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SIRT1-targeted miR-543 autophagy inhibition and epithelial–mesenchymal transition promotion in Helicobacter pylori CagA-associated gastric cancer

Gastric cancer is an important cause of death worldwide with Helicobacter pylori (H. pylori) considered a leading and known risk factor for its development. More particularly and despite the underlying mechanisms not being very clear, studies have revealed that the H. pylori cytotoxin-associated gene A (CagA) protein plays a key role in this process. In this study it was found that H. pylori increased the expression of miR-543 in human gastric cancer tissue when compared with H. pylori-negative gastric cancer tissue samples. In vitro experiments showed that increased expression of miR-543 induced by CagA is a strong promoter of cell proliferation, migration, and invasion. Conversely, a miR-543 inhibitor suppressed or reversed these effects. It was furthermore found that silencing miR-543 inhibited autophagy and led to epithelial–mesenchymal transition (EMT) under in vitro. The mechanisms by which miR-543 targets SIRT1 to downregulate autophagy was also described. The results suggest that in the progression of H. pylori-associated gastric cancer, CagA induces overexpression of miR-543, which subsequently targets SIRT1 to suppress autophagy. This may be followed by increased expression of EMT causing cell migration and invasion. Consequently, miR-543 might be considered a therapeutic target for H. pylori-associated gastric cancer.


Introduction
Gastric cancer, universally known as one of the most serious malignancies, is the fourth most commonly diagnosed cancer and the second most frequent cause of cancer-related deaths worldwide 1 . More than 1 million cases and 783,000 gastric cancer-associated deaths occurred worldwide in 2018 2 . Helicobacter pylori (H. pylori), a gram-negative pathogen, specifically colonizes the gastric epithelium 3 . This organism is present in approximately half of the global population 4 . A notable consequence of infection with H. pylori is an increased risk of atrophic gastritis and metaplastic gastritis. These can potentially progress to various diseases, such as peptic ulcers, gastric lymphoma or even gastric cancer 5,6 . Further studies have revealed that various virulence factors associated with H. pylori constitute a major cause of these diseases 7,8 . It has been reported that the cytotoxinassociated gene A (CagA), which is a virulence factor encoded by the cag pathogenicity island (cag-PAI) in 89% of H. pylori strains, plays critical roles in the pathogenicity of H. pylori 9,10 . However, the specific molecular mechanism(s) of H. pylori-associated gastric cancer is still unclear.
An increasing number of studies have suggested that microRNAs (miRNAs) play a key role in H. pylori-associated gastric diseases [11][12][13] . Approximately 19-25 nucleotides in length, miRNAs are short RNA transcripts that do not, or have a limited ability to, encode for proteins, and negatively regulate specific target gene expression [14][15][16][17] . In previous reports, miRNA microarrays have demonstrated that miR-543 expression is significantly increased in H. pylori-positive (HP+) gastric cancer tissue when compared with H. pylori-negative (HP−) cancer tissue samples 18 . It has also been demonstrated that miR-543 has the ability to accelerate cell proliferation, migration, and invasion in the development of various malignancies. These malignancies include colorectal, prostate, and lung cancer [19][20][21] .
Bioinformatics analysis has further predicted that miR-543 directly targets the 3ʹ-UTR of SIRT1 and leads to the repression of gene expression. Previous studies have shown that SIRT1-mediated deacetylation and activation of LKB1 results in increased phosphorylation of AMPK, which can trigger autophagy [22][23][24] . It has been revealed that autophagy is downregulated in human gastric mucosa with CagA-positive (CagA+) H. pylori infections and that the inhibition of autophagy increases cytokine production. This, in turn, promotes the H. pylori-induced inflammatory response 25 . When it comes to H. pyloriinduced cancer, further reports found that downregulation of p14ARF, as induced by H. pylori CagA, leads to the inhibition of autophagy 26 . We therefore postulated that miR-543 expression is increased by CagA, targets SIRT1, and inhibits autophagy in H. pylori-associated gastric cancer.
In the present study, expression of miR-543 and SIRT1 in human gastric cancer and paracarcinoma tissue samples were investigated. We analyzed cell proliferation, migration, and invasion in gastric cancer cell lines after infection with CagA+ H. pylori. Epithelial-mesenchymal transition (EMT) and autophagy in cells with different expression levels of miR-543 and SIRT1 were also studied. Our findings indicated that miR-543 may be considered a therapeutic target for H. pylori-associated gastric cancer.

