Article Figures & Data
Figures
- Figure 1.
Augmented hTERT expression in the specimens of progressive gastric diseases (from chronic inflammation to gastric cancer; GC) and H. pylori--infected gastric cancer cell lines, and high expression of hTERT predicted poor prognosis of gastric cancer patients. A, Expression of hTERT in primary gastric cancer and normal tissues from TCGA database. B, Expression of hTERT in different grades of primary gastric cancer and normal tissues from TCGA database. C, IHC staining of hTERT protein expression in superficial gastritis (SG), atrophic gastritis with intestinal metaplasia (AG/IM), and cancerous gastric tissues (GC). Scale bars, 50 μm. D, hTERT mRNA expression in Hp 26695-negative and -positive gastric epithelial cell line GES-1 from public database (GSE74577). E and F, Augmented expression of hTERT in H. pylori–infected AGS, BGC-823, and GES-1 cells. G, hTERT mRNA expression increased with the rising of H. pylori strains 26695 [MOI 1:25–1:100 (cell:H. pylori)] for 6 hours. H, hTERT mRNA expression in AGS cells infected by CagA-negative H. pylori at different time points. I, AGS and BGC-823 cells were infected with H. pylori strains 26695 (MOI 1:100) for 2 and 4 hours. hTERT protein expression was detected using western blot, respectively. J, hTERT protein was induced in AGS cells by H. pylori 26695 in a dose-dependent way. K, H. pylori CagA induced hTERT expression. AGS and BGC-823 cells were transfected with expression vectors encoding CagA for 72 hours and then analyzed hTERT expression using western blot. L, High expression of two different hTERT transcripts predicted poor prognosis of gastric cancer patients from public database. Error bars indicate mean ± SD; Student t test, *, P < 0.05; ***, P < 0.001.
- Figure 2.
NF-κB/LIN28A/let-7 axis was activated upon H. pylori infection. A, miR-let-7a expression in H. pylori–positive and H. pylori–negative patients from GEO database (GSE19769). B and C, qRT-PCR detection of miR-let-7a expression in AGS and BGC-823 cells infected with H. pylori strains 26695 and transfected by CagA expression plasmid, respectively. D, miR-let-7a downregulation by H. pylori strains 26695 in a dose-dependent way in AGS cells. E, Let-7a expression in BGC-823 cells infected by H. pylori 26695 at different time points. F, Let-7a expression in GES-1 cells infected by H. pylori 26695 at different time points. G, Let-7a expression in CagA-transfected and specific signaling pathway inhibitor–treated AGS cells. AGS cells were pretreated with DMSO or specific pathway inhibitors for 1 hour, and then transfected without or with CagA for 48 hours [(PPI, 10 μmol/L), (SB203580, 10 μmol/L), (BAY 11-7082, 5 μmol/L), (LY294002, 10 μmol/L)]. H, Let-7a expression in GES-1 cells infected by CagA-negative H. pylori at different time points. I and J, CagA induced Lin28A expression in AGS and BGC-823 cells. Lin28A mRNA and protein expression was analyzed using qRT-PCR and western blot, respectively. K, Immunofluorescence (IF) assay of NF-κB nuclear translocation in gastric cancer (GC) cells after CagA transfection. L, H. pylori 26695–induced LIN28A expression was blocked by BAY 11-7082. Shown is one representative of two independent experiments. Error bars, mean ± SD; Student t test, *, P < 0.05; ***, P < 0.001.
- Figure 3.
Let-7a directly targeted hTERT to inhibit its expression. A, Schematic diagram of the predicted let-7a target site in 3′-UTR of hTERT mRNA. B and D, hTERT mRNA expression was determined in control or let-7a–overexpressed or –suppressed AGS and BGC-823 cells. C and E, hTERT protein expression was determined in control mimics/inhibitors or miR-let-7a mimics/inhibitor-transfected AGS and BGC-823 cells. F, Telomerase activity in let-7a–overexpressed or –suppressed BGC-823 cells was analyzed using telomerase ELISA kit. G, Telomere length assay in let-7a–upregulated/downregulated BGC-823 cells was analyzed using Telo TAGGG telomere length assay kit. Shown is one representative of two independent experiments. H and I, Regulatory effect of let-7a on the 3′-UTR of hTERT mRNA. After 24-hour transfection with control or pSli-let-7a and anti-let-7a, a reporter plasmid containing wild-type 3′-UTR hTERT mRNA and a plasmid expressing Renilla luciferase (pRL-TK) were cotransfected into AGS and BGC-823 cells. Firefly luciferase activity was normalized to Renilla luciferase activity. J, Let-7a inhibited gastric cancer (GC) cell proliferation. Colony formation assay was used. K, Let-7a and hTERT expression in the progressive gastric diseased samples (SG, superficial gastritis; AG, atrophic gastritis). L and M, Let-7a and hTERT expression in differently treated gastric cancer cells, respectively. Cells were transfected with control (pcDNA3.1) or CagA expression plasmids for 24 hours and then were transfected with control (pSilencer) or let-7a expression plasmids for another 48 hours. N, Colony formation assay in differently treated BGC-823 cells. Cells were transfected with control (pcDNA3.1) or CagA plasmids for 24 hours, and then were transfected with control (pSilencer) or let-7a expression plasmids for another 48 hours. Then 300 cells were seeded into each well in 6-well plate and incubated for around 2 weeks. Error bars, mean ± SD; Student t test, *, P < 0.05; ***, P < 0.001. Shown is one representative of three independent experiments.
