Human GC cell lines MGC-803, SGC-7901, BGC-823, AGS and human normal gastric mucous epithelium cell GES-1 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were grown in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) (all from Gibco, Carlsbad, CA, USA). All cells were cultured in humidified incubator containing 5% CO₂ at 37°C.

Total RNA were isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. TaqMan miRNA assays were performed to determine the expression of miR-let-7a. TaqMan miRNA reverse transcription kit (Invitrogen) was used for cDNA synthesis and qRT-PCR was carried out by ABI StepOne Plus (Applied Biosystems, Foster City, CA, USA). U6 was an endogenous reference for miR-let-7a quantification using the 2^−ΔΔCt method. The relative expression of Rictor was quantifed using SYBR-Green Master Mix kit (Roche Diagnostics, Indianapolis, IN, USA) and β-actin was selected as control. The specific primers were as follows: miR-let-7a forward, 5′-GGTGAGGTAGTAGGTTGTATAGTT-3′ and reverse, 5′-CTCGCTTCGGCAGCACATATA-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; Rictor forward, 5′-ACCGGGCTTCTGACCATTAAA-3′ and reverse, 5′TTGTATGAACCGCCGACACT-3′; β-actin forward, 5′-AGAAAATCTGGCACCACACC-3′ and reverse, 5′-TAGCACAGCCTGGATAGCAA-3′. All data collected were in triplicate.

RIPA buffer (Beyotime, Shanghai, China) was used to isolate total proteins from cell lysates on ice. Equal amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis. Proteins on the gel were transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked with Tris-buffered saline with Tween-20 (TBST) containing 5% milk for 2 h and incubated overnight in special primary antibodies: anti-LC3, anti-p62, anti-Rictor, anti-Akt, anti-p-Akt, anti-mTOR, anti-p-mTOR and anti-GAPDH (dilution 1:1,000; Cell Signaling Technology, Beverly, MA, USA). After being washed with TBST three times 15-min each, the HRR-conjugated anti-mouse or anti-rabbit IgG antibodies (dilution 1:20,000; Jackson ImmunoResearch, Inc., West Grove, PA, USA) were incubated with bolted membranes at room temperature for 2 h and washed again. The enhanced chemiluminescence detection system was used to detect the expression of target bands.

Cells were seeded into glass bottom cell culture dish at 20,000 cells and cultured for 48 h. After being washed with PBS three times, cells were fixed in 4% paraformaldehyde for 20 min and permeabilized by 0.5% Triton X-100 for 10 min. Then, cells were blocked with 3% bovine serum albumin for 1 h and incubated overnight in special primary antibody: anti-LC3 (dilution 1:200). After removing primary antibody and washing again with PBS next day, cells were incubated with Alexa Flour 594 Rhodamine-conjugated goat anti-rabbit (dilution 1:200; Jackson ImmunoResearch) for 1 h and 30 min and mounted by staining with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Confocal microscopy (Carl Zeiss LSM710; Carl Zeiss, Jena, Germany) was used to photograph the stained cells in a ×40 oil lens and LC3 puncta was manually quantified.

The miR-let-7a mimics and miR-let-7a inhibitor vector lentiviral constructs (GenePharma, Shanghai, China) were modified to interfere with expression of miR-let-7a, as were siRictor and pcDNA3.1-Rictor to interfere with expression of Rictor from Santa Cruz Biotechnology (Dallas, TX, USA). Negative miRNA-let-7a and scramble Rictor were also transfected as negative controls. When MGC-803, SGC-7901 and GES-1 cells grew to 50–60% confluency, the miR-let-7a-NC, miR-let-7a mimics and miR-let-7a inhibitor lentiviral vectors were used to infect cells according to different multiplicity of infection (MOI) offered by the manufacturer. Puromycin (3 µg/ml) (Sigma, St. Louis, MO, USA) was used to screen out stable cell lines for one week and the expression of miR-let-7a was analyzed by qRT-PCR. siRNA against Rictor and pcDNA3.1-Rictor plasmids were transfected into GC cells using Lipofectamine 3000 reagent (Invitrogen).