H. pylori CagA increases expression of miR-543
The CagA protein has been considered a major cause of H. pylori-induced gastric carcinoma 27 . Most of the H. pylori hosts in China were infected with CagA+ strains 28,29 . We collected gastric cancer and paracarcinoma tissue samples from 50 patients and divided the samples into HP+ (all CagA+) and HP− groups. RT-PCR analysis demonstrated that, when compared with HP − tumor or normal tissue samples, the expression of miR-543 was significantly increased and SIRT1 expression decreased in HP+ tumor tissue (Fig. 1a, b). Immunohistochemistry (IHC) results confirmed the downregulated expression of SIRT1 and upregulated expression of CagA+ in HP+ tissue (Fig. 1c, d). Western blot analyses confirmed the overexpression of SIRT1 in HP+ tissues ( Fig. 1e, f). Consequently, the SNU1, AGS, MGC-803, and MKN1 gastric cancer cell lines were infected with H. pylori strain 26695 or strain 60190 (both CagA+). Following 24 h of incubation, western blot analyses showed that H. pylori strain 26695 is VacA-negative (Fig. 1g). As shown in Fig.1h, AGS, SNU1, MGC-803, or MKN1 cell lines after infected with H. pylori CagA+ strain 26695 in different hours. In most of cell lines, the expression of miR-543 in 24 h was highest. So we used 24 h for further experiment. RT-PCR results showed that expression levels of both CagA and miR-543 were increased in each cell line after infection with CagA+ H. pylori (strain 26695), especially in AGS and SNU1 cells (Fig. 1h-j). When compared with normal gastric mucosa cells (GES-1), RT-PCR data showed that expression of miR-543 was increased in CagA + H. pylori AGS, SNU1, MGC-803, and MKN1 cells.
The overexpression of miR-543 promotes the accelerating effect of CagA+ H. pylori on cell proliferation The miR-543 overexpression vector pCDH-miR-543 was constructed and transfected into MGC-803 and MKN1 cells (with or without H. pylori infected). Similarly, an anti-miR-543 vector, which is an inhibitor of miR-543, was constructed and transfected into SNU1 and AGS cells (with or without H. pylori infected). RT-PCR analysis showed that the expression of miR-543 was increased in MGC-803 and MKN1 cells, and decreased in SNU1 and AGS cells (Fig. 2a). Cell proliferation was determined by CCK-8 analysis after the cells were infected with H. pylori (CagA+) and transfected with mimics. Data showed that infection with H. pylori (CagA+) promoted cell proliferation in all four cell lines. Overexpression of miR-543 increased the promotion of cell proliferation, while anti-miR-543 inhibited proliferation (Fig. 2b). Colony formation assays showed similar results (Fig. 2e, f). The apoptosis rate was assessed by flow cytometry with Annexin V (AV)-fluorescein isothiocyanate (FITC) staining. Results showed that H. pylori (CagA+) blocked apoptosis and that pCDH-miR-543 enhanced this trend (Fig. 2c). Conversely, anti-miR-543 eliminated the inhibitory effect that CagA had on apoptosis (Fig. 2c). Transfection with pCDH-miR-543 alone can also promote cell proliferation and inhibit apoptosis, while anti-miR-543 had an opposite effect ( Fig. 2b-e). Therefore, AV/FITC apoptosis detection verified the aforementioned conclusions (see above).
Cell migration, invasion, and EMT is increased by miR-543 overexpression in CagA+ H. pylori infected cells The migration and invasion abilities of gastric cancer cells were assessed using Transwell assays. The results using MGC-803 and MKN1 cells showed that infection with the CagA+ H. pylori strain promoted cell migration and invasion. Cell migration and invasion effects were further promoted with the overexpression of miR-543 ( Fig. 3a, b). Similarly, although CagA+ H. pylori infection promoted migration and invasion of AGS and SNU1 cells, anti-miR-543 transfection reversed these effects (Fig. 3a,  b). To further confirm that miR-543 increased cell migration and invasion through the promotion of EMT, the expression of EMT-related proteins N-cadherin, Ecadherin, Snail, and Vimentin were examined by western blots and RT-PCR. Following infection with CagA+ H. pylori, the results showed that expression of N-cadherin and Snail were increased at both the protein and transcription levels. This trend was heightened by miR-543 overexpression and suppressed by anti-miR-543 ( Fig.  3c-e). Similarly, the inhibitory effect of CagA+ H. pylori on E-cadherin expression was enhanced by pCDH-miR-543, but suppressed by anti-miR-543 ( Fig. 3c-e). These results suggested that miR-543 had the ability to upregulate the EMT, and to promote cell migration and invasion.

