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. 2016 Dec 13;7(50):82770-82782.
doi: 10.18632/oncotarget.13165.

Helicobacter Pylori Infection Related Long Noncoding RNA (lncRNA) AF147447 Inhibits Gastric Cancer Proliferation and Invasion by Targeting MUC2 and Up-Regulating miR-34c

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Free PMC article

Helicobacter Pylori Infection Related Long Noncoding RNA (lncRNA) AF147447 Inhibits Gastric Cancer Proliferation and Invasion by Targeting MUC2 and Up-Regulating miR-34c

Xiaoying Zhou et al. Oncotarget. .
Free PMC article

Abstract

Long non-coding RNAs (lncRNAs) were shown to play critical roles in cancer biology. We investigated whether H. pylori infection could promote gastric cancer by regulating lncRNAs expression. Differentially expressed lncRNAs between H. pylori positive and negative tissues were identified by microarray and validated by qRT-PCR. Our results indicated that H. pylori positive tissues have a specific profile of lncRNAs. Cell biological assays with siRNA-mediated knockdown or lentivirus vector-mediated over-expression were performed to probe the functional relevance of the lncRNAs. We identified an lncRNA-AF147447 decreased expressed by H. pylori infection, which can inhibit GC proliferation and invasion in vitro and in vivo, act as a tumor suppressor in the development of H. pylori induced GC. LncRNA AF147447 could repress MUC2 expression by direct binding or increasing miR-34c expression. We also found that transcription factor E2F1 could be recruited to lncRNA AF147447 promoter by RNA immunoprecipatation and RNA pull down assays. These findings support a role of lncRNA AF147447 in tumor suppression. This discovery contributes to a better understanding of the importance of the deregulated lncRNAs by H. pylori infection and provides a rationale for the potential development of lncRNA-based targeted approaches for the treatment of H. pylori-related gastric cancer.

Keywords: MUC2; gastric cancer; helicobacter pylori infection; lncRNA.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of competing interest.

Figures

Figure 1
Figure 1. LncRNA AF147447 expression in H. pylori infected state
(A) AF147447 expression in H. pylori infected cells compared with controls. (B) AF147447 expression in H. pylori infected gastritis and GC tissues compared with their respective controls. (C) AF147447 expression in different histology type of H. pylori infected gastritis. (D) AF147447 expression in H. pylori infected gastric tissues in mice. (*p < 0.05, **p < 0.01).
Figure 2
Figure 2. LncRNA AF147447 inhibition promote GC proliferation and invasion in vitro and in vivo
(A) LncRNA Af147447 expression level after AF147447 siRNA transfection was validated by qRT-PCR. (B) MKN45 cells were seeded in 96-well plates, and cell proliferation was assessed daily for 3 days using the Cell Counting Kit-8 (CCK-8) assay. (C) Cells were treated with either AF147447 siRNA or siRNA-NC for 24 h. The representative images of invasive cells at the bottom of the membrane stained with crystal violet were visualized as shown. All experiments were performed in triplicate. (D) The in vivo models used were xenograft-transplanted nude mouse tumor models of human gastric cancer established with Af147447 siRNA or siRNA control. The left arms of the mice were injected with control GC cell and the right arms of the mice were injected with AF147447 siRNA GC cells. Photographs of tumors are presented. Effects of lncRNA AF147447 expression in nude mouse models are shown. (*p < 0.05, **p < 0.01).
Figure 3
Figure 3. LncRNA AF147447 over-expression inhibit GC proliferation and invasion in vitro and in vivo
(A) Af147447 expression level after AF147447 over-expression was validated by qRT-PCR. (B) 7901 cells were seeded in 96-well plates, and cell proliferation was assessed daily for 3 days using the Cell Counting Kit-8 (CCK-8) assay. (C) Cells were treated with either pcDNA-AF147447 or pLV-AF147447 for 24 h. The representative images of invasive cells at the bottom of the membrane stained with crystal violet were visualized as shown. All experiments were performed in triplicate. (D) The in vivo models used were xenograft-transplanted nude mouse tumor models of human gastric cancer established with pLV-AF147447 or pLV-NC. The left arms of the mice were injected with pLV-AF147447 GC cells and the right arms of the mice were injected with pLV-NC cells. Photographs of tumors are presented. Effects of lncRNA AF147447 expression in nude mouse models are shown. (*p < 0.05, **p < 0.01).
Figure 4
Figure 4. Identification and validation of lncRNA-AF147447 targets
(A) Biotinylated lncRNA-AF147447 or antisense RNA were incubated with nuclear extracts, targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of MUC2 and lncRNA-AF147447. Another MUC family: MUC1 is shown as a control. (B) Diagram of MUC2 promoter region constructs. (C) Luciferase reporter assays in 7901 cells, with co-transfection of wt or mt and lncRNA as indicated. (D) MUC2 expression was validated by qRT-PCR after transfecting with pLV-AF147447 or siRNA or their respective controls. (E) MUC2 expression was validated by western blot after transfecting with pLV-AF147447 or siRNA or their respective controls. (*p < 0.05; **p < 0.01).
Figure 5
Figure 5. MUC2 expression under H. pylori infected state
(A) MUC2 mRNA expression in H. pylori infected cells compared with controls. (B) MUC2 protein expression in H. pylori infected cells compared with controls. (C) MUC2 mRNA expression in H. pylori infected control and tumor tissues. (D) MUC2 protein expression in H. Pylori positive and negative tissues was detected by immunohistochemical staining. (E) A statistically significant inverse correlation between MUC2 and lncRNA-AF147447 levels in clinical specimens (Spearman's correlation analysis, r = −0.864, p = 0.000) (*p < 0.05; **p < 0.01).
Figure 6
Figure 6. LncRNA-AF147447 interact with increased miR-34c expression level
(A) Diagram of MUC2 3′UTR region constructs. Luciferase reporter assays in 7901 cells, with co-transfection of miR-34c mimics and wt or mut 3′UTR. (B) Diagram of AF147447 wt and mut constructs. Luciferase reporter assays in 7901 cells, with co-transfection of miR-34c mimic and AF147447 wt or mut. (C) MUC2 mRNA expression was validated by qRT-PCR after cells transfecting with mimic or inhibitor. (D) MUC2 protein expression was validated by western blot after cells transfecting with mimic or inhibitor. (E) AF147447 expression was validated by qRT-PCR after cells transfecting with mimic or inhibitor. (F) MiR-34c expression was validated by qRT-PCR after cells transfecting pLV-AF147447 or si-AF147447. (G) Pearson analysis showed that miR-34c and lncRNA AF147447 was positively correlated. (*p < 0.05; **p < 0.01).
Figure 7
Figure 7. LncRNA AF147447 physically associates with E2F1
(A) RIP experiments were performed using the E2F1 antibody to immunoprecipitate (IP) and a primer to detect lncRNA-AF147447. RIP enrichment was determined as RNA associated with E2F1 IP relative to an input control. (B) Diagram of lncRNA-AF147447 promoter region constructs. Luciferase reporter assays in 7901 cells, with co-transfection of lncRNA-AF147447, with siRNA-E2F1 or siRNA-control. (C) LncRNA-AF147447 expression was detected by qRT-PCR after cells transfecting with si-E2F1 or pcDNA-E2F1. (D) MUC2 mRNA and protein were detected by qRT-PCR and western blot. (*p < 0.05; **p < 0.01).

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