The wild-type (WT) or mutated-type (MUT) 3′-UTRs of Rictor containing miR-let-7a binding sites were amplified by PCR and subcloned into pmirGLO Dual-Luciferase miRNA Target Expression vectors (Promega, Madison, WI, USA). For luciferase activity assays, HEK293T cells were seeded into a 24-well plate, then co-transfected with Rictor 3′-UTR construct and miR-let-7a or NC using Lipofectamine 3000 (Invitrogen). Luciferase activity was measured by Dual-Luciferase Reporter assay kit (Promega, USA) and normalized by Renilla luciferase overnight.

Data are presented as mean ± standard deviation (SD) from at least three independent experiments and the Students two-tailed t-test was used to determine whether these data are statistically significant. Values of P<0.05 were considered significant.

To elucidate the role of miR-let-7a on autophagic activity in GC, we firstly constructed miR-let-7a mimics and miR-let-7a inhibitors using lentiviral transfection in GC cells (MGC-803 and SGC-7901). miRNA real-time polymerase chain reaction (RT-PCR) was used to determine the expression of miR-let-7a (Fig. 1A and B). As shown, miR-let-7a was knocked downed ~65 and 60%, increased appoximately 9- and 14-fold in MGC-803 and SGC-7901 cells, respectively. Next, we explored the effect of miR-let-7a on autophagy, confocal imaging was employed to evaluate the number of LC3 puncta (Fig. 1C and D). It showed that the number of LC3 puncta were markedly elevated in MGC-803 and SGC-7901 cells which were overexpressed of miR-let-7a. However, the LC3 dot formation was significantly reduced after knocking down miR-let-7a both in MGC-803 and SGC-7901 cells. These results recognized miR-let-7a as an enhancer of autophagy and it contributes to the promotion of autophagic responses in GC cells.

The conversion of LC3-I to LC3-II and p62 protein expression were detected by western blotting. LC3-I scattered in the cytoplasm, and upon autophagy induction, LC3-I was conjugated with phosphatidylethanolamine (PE) to form LC3-II and localized to autophagosomes and adventitia. LC3-II is always retained on the autophagic membrane until it is fused to lysosomes. We found that LC3-II/GAPDH ratios were increased after overexpressing miR-let-7a and were decreased after downregulating miR-let-7a, these results were consistent with previous LC3 puncta formation assay (Fig. 2A and B, and D and E). P62 is a selective autophagic-lysosomal degradation substrate, total cellular p62 protein levels reflect autophagic activity. When autophagy occurs, p62 levels are decreased, while autophagic activity is inhibited, p62 protein is accumulated. As expected, overexpression of miR-let-7a led to reduced p62 protein levels, but inhibition of miR-let-7a resulted in an increase of p62 expression (Fig. 2A, C, D and F). Then, we performed an autophagic flux assay to determine whether autophagosome accumulation resulted from increased autophagic activity or due to a block in downstream degradation. Chloroquine (CQ), a lysosomotropic reagent, was used to block autophagosome degradation in MGC-803 and SGC-7901 cells. As shown, lipidated LC3-II was significantly increased both in NC and miR-let-7a transfected cells by CQ (Fig. 2A and B and D and E). Similarly, p62 protein levels were also increased in miR-let-7a transfected cells treated with CQ (Fig. 2A, C, D and F). These results demonstrate that miR-let-7a affected autophagic activity in GC cells.

In order to clarify the specific mechanism of miR-let-7a-induced autophagy, bioinformatics software including TargetScan and miRanda were employed to predict the potential binding sequences in the 3′-untranslated region (3′-UTR) of target genes. As shown, the 3′-UTR of human Rictor mRNA resides underlying miR-let-7a binding sequences (Fig. 3A). Furthermore, the wild-type (WT) or mutated-type (MUT) sequences in the 3′-UTR of Rictor that miR-let-7a targets were designed to construct luciferase reporter vector and co-transfected with either miR-let-7a or NC control. As shown, overexpression of miR-let-7a markedly reduced luciferase activity when HEK293T cells were co-transfected with WT 3′-UTR of Rictor compared to NC. In contrast, this inhibitory effect of miR-let-7a on luciferase activity was abrogated when HEK293T cells were co-transfected with MUT (Fig. 3B).