SIRT1 is a direct target of miR-543
Bioinformatics analyses (http://www.targetscan.org) predicted that SIRT1 was a potential functional target of miR-543 (Fig. 4a). Dual-luciferase reporter assays showed that overexpression of miR-543 reduced the intensity of fluorescence in SNU1 and AGS cells transfected with SIRT1 wild-type (WT) vectors but had no effect on the cells that were transfected with SIRT1-mutant (MUT) vectors (Fig. 4b). Subsequent analyses confirmed that miR-543 significantly inhibited SIRT1 expression at both the transcript and protein levels in gastric cancer cells ( Fig. 4c-e). Collectively, the results suggested that miR-543 directly targeted SIRT1 and caused translational repression in gastric cancer cells.
SIRT1 is targeted by miR-543 to suppress autophagy in gastric cancer cells SIRT1-silencing vectors (siSIRT1) were constructed and transfected into AGS and SNU1 cells. While RT-PCR analysis showed that transfection with anti-miR-543 increased the expression of SIRT1 (Fig. 3c), siSIRT1 effectively suppressed the increased expression of SIRT1 (Fig. 5a). Cell proliferation was determined by CCK-8 and colony formation assays after the cells had been infected with H. pylori (CagA+) and transfected with different mimics. The results indicated that, while both the anti-miR-543 vector and autophagy enhancer rapamycin (RAPA) inhibited proliferation of AGS and SNU1 cells, siSIRT1 reversed the inhibition of AGS and SNU1 cell proliferation (Fig. 5b). Colony formation assays mirrored these results (Fig. 5c, d). Analyses using transwell assays revealed that siSIRT1 mimics had the ability to reverse the inhibition of cell migration and invasion caused by anti-miR-543 or RAPA (Fig. 5e, f). Western blot analyses of EMT-related N-cadherin, E-cadherin, Snail, and Vimentin proteins indicated that both transfection of anti-miR-543 and addition of RAPA suppressed the EMT caused by H. pylori CagA. Western blot analyses also showed that siSIRT1 reversed the inhibitory effect ( Fig. 5g-i).
In detecting and quantifying autophagy, mRFP-GFP-LC3 constructs were used. The results showed that both transfection of anti-miR-543 and addition of RAPA suppressed the inhibition of autophagy caused by H. pylori CagA. We use mRFP-GFP-LC3 adenovirus to monitor autophagic flux. The green puncta indicate the autophagosomes, while the red puncta represent the autolysosomes ( Fig. 6a, b). And the results indicated that siSIRT1 downregulated the expression of autophagy to levels similar to those observed in the control group. Western blot analyses of autophagy-related proteins LC3 and p62, and transmission electron microscope (TEM) results were similar to the results obtained from the mRFP-GFP-LC3 experiments ( Fig. 6c-f). Collectively, these results suggested that miR-543 targeted SIRT1, thus inhibiting CagA-mediated autophagy and EMT. Downregulation of autophagy and elevated levels of EMT significantly increased the rates of cell proliferation, migration, and invasion.

SIRT1 is targeted by miR-543 to suppress autophagy in vivo
To determine if the above results could be recapitulated in vivo, C57BL/6 mice were inoculated with H. pylori strain 26695 (CagA+), anti-miR-543, and siSIRT1. Gastric epithelium tissue samples were then obtained and analyzed. Rapid urease tests showed that H. pylori had infected the samples (Fig. 7a). Hematoxylin-eosin staining confirmed the infection (Fig. 7b). Consequently, western blot analyses showed that the expressions of LC3-I and LC-II were decreased by H. pylori CagA. Western blot analyses also showed that the miR-543 inhibitor reversed the reduction effect of miR-543 on LC3, while siSIRT1 suppressed the reversal effect of anti-miR-543 (Fig. 7c, d). In contrast to the results observed for LC3-I and LC-II, an increased expression of p62, a protein that negatively regulates autophagy, was observed (Fig. 7c, d). However, these results validated the hypothesis that miR-543 targets SIRT1 thereby inhibiting CagA-mediated autophagy in vivo.

