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

Abstract

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.

Fig. 1. H. pylori CagA increased expression of miR-543.

a RT-PCR results of miR-543 expression. b RT-PCR results of SIRT1 expression. c IHC experiments of CagA and SIRT1 in normal gastric tissue; HP+ or HP−. d Quantified IHC results. e Western blot results of SIRT1 in tumor tissues; HP+ or HP−. f Quantified western blot results. g Western blot results of CagA and VacA in AGS, SNU1, MGC-803, and MKN1; infected with strain 60190 or 26695. h RT-PCR results of miR-543 in AGS, SNU1, MGC-803, or MKN1 cell lines after infected with H. pylori CagA+ strain 26695 in different hours. i RT-PCR results of CagA in AGS, SNU1, MGC-803, or MKN1 cell lines after infected with H. pylori CagA+ or CagA− strain in 24 h. j RT-PCR results of miR-543 in AGS, SNU1, MGC-803, or MKN1 cell lines after infected with H. pylori CagA+ or CagA− strain in 24 h. k RT-PCR results of miR-543 in GES-1, AGS, SNU1, MGC-803, or MKN1 cells. Data are expressed as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 compared with control

Fig. 2. miR-543 overexpression promoted the accelerating effect of CagA+ H. pylori on cell proliferation.

a RT-PCR results of miR-543 expression in MGC-803 and MKN1 cells transfected with pCDH-miR-543 vectors or AGS and SNU1 transfected with the anti-miR-543 vector; infected with HP or not. b CCK-8 cell proliferation analyses of MGC-803 (HP (CagA+) or pCDH-miR-543 or both HP (CagA+) and pCDH-miR-543), MKN1 (HP (CagA+) or pCDH-miR-543 or both HP (CagA+ ) and pCDH-miR-543), AGS (HP (CagA+) or anti-miR-543 or both HP (CagA+) and anti-miR-543), and SNU1 (HP (CagA+) or anti-miR-543 or both HP (CagA+) and anti-miR-543). c AV-FITC staining apoptosis results of four kinds of cell lines processed as shown in Fig. 2c. d Quantitative analysis of apoptosis. e Colony formation cell proliferation analyses results. f Quantitative analysis of cell proliferation. Data are expressed as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 versus control group; ^##P < 0.01, ^###P < 0.001 versus CagA+ HP group

Fig. 3. Promotion of cell migration, invasion, and EMT was increased by miR-543 overexpression in CagA + H. pylori cells.

a Transwell chamber results of MGC-803 (HP (CagA+) or both HP (CagA+) and pCDH-miR-543), MKN1 (HP (CagA+) or both HP (CagA+) and pCDH-miR-543), AGS (HP (CagA+) or both HP (CagA+) and anti-miR-543), and SNU1 (HP (CagA+) or both HP (CagA+) and anti-miR-543). b Quantitative analysis of cell migration and invasion of four kinds of cell lines processed as shown in Fig. 3a. c Western blot results of EMT-related N-cadherin, E-cadherin, Snail, and Vimentin proteins. d Quantified western blot results and RT-PCR results of EMT-related genes. 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 CagA+HP group

Fig. 4. SIRT1 is a direct target of miR-543.

a Complementary sequences between miR-543 and the 3ʹ-UTR of SIRT1 or a mutated version of SIRT1 were obtained using publicly available algorithms. b Dual-luciferase reporter assays of SNU1 and AGS transfected by a mimic of miR-543 (with SIRT1-WT or SIRT1-MUT). c, d Western blot results of SIRT1 and quantified. e RT-PCR detection of SIRT1 expression in MGC-803 and MKN1 cells transfected with pCDH-miR-543, or AGS, and SNU1 cells transfected with anti-miR-543. Data are expressed as the mean ± SD (n = 3). ***P < 0.001 versus control group

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

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 Hematoxylin-eosin 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