determined by the CCK-8 assay kit (Dojindo Molecular Technologies, Rockville, MD, USA) following the manufacturer's instructions. Briefly, 10 µl of CCK-8 solution was added into each well of the 96-well plates. After 3 h of incubation, cell absorbance was examined at 450 nm.

Total proteins were extracted using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) and quantified using Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Subsequently, equal amounts of protein (50 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes (Pall, New York, NY, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were probed overnight at 4°C with rabbit monoclonal antibody against ATG5 (ab108327, 1:2,000 dilution; Abcam, Cambridge, UK), mouse monoclonal antibody against p62 (ab56416, 1:1,000 dilution; Abcam), rabbit polyclonal antibody against LC3B (ab51520, 1:3,000 dilution; Abcam, containing LC3-I and LC3-II), or rabbit polyclonal antibody against β-actin (ab8227, 1:2,000 dilution; Abcam). Next, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit (ab6721, 1:5,000 dilution; Abcam) or goat anti-mouse (ab6789, 1:5,000 dilution; Abcam) secondary antibody for 1 h at room temperature. Finally, protein signals were detected by Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and analyzed by Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Partial sequences of MALAT1 or ATG5 3'-untranslated region containing predicted miR-30b-binding sites were constructed into a psiCHECK-2 luciferase vector (Promega Corporation, Madison, WI, USA) to generate MALAT1 wild-type (WT) or ATG5 WT luciferase reporter, respectively. Also, MALAT1 mutant (MUT) or ATG5 MUT luciferase reporter containing mutant miR-30b-binding sites were also produced using GeneArt™ Site-Directed Mutagenesis System (Invitrogen; Thermo Fisher Scientific, Inc.). Then, the constructed luciferase reporters were transfected into 293T cells in combination with mimics or plasmids. At 48 h after transfection, luciferase activities were determined using a dual luciferase reporter assay kit (Promega Corporation).

RIP assay was performed using Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (EMD Millipore, Bedford, MA, USA) and primary antibody against IgG (EMD Millipore) or Argonaute 2 (Ago2; EMD Millipore) according to the instructions of the manufacturer.

GC cells and CDDP-resistant GC cells were transfected with pSelect-GFP-hLC3 plasmid (InvivoGen, San Diego, CA, USA) and other plasmids or oligomers (mimics, inhibitors or siRNAs), followed by treatment with CDDP (5 µg/ml) for 48 h. The cells were then fixed using 4% paraformaldehyde for 20 min at 4°C and stained using DAPI solution (1 µg/ml; Beyotime Institute of Biotechnology) for 15 min. Finally, the cells were visualized using a confocal laser scanning microscope (TCS-TIV; Leica, Nussloch, Germany). The percentage of GFP-LC3-positive cells containing >20 puncta was counted in 50 randomly selected fields.

Data were obtained from at least 3 independent experiments with the results expressed as mean ± standard deviation. Student's t-test was used to compare the difference between two groups, and one-way analysis of variance followed by Tukey's post-host test was employed to evaluate the differences among multiple (>2) groups. In all cases, P<0.05 indicated that the difference was statistically significant.

First, CDDP-resistant GC cell lines (AGS/CDDP and HGC-27/CDDP) were established to explore whether MALAT1 was associated with CDDP resistance in GC. As shown in Fig. 1A, the IC₅₀ of CDDP was markedly increased in AGS/CDDP and HGC-27/CDDP cells when compared with that in the corresponding parental cell lines (AGS and HGC-27, respectively), indicating that CDDP-resistant GC cell lines were successfully established. Then, it was further demonstrated that MALAT1 expression was notably upregulated in AGS/CDDP and HGC-27/CDDP cell lines relative to the respective parental cell lines (Fig. 1B), indicating that MALAT1 may be correlated with CDDP resistance in GC.