Discussion
H. pylori-induced chronic gastritis is the leading known risk factor of gastric cancer 30 . The pathogenicity of H. pylori is largely due to its virulent components, such as flagella, lipopolysaccharides, VacA, and cytotoxin-related gene pathogenic island (cagPAI). CagPAI is one of the An increasing number of studies have suggested that miRNAs play a key role in gastric diseases that are induced by CagA+ strains of H. pylori [19][20][21]31 . As revealed by miRNA microarray analyses, miR-543 expression is significantly increased in HP+ gastric cancer tissue when compared with HP− cancer tissue 18 . The purpose of this study was therefore to determine the role that miR-543 plays in H. pylori-induced gastric cancer and also to assess its specific molecular mechanism.
In this study it was confirmed that H. pylori CagA increased the expression of miR-543 in human gastric cancer tissue and four different gastric cancer cell lines (SNU1, AGS, MGC-803, and MKN1). To investigate whether the expression of miR-543 was responsible for the pathogenicity of H. pylori CagA, we constructed and transfected gastric cancer cells with a miR-543 overexpression vector (pCDH-miR-543) or miR-543 inhibitor (anti-miR-543). Our in vitro experiments indicated that miR-543 overexpression increased cell proliferation induced by H. pylori CagA. In addition, the examination of EMT-related N-cadherin, E-cadherin, and Snail proteins showed that H. pylori CagA increased the EMT, thereby causing an increase in cell migration and invasion. Lastly, it was found that the effects of CagA were enhanced by miR-543 overexpression.
To further investigate the molecular mechanism of miR-543, bioinformatics analyses predicted that SIRT1 served as a potential functional target. Dualluciferase reporter assays revealed that miR-543 directly targeted SIRT1 and caused translational repression in gastric cancer cells. SIRT1 has been associated with autophagy through the activation of AMPK 22-24 . Conversely, H. pylori CagA has been shown to downregulate autophagy 26 . Further reports have revealed that autophagy can suppress EMT under several circumstances [32][33][34] . To identify whether miR-543 targets SIRT1 to inhibit autophagy and facilitate EMT, siSIRT1 mimics were constructed and cells transfected. CCK-8, colony formation, and western blot assays collectively revealed that siSIRT1 reversed the inhibiting effect of anti-miR-543 or RAPA on EMT-induced cell migration and invasion. Autophagy detection by mRFP-GFP-LC3, TEM, and western blot analyses of autophagy-related proteins confirmed that siSIRT1 downregulated autophagy. Follow-up in vivo experiments supported these results.
Collectively, this study showed that in the process of H. pylori CagA-inducing gastric cancer, miR-543 promoted cell proliferation. We also found that, when induced by H. pylori CagA, miR-543 overexpression targeted SIRT1, thereby leading to the inhibition of autophagy. The inhibition of autophagy promoted EMT, which led directly to cell migration and invasion. These results provide new insights into the molecular mechanism(s) of gastric cancer, with miR-543 serving as a potential therapeutic target for gastric cancer linked to H. pylori.

Clinical specimens and cell lines
The current study included 50 gastric cancer patients who attended Peking University Third Hospital, China from 2015 to 2017. In total, 20 HP−, 30 HP+, and 50 paracarcinoma tissue samples were collected via biopsy or surgical resection. Samples were divided and either frozen in liquid nitrogen and stored at −80°C or preserved in RNAlater (Ambion, Austin, TX, USA) at −20°C. The study was conducted in accordance with the Declaration of Helsinki and approved by Peking University Third Hospital. Informed consent was obtained for study participation from all patients or their direct relatives.

Immunohistochemistry (IHC)
Thawed samples were fixed in 4% formalin and embedded in paraffin for histopathological analyses. Samples were deparaffinized with xylol and then sliced into 4-µm sections. Sections were rehydrated using a graded ethanol series. A heat-induced epitope protocol was used for antigen-retrieval (95°C for 40 min). Samples were incubated in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase. Samples were blocked with serum (Vectastain Elite ABC kit; Vector Laboratories Inc., Burlingame, CA, USA) and then incubated overnight at 4°C with polyclonal rabbit anti-human SIRT1 antibody at a concentration of 1:1000 (Abcam, Shanghai, China). After washing three times in TBST (150 mM NaCl, 10 mM Tris-HCl, pH 7.6), sections were incubated with a secondary antibody for 20 min at room temperature. Peroxidase-conjugated biotin-streptavidin complex (Dako, Glostrup, Denmark) was then applied to the sections for 20 min. Sections were visualized with 3, 3ʹ-diaminobenzidine and counterstained with hematoxylin. The negative control used nonimmune serum instead of primary antibody.

