Figure 1.
Screening and identification of miRNA-584 and miRNA-1290 in CagA-transfected cells. A. Detection of cagA DNA fragments in transfected cells.
The standard calcium phosphate precipitation method was used to establish stable CagA-expressing gastric carcinoma AGS cells. Transfection was validated using PCR and DNA sequencing after cell clones had grown. AGS cells stably expressing the empty pEGFP-c1 vector were also established as a control. B. Detection of CagA protein in transfected cells. Stable CagA-expressing gastric carcinoma AGS cells were lysed with RIPA buffer. The lysates were subjected to SDS-PAGE and subsequently to western blot analysis to probe with anti-GFP antibodies. AGS cells stably expressing the empty pEGFP-c1 vector were also established as a control. C. Screening and identification of miRNA-84 and miRNA-290 in stable CagA-expressing AGS cells. Total RNA was extracted from stable CagA-expressing AGS cells and control cells. Mammalian miRNA expression profile microarray scanning and TaqMan real-time PCR were performed. The results showed that both miRNA-584 and miRNA-1290 were up-regulated in stable CagA-expressing AGS cells. D. Identification of miRNA-584 and miRNA-1290 in gastric carcinoma SGC7901 cells transiently expressing CagA. Total RNA from gastric carcinoma SGC7901 cells transfected with cagA/pEGFP-c1 plasmid was extracted and real-time PCR was performed. Both miRNA-584 and miRNA-1290 were up-regulated in transiently CagA-expressing SGC7901 cells. E. Identification of miRNA-584 and miRNA-1290 in gastric carcinoma AGS cells infected with H.pylori. Total RNA was extracted from AGS cells infected with H.pylori including NCTC11637 and 9 cagA– isolates for 7 h (MOI≈50) respectively, and TaqMan real-time PCR was performed. Both miRNA-584 and miRNA-1290 were up-regulated after infection of cagA+ H.pylori (all P <0.01, ANOVA). Data are represented as mean +/− s.e.m.
Figure 2.
miRNA-1290 was up-regulated in an Erk1/2-dependent manner and miRNA-584 was activated indirectly by miRNA-1290. A. Determination of transactivities of Elk-1.
AGS cells were transiently transfected with pGL3-Elk-1 plasmid for 24 h and subsequently infected with H.pylori including NCTC11637 and 7 cagA+ isolates for additional 7 h (MOI≈50) respectively. Cells were lysed and the lysates were collected for determination of luminescent intensities by dual-luciferase ÿreporter assay. The results showed that transactivities of Elk-1 was up-regulated after infection of cagA+ H.pylori (P <0.01, ANOVA). B. Determination of luciferase activations containing promoter region of miRNA-584 and miRNA-1290. Plasmids containing Elk-1 or cagA were co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 and miRNA-1290 (≈2000 bp) respectively into 293T cells for 24 h (Elk-1/cagA: luciferase reporters: Renilla luciferase = 3∶1∶0.01). Cells were lysed and the lysates were collected for determination of luminescent intensities by dual-luciferase_reporter assay. The results showed that miRNA-1290 was up-regulated by both Elk-1 and CagA (P <0.01, ANOVA), whereas miRNA-584 was not affected. C. Determination of miRNA-584 in stable miRNA-1290–expressing cells. To determine miRNA-584 in stable miRNA-1290–expressing cells, total RNA from stable miRNA-1290–expressing cells were isolated and real-time PCR was performed for miRNA-584. The results showed that miRNA-584 was up-regulated in stable miRNA-1290–expressing cells. D. Expression of miRNA-1290 enhanced NF-κB activities. miRNA-1290 was co-transfected with NF-κB reporter into SGC7901 cells for 24 h (miRNA-1290: luciferase reporters: Renilla luciferase = 3∶1∶0.01). Cells were lysed and the lysates were collected for determination of luminescent intensities by dual- luciferase_reporter assay. The results showed that miRNA-1290 enhanced NF-κB activities (P <0.01, ANOVA). E. Detection of NKRF protein in transfected cells. miRNA-1290-expressing gastric carcinoma SGC7901 cells were lysed and subsequently were subjected to western blot analysis to probe with anti-NKRF antibodies. SGC7901 cells expressing the empty vector were also established as a control. F. Knockdown of NKRF increased NF-κB activities. NKRF shRNA plasmid (Santa Cruz) was co-transfected with NF-κB reporters into SGC7901 cells for 48 h (NKRF shRNA plasmid: NF-κB reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that knockdown of NKRF increased NF-κB activities (P <0.01, ANOVA). G. Activation of NF-κB upregulated miRNA-584. NKRF shRNA plasmid was co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 into SGC7901 cells for 48 h (NKRF shRNA plasmid: luciferase reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that NF-κB up-regulated miRNA-584 (P <0.01, ANOVA). H. Overexpression of c-Rel upregulated miRNA-584. c-Rel plasmid was co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 into SGC7901 cells for 48 h (c-Rel plasmid: luciferase reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that Overexpression of c-Rel upregulated miRNA-584 (P <0.01, ANOVA). Data are represented as mean +/− s.e.m.
Figure 3.
cagA+ H.pylori activated Erk1/2 kinases and miRNA-584 sustained Erk1/2 activities through inhibition of PPP2a. A. Erk1/2 kinases were activated by CagA.
