1. Volume 156, Issue 4, Pages 1127-1139.e8 (March 2019)
Integrated Analysis of Mouse and Human Gastric Neoplasms Identifies Conserved microRNA Networks in Gastric Carcinogenesis 
Zheng Chen, Zheng Li, Mohammed Soutto, Weizhi Wang, M. Blanca Piazuelo, Shoumin Zhu, Yan Guo, Maria J. Maturana, Alejandro H. Corvalan, Xi Chen, Zekuan Xu, Wael M. El-Rifai 
Gastroenterology 
Volume 156, Issue 4, Pages e8 (March 2019)
DOI: /j.gastro
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2. Gastroenterology 2019 156, 1127-1139. e8DOI: (10. 1053/j. gastro. 2018
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3. Figure 1
miRNA signature in mouse and human gastric cancer. (A) The number of significantly deregulated miRNAs in stage I or II human GC compared with NG tissues from non-cancer patients (NG) (P < .05). (B) The number of significantly deregulated miRNAs in human TANG tissue samples compared with NG (P < .05). (C) A Venn diagram analysis of A and B depicts 676 miRNAs that are similarly up-regulated in GC and TANG compared with NG (P < .05). (D) A Venn diagram analysis of panels A and B depicts 46 miRNAs that are similarly down-regulated in GC and TANG compared with NG (P < .05). (E) Hierarchical cluster analysis of miRNA expression in GC (stages I and II), NG) and TANG tissues. (F) The number of significantly deregulated miRNAs in mGC from the TFF1-KO mice model compared with mNG tissues (P < .05). (G) Hierarchical cluster analysis of miRNA expression in mouse tissues (mGC and mNG). (H) A Venn diagram analysis of significantly deregulated miRNA in human gastric cancer and mGC samples shows consistent and conserved up-regulation of 58 miRNAs and down-regulation of 5 miRNAs in both mouse and human amples (P < .05). Upper panel, up-regulated miRNAs. Lower panel, down-regulated miRNAs. (I) Circular plot from Ingenuity Pathway Analysis of sequencing data identified (inner circle) 18 significantly deregulated signaling pathways in (middle circle) human and (outer circle) mGC samples. The number of miRNAs regulating each pathway is denoted.
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4. Figure 2
qRT-PCR validation of expression levels of up-regulated miRNAs in human and mouse gastric tumors. (A) Expression analysis of MIR135B-5p analysis in TFF1-KO gastric LGD and cancer, compared with NG tissues. (B) Expression analysis of MIR135B-5p in human GC compared with TANG tissues, cohort 1 (United States). (C) Expression analysis of MIR135B-5p in GC compared with NG tissues from non-cancer patients, cohort 2 (Chile). (D) Expression analysis of MIR135B-5p in NG, TANG, and GC tissues, cohort 3 (China). (E–H) Expression analysis of MIR196B-5p (E–H) and MIR92A-5p (I–L) in the same samples as in A–D. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Mann-Whitney test.
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5. Figure 3
qRT-PCR validation of expression levels of down-regulated miRNAs in human and mouse gastric tumors. (A) Expression analysis of MIR143-3p analysis in TFF1-KO gastric LGD and cancer, compared with NG tissues. (B) Expression analysis of MIR143-3p in human GC compared with TANG tissues, cohort 1 (United States). (C) Expression analysis of MIR143-3p in GC compared with NG tissues from non-cancer patients (NG), cohort 2 (Chile). (D) Expression analysis of MIR143-3p in NG, TANG, and GC tissues, cohort 3 (China). (E–H) Expression analysis of MIR204-5p (E–H) and MIR133-3p (I–L) in the same samples as in A–D. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Mann-Whitney test.
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6. Figure 4
(A–H) MIR143-3p suppresses cellular proliferation and gastric organoid growth. Reconstitution of MIR143-3p was established by using lentivirus infection, followed by puromycin selection. (A) qRT-PCR analysis of MIR143-3p expression level after its stable reconstitution in STKM2 cells, compared with control (left panel). ATP-glo cell viability assay analysis of MIR143-3p stably reconstituted STKM2 cells and control cells (right panel).