Plasmid construction
The human miR-543 precursor and human SIRT1 coding sequence were cloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen, Shanghai, China) at the KpnI and XhoI cut sites (TaKaRa, Dalian, China) to generate stably-transfected gastric cancer cell lines. Virus was produced in 293 T cells cotransfected with the pCDH lentiviral vector and associated packaging plasmid.

Protein extraction and western blotting
Proteins were extracted from subconfluent cell cultures and subjected to western blot analyses. After blocking with 5% nonfat milk in PBS-T for 1 h at room temperature, the membranes were blotted with primary antibody, followed by blocking, and incubation with a peroxidaseconjugated secondary antibody. Bound antibodies were visualized using enhanced chemiluminescence (Bio-Rad, Hercules, CA, USA). The primary antibodies against SIRT1, Snail, N-cadherin, E-cadherin, p62, LC3, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Signals were detected using a FluorChem E system (Alpha Innotech Corp, Santa Clara, CA, USA).

RNA extraction and real-time PCR
Total RNA was extracted using TRIzol (Life Technologies) as per the manufacturer's protocol. RNA was reverse-transcribed into cDNA with the Super-Script First-Strand cDNA System (Invitrogen, Carlsbad, CA, USA), and amplified with the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). We prepared a master mix for qPCR testing using Platinum SYBR Green qPCR SuperMix-UDG, forward and reverse primers, and template cDNA (10 ng). The reaction conditions were 95°C for 5 min, and then 32 cycles at 95°C for 15 s and 1 min at 60°C and 30 s at 72°C. Beta-actin was used as an internal control. The relative expression levels of mRNA were quantified using the ΔΔ Ct method.

Cell proliferation assays
Cell proliferation was determined using CCK-8 assays (Dojindo Laboratories, Kumamoto, Japan). Briefly, gastric cancer cells were seeded into 96-well plates at an initial density of 5000 cells per well. Cells transfected with anti-miR-543 were plated on day 1. Ten microliters of the kit reagent were added to each well at 0, 24, 48, or 72 h after seeding. All plates were scanned using a microplate reader (Thermo Scientific) after a further 2 h. Cell proliferation was evaluated by absorbance at 450 nm.

Apoptosis assay
Apoptosis was measured using an AV/FITC apoptosis detection kit (Bender Medsystem, Vienna, Austria). Briefly, cells cultured in 6-cm dishes were trypsinized, washed, and stained with FITC-conjugated anti-AV antibody in darkness for 15 min at room temperature, and then analyzed with a flow cytometer (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA).

Colony formation assay
Cell proliferation was assessed using a soft agar colony formation assay. A six-well plate containing a 1.5 mL bottom layer and 0.5 mL top layer of agar was used (5.1 mg/mL; Difco Laboratories, Detroit, MI, USA). In total, 104 cells were transferred onto the bottom layer of each well, overlaid with a top layer of agar, and cultured at 37°C in 5% CO 2 . Giemsa staining was used to quantify the formation of colonies on the 7th day.

Cell migration and invasion assays
The migration and invasion abilities of gastric cancer cells were assessed using Transwell assays (Millipore, Billerica, MA, USA). GCs were seeded into uncoated or Matrigel-coated (BD Bioscience, Bedford, MA, USA) plates with 8-μm diameter pores in order to perform migration and invasion assays, respectively. The top chambers were seeded with cells at a density of 2 × 10 4 cells/well in serum-free medium, while FBS with 10% serum was added to the lower chamber. After 24 h of incubation, nonmigratory cells found on the top surface of the filter were removed by rubbing with a cotton swab. Cells that had migrated to the lower chamber were quantified in five random fields using an optical inverted microscope at ×200 magnification (Nikon, Tokyo, Japan).