RT-qPCR analysis further demonstrated that the level of MALAT1 was notably increased in AGS/CDDP cells transfected with MALAT1 overexpression plasmid, but was strikingly reduced in HGC-27/CDDP cells transfected with si-MALAT1 (Fig. 2A). Therefore, MALAT1 overexpression plasmid and si-MALAT1 were used for the following gain-of-function and loss-of-function assays, respectively. Moreover, MALAT1 overexpression induced an obvious increase in AGS/CDDP cell viability (Fig. 2B). Conversely, the viability of MALAT1-silenced HGC-27/CDDP cells was markedly reduced (Fig. 2B). The CCK-8 assay further demonstrated that AGS/CDDP and HGC-27/CDDP cells were more resistant to CDDP compared with their respective parental cells (Fig. 2C). Moreover, ectopic expression of MALAT1 markedly enhanced the resistance of AGS/CDDP cells to CDDP (Fig. 2C). Conversely, MALAT1 knockdown weakened CDDP resistance of HGC-27/CDDP cells (Fig. 2C). Previous studies indicated that MALAT1 improved drug resistance of cancer cells by promoting autophagy (19,20). Hence, the effect of MALAT1 on autophagy was further investigated. The results revealed that cell autophagic activity was markedly increased in CDDP-resistant GC cell lines, as evidenced by an increased percentage of GFP-LC3-positive cells (Fig. 2D), the LC3-II/LC3-I protein ratio was increased (Fig. 2E) and p62 protein expression was reduced (Fig. 2E)in AGS/CDDP and HGC-27/CDDP cells relative to their respective parental cells. Moreover, MALAT1 overexpression facilitated cell autophagy in AGS/CDDP cells, while MALAT1 knockdown suppressed autophagy in HGC-27/CDDP cells (Fig. 2D and E). In summary, these results demonstrated that MALAT1 potentiated CDDP resistance by promoting autophagy in CDDP-resistant GC cells.

Accumulating evidence supports the hypothesis that lncRNAs may act as competing endogenous RNAs (ceRNAs) to regulate the expression of miRNAs and miRNA target genes in GC (21). Hence, bioinformatics analysis was performed by miRcode online website to identify miRNAs that may interact with MALAT1. Among candidate miRNAs, miR-30b was selected due to its critical role in GC progression and autophagy (22-24) (Fig. 3A). Subsequent luciferase assay demonstrated that miR-30b overexpression markedly reduced luciferase activity of the wild-type MALAT1 reporter, but did not affect luciferase activity of the mutant MALAT1 reporter, suggesting that MALAT1 may interact with miR-30b by putative binding sites (Fig. 3B). To further investigate the spatial interaction between MALAT1 and miR-30b, RIP assay was performed in AGS/CDDP cell lysates using an antibody against Ago2, which is an essential component of the RNA-induced silencing complex (RISC). The results revealed that miR-30b and MALAT1 were substantially enriched in Ago2 immunoprecipitation complexes (Fig. 3C), indicating that MALAT1 was able to spatially interact with miR-30b. RT-qPCR assay further demonstrated that miR-30b expression was notably downregulated in MALAT1-overexpressing AGS/CDDP cells, but was markedly upregulated in MALAT1-silenced AGS/CDDP cells (Fig. 3D). In summary, these results indicate that MALAT1 inhibits miR-30b expression by direct interaction.