Experiments similar to those in A were performed in (B) MKN45, (C) AGS, and (D) MKN28. (E, left) EdU immunofluorescence staining in MIR143-3p stably reconstituted STKM2 cells and controls, and (right) quantification of data are shown. Experiments similar to those in E were performed in (F) MKN45, (G) AGS, and (H) MKN28. (I–N) Reconstitution of MIR143-3p in gastric organoids from mouse LGD lesions in TFF1-KO mouse. (I) Light-field images of mouse gastric organoids (days 1, 3, 5, and 7). (J) Light-field images of mouse gastric organoids, 3 days after MIR143-3p reconstitution using lentivirus infection or control lentivirus. (K) Quantification data of organoid diameters from J, Mann-Whitney test. (L) qRT-PCR analysis of MIR143-3p expression levels, after reconstitution of MIR143-3p in gastric organoids and controls. (M) Ki-67 immunofluorescence staining in gastric organoids, 3 days after MIR143-3p reconstitution with lentivirus infection or control lentivirus. (N) Quantification data of Ki67-positive cells in M. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Student t test.
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7. Figure 5
BRD2 is a direct downstream target of MIR143-3p. (A) BRD2 is a predicted downstream target of MIR143-3p, based on 3 online databases. (B) Two predicted MIR143-3p binding sites on human BRD2 3′-UTR are shown. (C) Western blot analysis of BRD2, MYC, and β-actin protein expression levels in 4 normal human gastric tissues (NG) and AGS, MKN28, MKN45, and SNU-1 gastric cancer cell lines. (D) Western blot analysis of BRD4, BRD2, and β-actin protein expression levels in AGS cell, after transient reconstitution of MIR143-3p using a mimic (2.5–40 pmol). BRD2, not BRD4, is down-regulated after reconstitution. Western blot analysis after reconstitution of MIR143-3p in (E) AGS, (F) STKM2, (G) MKN45, and (H) mouse LGD gastric organoids. WT or mutant (missing both MIR143-3p bindings sites) BRD2 3′-UTR luciferase reporter analysis in (I) AGS cells or (J) MKN45 after stable reconstitution of MIR143-3p or control. (K) c-MYC promoter (4×TBE1 wt) luciferase reporter analysis in AGS cells after reconstitution of MIR143-3p or control, with and without BRD2 transient transfection. (L) qRT-PCR analysis of c-MYC gene expression level in mouse LGD gastric organoids after reconstitution of MIR143-3p or control. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Student t test or 1-way ANOVA.
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8. Figure 6
MIR143-3p sensitizes GC cells to CDDP treatment. (A) IC50 analysis using ATP-glo assay in STKM2 cells with stable reconstitution of MIR143-3p or control treated with CDDP (0, 0.75, 1.55, 3.1, 6.25, 12.5, 25, or 50 μmol/L). (B–D) Similar experiments in (B) MKN45, (C) AGS, and (D) MKN28 cells. IC50s were calculated using Prism 5 software. (E) Representative clonogenic survival assay in AGS cells with stable reconstitution of MIR143-3p or controls with or without 2.5 μmol/L of CDDP treatment. (F) Quantification of surviving colonies. (G) IC50 analysis based on clonogenic assay similar to E (CDDP: 10, 5, 2.5, 1.25, or μmol/L) for 24 hours and measurements at day 7. (H) Western blot analysis of BRD2, total PARP (PARP), cleaved PARP (c-PARP), c-MYC, and β-actin protein expression level in MKN45 cells with stable-reconstitution MIR143-3p or control after treatment with CDDP or JQ-1 (BRD2 inhibitor) alone or in combination. ∗∗∗P < .001, Student t test. M, mol/L.
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9. Figure 7
MIR143-3p inhibits GC tumor xenograft growth and sensitizes to CDDP treatment in vivo. (A) Representative images of gastric cancer cell (AGS) tumor xenografts using stable reconstitution of MIR143-3p or control with and without treatment with CDDP. (B) qRT-PCR analysis of MIR143-3p expression in tumor xenografts. (C) Xenograft tumor growth rates. (D) Western blot analysis of BRD2, total PARP (PARP), cleaved PARP (c-PARP), c-MYC, and β-actin protein expression levels of tumor xenografts. (E) Immunohistochemistry staining of Ki-67 or cleaved caspase 3 in tumor xenografts (left panel) with quantification of immunohistochemistry data (right panel). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, Student t test or 1-way ANOVA.