Luciferase reporter assays
Luciferase reporter assays were conducted using the Dual-Luciferase Reporter Assay System (psiCHECK-2 vector; Promega, Madison, WI, USA). A fragment of the SIRT1 3ʹ-UTR containing either the predicted binding site for miR-543 or a mutated 3ʹ-UTR was inserted into the psiCHECK-2 vector. After verification by Sanger sequencing and using the Lipofectamine RNAiMAX kit (see figure on previous page) Fig. 5 SIRT1 was targeted by miR-543 to suppress cell proliferation, migration and invasion. a RT-PCR analyses of SIRT1 in AGS and SNU1 cells transfected with anti-miR-543, siSIRT1, or both anti-miR-543 and siSIRT1. b CCK-8 cell proliferation analyses of AGS and SNU1 cells transfected with anti-miR-543, siSIRT1, or both anti-miR-543 and siSIRT1. Autophagy enhancer RAPA addition and siSIRT1 transfection results were also studied and compared. c Colony formation assays show the results of cell proliferation. d Quantitative analysis of colony formation. e Transwell chamber experiments on cell migration and invasion. f Quantitative results of migration and invasion. g RT-PCR detection of N-cadherin, E-cadherin, and Snail proteins in AGS and SNU1 cells. h, i Western blot detection of N-cadherin, E-cadherin, Vimentin, and Snail in AGS and SNU1 cells and quantified. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus siSIRT1 group (see figure on previous page) Fig. 6 SIRT1 was targeted by miR-543 to suppress autophagy. a Detection of autophagy using mRFP-GFP-LC3 in AGS and SNU1 cells transfected with anti-miR-543, siSIRT1, or both anti-miR-543 and siSIRT1. Green puncta indicate the autophagosomes, while the red puncta represent the autolysosomes. Autophagy enhancer RAPA addition and siSIRT1 transfection results were also studied and compared. b Quantitative analysis of mRFP-GFP-LC3 detection. c The autophagic vacuoles (autophagosomes) were detected by transmission electron microscopy (TEM). The representative TEM images are shown and the typical autophagosomes are marked with arrows. d Quantitative analysis of autophagy detection by TEM. e, f Western blot detection of autophagy-related proteins LC3-I, LC3-II, and p62 and Quantified. Data are expressed as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus control group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus siSIRT1 group Fig. 7 SIRT1 was targeted by miR-543 to suppress autophagy in vivo. a Rapid urease test of H. pylori infection on C57BL/6 mice. b Hematoxylineosin staining image of control and HP (CagA+) group in mice gastric epithelium. c, d Western blot detection of SIRT1 and autophagy-related proteins LC3-I, LC3-II, and p62 and quantified. Data are expressed as the mean ± SD (n = 8). **P < 0.01, ***P < 0.001 versus control group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus HP(CagA+) group, $$ P < 0.01, $$$ P < 0.001 versus HP(CagA+) + miR-543 inhibitor group (Invitrogen) according to the manufacturer's instructions, the psiCHECK-2 vectors containing either the WT, or mutated SIRT1 3ʹ-UTR were transfected into gastric cancer cells. Gastric cancer cell lines presented either with or without a synthetic miR-543 mimic. At 36 h after transfection, luciferase activity was detected using a dualluciferase reporter assay system and normalized against Renilla activity. Data were normalized to the luciferase activity of cells transfected with miR-control elements.

Autophagic flux analysis
The mRFP-GFP-LC3 gastric cells were fixed with 4% paraformaldehyde and stained with 10 μM Hoechst 33342. Cell images were obtained from the Operetta High Content Imaging System (Perkin-Elmer, Waltham, MA, USA) and analyzed using Harmony Analysis Software (Perkin-Elmer). Cells were detected using green fluorescent protein (GFP) or red (mRFP) fluorescence. In merged images, autophagosomes stained puncta yellow, while autolysosomes stained puncta red. Autophagic flux was determined by the increased percentage of red only puncta in the merged images.

Electron microscopy
Cells were fixed with 2.5% glutaraldehyde in phosphate buffer and stored at 4°C until embedded. Cells were postfixed with 1% osmium tetroxide followed by an increasing gradient dehydration step using ethanol and acetone. Cells were then embedded in Araldite, following which ultrathin (50-60 nm) tissue sections were obtained, placed on uncoated copper grids, and stained with 3% lead citrate-uranyl acetate. Images were examined with a CM-120 electron microscope (Philips Microscopy, Hillsboro, OR, USA).

H. pylori infection in vivo
Six to eight-week-old C57BL/6 mice (Institute of Zoology, Chinese Academy of Sciences, Shanghai, China) were inoculated orally with broth-cultured H. pylori strain 26695 (CagA+) at a concentration of 1 × 10 8 CFU. Cultures were grown in 0.15 mL broth. Control mice were given PBS. A lentivirus expressing SIRT1 and miR-543 was purchased from HanBio. One hundred microliters of filter-purified lentivirus cocktail (1 × 10 5 IU/μl) or PBS was administered by intravenous injection weekly. Mice were killed 4 months after inoculation and gastric tissue collected for western blot analyses.

Statistical analysis
The results are expressed as the mean ± SD. Statistical significance was evaluated by ANOVA, followed by Tukey-Kramer multiple comparison tests and by Student's t-tests. A p < 0.05 denotes statistical significance.