AGS cells were infected with H.pylori including NCTC11637 and 9 cagA– and 7 cagA+ isolates for 7 h (MOI≈50) respectively. Cells were collected and lysed at 24 h after transfection. The lysates were co-precipitated with agarose-conjugated anti-Erk1/2 antibodies, and after washing 5 times, the beads were mixed with recombinant Elk-1 and ATP. Mixtures were subjected to SDS-PAGE after denaturing and subsequently western blot analysis probed with antibodies against phosph-Elk-1. Uninfected AGS cells were also established as a control. B. Determination of miRNA-584 and miRNA-1290 in transiently transfected cells. Total RNA was extracted from gastric carcinoma AGS cells transiently transfected with plasmids containing miRNA-584 or miRNA-1290 at 24 h after transfection. Real-time PCR was performed for determination of miRNA-584 and miRNA-1290. C. Detection of Erk1/2 activities. Gastric carcinoma AGS cells were transiently transfected with plasmids containing miRNA-584 or miRNA-1290. Cells were collected and the lysates were co-precipitated with agarose-conjugated anti-Erk1/2 antibodies, and after washing 5 times, the beads were mixed with recombinant Elk-1 and ATP at 30°C for 30 min. Mixtures were subjected to SDS-PAGE after denaturing and subsequently western blot analysis probed with antibodies against phosph-Elk-1. The empty vector was also established as a control. D. Detection of PPP2ca expression. Gastric carcinoma SGC7901 cells were transiently transfected with plasmids containing miRNA-584 or miRNA-1290. Cells were collected and lysed at 24 h after transfection. Lysates were subjected to SDS-PAGE after denaturing and subsequently western blot analysis probed with antibodies against PPP2ca. Data are represented as mean +/− s.e.m.
Figure 4.
Foxa1 is a target of miRNA-584 and miRNA-1290 and knockdown of Foxa1 promotes EMT. A. Schematic of potential target screening strategy. B. Transcription factor targets of miRNA-584 and miRNA-1290.
Transcription factors targeted by miRNA-584 and miRNA-1290 expressed in AGS cells were predicted by TargetScan (www. targetscan.org). C. Determination of pMIR-report luciferase activity. The recombinant plasmid pMIR-Foxa1 report was co-transfected with pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or miRNA-1290 into AGS gastric cancer cells for 48 h. Cells were lysed and luciferase activity was determined by dual-luminescence reporter assay. Co-transfection of miRNA-584 or miRNA-1290 with pMIR-Foxa1 inhibited more than 90% of firefly luciferase activity (all P <0.001, ANOVA), indicating that miRNA-584 and miRNA-1290 target Foxa1 mRNA. D. Detection of Foxa1 in cells transiently expressing miRNA-584 and miRNA-1290. pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or miRNA -1290 was transfected into SW620 colon cancer cells for 48 h. Cells (1×106) were lysed and the supernatant was subjected to western blot analysis. The results showed that the expression of Foxa1 protein was down-regulated about 50% after transfection of miRNA-584 or miRNA-1290. These results indicate that Foxa1 is a target of miRNA-584 and miRNA-1290. E. Analysis of the percentage of CD44+CD133+ cells. SW620 colon cancer cells were infected with recombinant Foxa1 shRNA lentivirus. Infected SW620 cells were digested and resuspended at 1×106 per 100 µL of PBS containing 2% FBS, and labeling antibodies (APC-CD44 and PE-CD133) were added at 4°C for 30 min in the dark. After two washes, cells were analyzed using a FACSAria. An empty vector control was also established. The percentage of CD44+CD133+ cells significantly decreased after Foxa1 knockdown (P <0.05, ANOVA). The supernatant from SW620 colon cancer cells with Foxa1 knockdown was subjected to western blot for detection of Bmi1. F. Foxa1 knockdown promotes EMT. The supernatant from SW620 colon cancer cells with Foxa1 knockdown was subjected to SDS-PAGE, and proteins were transferred onto a nitrocellulose membrane for western blotting for Foxa1, E-cadherin, and vimentin. G. Detection of E-cadherin in cells with stable expression of miRNA-584 or/and miRNA-1290. pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or/and miRNA -1290 was transfected into SW620 colon cancer cells. Cells were separated at 24 h after transfection and screened by 5 µg/mL puromycin for 7 days. Survival clones were picked up and amplified respectively for 4 weeks. Cells were lysed and the supernatant was detected for E-cadherin. GAPDH was as controls.
Figure 5.
Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia in knock-in mice. A. Determination of miRNA-584 and miRNA-1290 in knock-in mice.
Total RNA was extracted from minced tissues of mice by Trizol reagent. Real-time PCR was performed for determination of miRNA-584 and miRNA-1290. The results showed miRNA-584 and miRNA-1290 increased in knock-in mice. Data are represented as mean +/− s.e.m. B. Morphological observation of knock-in mice. Obvious morphological alterations were not observed at the 12th week in knock-in mice. However, gastric mucosa layers were significantly thinned and flattened, and rugal folds had disappeared at the 72nd week C. H&E staining of gastric mucosa. Obvious morphological alterations were not observed at the 12th week in knock-in mice. However, gastric epitheliums were replaced by intestinal metaplasia under light microscopy (×100). D. Determination of Muc 2 and Muc 6. Total RNA was extracted from minced gastric tissues of mice by Trizol reagent. Real-time PCR was performed for determination of Muc2 and Muc6. The results showed Muc2 was significantly increased while Muc6 was significantly decreased in transgenic mice (all P <0.01, ANOVA). Data are represented as mean +/−s.e.m. E. PAS staining. Gastric mucosa from transgenic mice at 72nd W were checked by PAS staining (×100). The results showed that PAS-positive cells were increased in in transgenic mice.