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10. Supplementary Figure 1
Top deregulated miRNAs in both TFF1-KO mouse and human gastric cancers. (A) Top up-regulated miRNAs in human (left side) and mouse (right side) gastric cancer samples. (B) Top down-regulated miRNAs in human (left side) and mouse (right side) gastric cancer samples.
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11. Supplementary Figure 2
qRT-PCR validation of miRNAs in non-cancer normal (NG), stages I and II and stages III and IV gastric cancer tissues samples. qRT-PCR analysis of (A) MIR135b-5p, (B) MIR196B-5p, (C) MIR92A-5p, (D) MIR143-3p, (E) MIR204-5p, and (F) MIR133-3p. Expression levels were measured in non-cancer normal (NG), stages I and II, and stages III and IV gastric cancer (GC) tissues samples (cohort 3). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001; Mann-Whitney test.
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12. Supplementary Figure 3
MIR143-3p expression in normal human gastric tissue samples and gastric cancer cell lines. (A) qRT-PCR analysis of MIR143-3p expression level in 4 normal gastric tissues and AGS, MKN28, MKN45, and STKM2 cell lines. (B) Quantification and data analysis of A. ∗P < .05, Mann-Whitney test.
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13. Supplementary Figure 4
BRD2 and MYC protein expression levels were significantly up-regulated in TFF1-KO mice gastric cancer (GC) samples. (A) Western blot analysis of BRD2, MYC, and β-actin (Actin) protein expression levels in WT (normal) and TFF1-KO mice (gastric cancer) tissue samples. (B) Quantification of BRD2 and MYC protein expression levels in A. Data were normalized to respective β-actin expression level with Image Lab software (Bio-Rad). (C) qRT-PCR analysis of (left panel) MIR143-3p expression levels in same samples as in A relative to the expression levels of MIR101-3p and MIR140-3p; (right panel) Tff1 mRNA expression level in the same samples as in A. ∗∗, P < .01, Mann-Whitney test.
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14. Supplementary Figure 5
MIR143-3p binding sites were mutated in mutant BRD2 3′ UTR reporter plasmid. BRD2 3′ UTR sequences (BRD2 3′ UTR) containing MIR143-3p binding sites as compared with the mutant BRD2 3′ UTR reporter DNA sequencing results of MIR143-3p binding sites (sequencing 1 and sequencing 2).
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15. Supplementary Figure 6
MIR143-3p reconstitution significantly decreased MYC mRNA expression levels in human gastric cancer cells. qRT-PCR analysis showed that MIR143-3p stable reconstitution has no effect on regulating BRD2 mRNA expression in (A) STKM2 and (B) AGS cells. (C, D) MIR143-3p stable reconstitution significantly decreased MYC mRNA expression levels in STKM2 and AGS cells, as compared with control cells. ∗P < .05, Student t test.
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16. Supplementary Figure 7
Strong inverse correlation between MIR143-3p and c-MYC mRNA expression levels in human gastric tissue samples. qRT-PCR analysis of MIR143-3p and MYC mRNA expression levels in normal and gastric cancer tissue samples from human cohort 1. Data analyzed using linear regression.
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17. Supplementary Figure 8
BRD2 expression predicts poor overall survival outcome in gastric cancer patients. BRD2 mRNA expression and gastric cancer patients’ overall survival data analysis of 320 intestinal gastric cancers were obtained from an online database (
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18. Supplementary Figure 9
BRD2 expression correlates with poor overall survival outcome in gastric cancer patients. Clinical, survival, and MIR143-3p expression data of 60 gastric cancer patients were obtained from the First Affiliated Hospital of Nanjing Medical University. Kaplan-Meier survival analysis was performed using SPSS, version 24. Cum, cumulative; Suv, survival.
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