We performed RT-PCR assay and western blot analysis to further confirm whether miR-let-7a suppressed the expression of Rictor in GC cells. As shown, overexpression of miR-let-7a led to a significant decrease both in mRNA and protein levels in MGC-803 and SGC-7901 cells compared with NC. However, inhibition of miR-let-7a notably increased the levels of Rictor mRNA and protein (Fig. 3C-H). Therefore, the data above provided strong evidence that Rictor is the target of miR-let-7a, and miR-let-7a inhibits the expression of Rictor by directly binding to its 3′-UTR.

To explore whether the effect of miR-let-7a on GC cell autophagic activity was mediated by Rictor, we performed ‘rescue experiments’. GC cell lines MGC-803 and SGC-7901 were co-transfected with miR-let-7a overexpression lentivirus and either pcDNA3.1-Rictor plasmids or empty vector, worthwhile, other MGC-803 and SGC-7901 cells were co-transfected with miR-let-7a supression lentivirus and either siRictor or empty vector, respectively. As shown, upregulated Rictor expression reversed the promotion of autophagic activity caused by the overexpression of miR-let-7a, the results suggested that LC3-II/GAPDH ratios were decreased and p62 protein levels were increased in MGC-803 and SGC-7901 cells (Fig. 4A and B). Simultaneously, we found that the similar rescue effect was observed in MGC-803 and SGC-7901 cells where downregulated Rictor expression reversed the supression of autophagic activity caused by the knockdown of miR-let-7a (Fig. 4A and B).

Rapamycin, as a potent immunosuppressive and antitumor agent (24), is commonly used in autophagy study. As a control, we found that miR-let-7a enhanced the activity of autophagy as well as rapamycin by confocal imaging in MGC-803 cells, and LC3 dots formation was reversed upon co-expression with Rictor (Fig. 4C). Inhibition of endogenous miR-let-7a led to the opposite effect (Fig. 4D). These results show that Rictor is the vital functional element in miR-let-7a-mediated autophagic response.

It has been reported that Akt-mTOR signaling pathway is involved in autophagic activity and Rictor plays an important role in the Akt-mTOR signaling pathway (21). We confirmed that Akt and mTOR phosphorylations were involved in miR-let-7a-mediated autophagy in GC cells by western blot analysis. As shown, both the protein levels of phosphorylated Akt and phosphorylated mTOR were significantly decreased by upregulating miR-let-7a and increased by downregulating miR-let-7a in MGC-803 and SGC-7901 cells. However, the total protein levels were almost unchanged (Fig. 5A and B). These data indicated that there is a direct link between miR-let-7a and Akt-mTOR signaling pathway. Therefore, we confirm that miR-let-7a regulates autophagic activity throughout the Akt-mTOR signaling pathway in GC cells.

Knockdown of miR-let-7a suppresses human normal gastric mucous epithelium cell autophagic activity. To investigate whether miR-let-7a affects autophagy in human normal gastric mucous epithelium cells (GES-1), we detected the expression level of miR-let-7a in GC cells and GES-1. We found that the expression of miR-let-7a in GES-1 cells was relatively higher than that in GC cells (Fig. 6A). Thus, we transfected GES-1 cells with miR-let-7a inhibitor or NC. Successful knockdown of miR-let-7a was confirmed by RT-PCR (Fig. 6B). The expression of LC3-II was reduced, and P62 was relatively higher compared with control or NC after knockdown of miR-let-7a by western blotting (Fig. 6C-E). These results suggest that miR-let-7a affects autophagy in human normal gastric mucous epithelium cells.