Next, restoration experiments were performed to determine whether MALAT1 enhanced CDDP resistance by regulating miR-30b expression in CDDP-resistant GC cells. First, it was demonstrated that the introduction of miR-30b mimic markedly ablated the inhibitory effect of MALAT1 on miR-30b expression in AGS/CDDP cells. Conversely, the transfection of miR-30b inhibitor notably abolished si-MALAT1-induced miR-30b upregulation in HGC-27/CDDP cells (Fig. 4A). Functional analyses revealed that miR-30b overexpression effectively reduced MALAT1-mediated CDDP resistance in AGS/CDDP cells (Fig. 4B). By contrast, the resistance of HGC-27/CDDP cells to CDDP was notably enhanced in si-MALAT1-transfected HGC-27/CDDP cells following miR-30b downregulation (Fig. 4B). Therefore, miR-30b depletion abrogated the suppressive effect of MALAT1 knockdown on CDDP resistance in HGC-27/CDDP cells. Rescue assays further demonstrated that the restoration of miR-30b attenuated MALAT1-induced autophagy in AGS/CDDP cells, as evidenced by a reduced GFP-LC3-positive cell percentage (Fig. 4C), decreased the LC3-II/LC3-I protein ratio and increased p62 protein expression (Fig. 4D) in MALAT1-enforced AGS/CDDP cells after the introduction of miR-30b mimic. By contrast, the loss of miR-30b relieved si-MALAT1-mediated autophagy inhibition in HGC-27/CDDP cells (Fig. 4C and D). In summary, these data demonstrated that miR-30b weakened MALAT1-induced CDDP resistance by inhibiting autophagy in CDDP-resistant GC cells.

The TargetScan online website was then employed to search for targets of miR-30b. Among candidate targets, ATG5 was selected on account of its key roles in autophagy and chemoresistance (11,25) (Fig. 5A). A luciferase assay revealed that the introduction of miR-30b mimic resulted in a marked reduction of luciferase activity of the wild-type ATG5 reporter (Fig. 5B). However, miR-30b expression exerted no effect on luciferase activity of the mutant ATG5 reporter (Fig. 5B). These data indicated that miR-30b may interact with ATG5 by predicted binding sites. Moreover, miR-30b upregulation strikingly inhibited ATG5 expression in AGS/CDDP cells (Fig. 5C). Conversely, miR-30b depletion induced a marked increase of the ATG5 level in AGS/CDDP cells (Fig. 5C). Generally, these results confirmed that ATG5 is a target of miR-30b.

It was next demonstrated that ATG5 overexpression reversed the miR-30b-mediated inhibitory effect on ATG5 expression, and ATG5 silencing weakened anti-miR-30b-induced ATG5 upregulation (Fig. 6A). Functional analyses revealed that miR-30b upregulation resulted in reduced CDDP resistance (Fig. 6B), reduced GFP-LC3-positive cell percentage (Fig. 6C), decreased the LC3-II/LC3-I protein ratio and increased p62 protein expression (Fig. 6D) in AGS/CDDP cells. Therefore, miR-30b overexpression alleviated CDDP resistance and inhibited autophagy in AGS/CDDP cells, while these effects were abolished by increased ATG5 expression (Fig. 6B-D). By contrast, ATG5 knockdown rescued anti-miR-30b-induced CDDP resistance and autophagy in HGC-27/CDDP cells (Fig. 6B-D). In summary, these results revealed that miR-30b reduced CDDP resistance and autophagic activity of AGS/CDDP and HGC-27/CDDP cells via targeting ATG5.

We further investigated whether MALAT1 could act as a ceRNA of miR-30b to increase ATG5 expression in AGS/CDDP and HGC-27/CDDP cells. As shown in Fig. 7A, miR-30b overexpression attenuated luciferase activity of the wild-type ATG5 reporter, while this effect was rescued by increased MALAT1 expression in 293T cells. Western blot assay further revealed that ectopic expression of MALAT1 induced the upregulation of the ATG5 protein expression, while this effect was weakened by increased miR-30b expression in AGS/CDDP cells (Fig. 7B). By contrast, ATG5 expression was decreased in MALAT1-silenced HGC-27/CDDP cells and the downregulation of miR-30b alleviated the inhibitory effect of MALAT1 knockdown on ATG5 expression in HGC-27/CDDP cells (Fig. 7C). In conclusion, these data demonstrated that MALAT1 sequestered miR-30b from ATG5, resulting in the upregulation of ATG5 expression in CDDP-resistant GC cells.