- Figure 4.
hTERT mediated the effects of let-7a on gastric cancer (GC) cell proliferation. A, AGS and BGC-823 cells transfected with control siRNA or two specific hTERT siRNAs (si1 and si2) for 72 hours; protein expression was detected by western blot. B and C, Colony formation and EdU incorporation assays showed gastric cancer cell proliferation in different groups. The cells were transfected with control or hTERT siRNA for 48 hours and then seeded into six-well plate for colony formation assay and into 96-well plate for staining EdU. D, Colony formation assay in differently treated gastric cancer cells. The cells were transfected with control or hTERT siRNA for 24 h, and then were transfected with control or anti-let-7a plasmids for another 48 h. Then 300 cells were seeded into each well in 6-well plate and incubated for around 2 weeks. E–H, Gastric cancer cell proliferation in differently treated groups. Colony formation assay and EdU incorporation assay were used. Error bars, mean ± SD; Student t test, *, P < 0.05; ***P, < 0.001. Shown is one representative of three independent experiments.
- Figure 5.
hTERT positively regulated LIN28A expression and downregulated let-7a expression. A, Let-7a, hTERT, and Lin28A expression were determined using qRT-PCR in hTERT-depleted gastric cancer (GC) cells. B, LIN28A and hTERT expression were determined by western blot in differently treated gastric cancer cells. AGS cells were pretreated with DMSO or BAY 11-7082 (5 μM) for 1 hour and then transfected with control or hTERT overexpression plasmids for 48 hours. C, LIN28A and hTERT expression was determined by western blot in differently treated BGC-823 cells. The cells were transfected with control or hTERT siRNA for 24 hours, and then were transfected with control or anti-let-7a plasmids for another 24 hours. D and E, LIN28A and let-7a expression levels in tumors derived from control- or hTERT-depleted BGC-823 cell injected mice (n = 7). Error bars, mean ± SD; Student t test, **, P < 0.01; ***, P < 0.001.
- Figure 6.
Confirmation of positive correlation between hTERT and LIN28A, and negative correlation between hTERT and let-7a in mouse and human tissues. A, IHC staining of mTERT (mouse TERT) and LIN28A in mice mucosal epithelial tissue with control and H. pylori infection. Scale bars, 50 μm. B, IHC staining of hTERT and LIN28A in different stages of clinical gastric samples. Scale bars, 50 μm. C–E, Let-7a, hTERT, and LIN28A expression levels in AG/IM (n = 26) and gastric cancer (GC; n = 14) clinical samples were analyzed using qRT-PCR. F, Schematic model of this study. H. pylori infection led to the translocation of NF-κB subunit p65 into nucleus. Then the activated NF-κB transcriptionally enhanced LIN28A expression, which suppressed let-7a biogenesis, resulting downregulation of let-7a upon H. pylori infection. Decreased let-7a lost the inhibitory effect on the translation of hTERT, leading to more hTERT protein translation and telomerase reactivation, which facilitated gastric cancer formation. hTERT in turn enhanced LIN28A expression, forming the positive feedback regulation between NF-κB/LIN28A/let-7a axis and hTERT to sustain its overexpression in gastric cancer. Error bars, mean ± SD; Student t test, ***, P < 0.001.
Tables
- Table 1.
The relevance between hTERT expression and clinicopathologic parameters in human gastric tissues.
Staging Number Age (years) Sex (M/F) hTERT qRT-PCRa hTERT IHCb SG 19 45.8 ± 16.2 9/10 — — n = 15 3.23 ± 2.17 AG/IM 32 49.7 ± 19.1 20/12 n = 26 16.58 ± 1.91 n = 15 20.06 ± 5.89 Dysplasia 19 57.8 ± 14.8 12/7 — — n = 16 39.89 ± 6.54 Cancer 27 59.1 ± 15.3 19/8 n = 20 29.81 ± 2.58 n = 25 58.82 ± 7.32
Supplementary Data
- Supplementary Figure 1 - Figure S1. IF staining of p65 and hTERT in CagA transfected BGC-823 cells.
- Supplementary Figure 2 - Figure S2. (a) hTERT and Lin28A expression were determined using western blot in hTERT depleted GC cells. (b) Co-expression of hTERT and Lin28A in GC cells.
- Supplementary Figure Legends - Supplementary Figure Legends 1 and 2
Volume 19, Issue 1
