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Published in final edited form as: Gastroenterology. 2018 Nov 28;156(4):1127–1139.e8. doi: 10.1053/j.gastro.2018.11.052

Integrated Analysis of Mouse and Human Gastric Neoplasms Identifies Conserved microRNA Networks in Gastric Carcinogenesis

Zheng Chen 1,2,#, Zheng Li 1,#, Mohammed Soutto 2,3, Weizhi Wang 1, M Blanca Piazuelo 4, Shoumin Zhu 2, Yan Guo 5, Maria J Maturana 6, Alejandro H Corvalan 6, Xi Chen 7, Zekuan Xu 1,8,*, Wael M El-Rifai 2,3,9,*
PMCID: PMC6409191  NIHMSID: NIHMS1515208  PMID: 30502323

Abstract

Background & Aims:

microRNAs (miRNAs) are small non-coding RNAs that bind to 3′UTR regions of mRNAs to promote their degradation or block their translation. Mice with disruption of the trefoil factor 1 gene (Tff1) develop gastric neoplasms. We studied these mice to identify conserved miRNA networks involved in gastric carcinogenesis.

Methods:

We performed next-generation miRNA sequencing analysis of normal gastric tissues (based on histology) from subjects without evidence of gastric neoplasm from patients (n=64) and TFF1-knockout mice (n=22). We validated our findings using 270 normal gastric tissues (including 61 samples from patients without evidence of neoplastic lesions) and 234 gastric tumor tissues from 3 separate cohorts of patients and from mice. We performed molecular and functional assays using cell lines (MKN28, MKN45, STKM2, and AGS cells), gastric organoids, and mice with xenograft tumors.

Results:

We identified 117 miRNAs that were significantly deregulated in mouse and human gastric tumor tissues, compared with non-tumor tissues. We validated changes in levels of 6 miRNAs by quantitative real-time PCR analyses of neoplastic gastric tissues from mice (n=39) and 3 independent cohorts patients (332 patients total). We found levels of MIR135B-5p, MIR196B-5p, and MIR92A-5p to be increased in tumor tissues whereas levels of MIR143-3p, MIR204-5p, and MIR133–3p were decreased in tumor tissues. Levels of MIR143-3p were reduced not only in gastric cancer tissues, but also in normal tissues adjacent to tumors in humans and low-grade dysplasia in mice. Transgenic expression of MIR143-3p in gastric cancer cell lines reduced their proliferation and restored their sensitivity to cisplatin. AGS cells with stable transgenic expression of MIR143-3p grew more slowly as xenograft tumors in mice than control AGS cells; tumor growth from AGS cells that expressed MIR143-3p, but not control cells, was sensitive to cisplatin. We identified and validated bromodomain containing 2 (BRD2) as a direct target of MIR143-3p; increased levels of BRD2 in gastric tumors associated with shorter survival times of patients.

Conclusions:

In an analysis of miRNA profiles of gastric tumors from mice and human patients, we identified a conserved signature associated with early stages of gastric tumorigenesis. Strategies to restore MIR143-3p or inhibit BRD2 might be developed for treatment of gastric cancer.

Keywords: stomach, transcription factor, tumor suppressor, progression

Graphical Abstract

graphic file with name nihms-1515208-f0001.jpg

Introduction

Gastric cancer is the third leading cause of cancer related deaths worldwide, leading to more than 700,000 deaths each year1. In 2014, there were an estimated 95,764 people living with stomach cancer in the United States2. Almost one-third of gastric cancer patients are diagnosed at a late stage (Stage III or IV) with a five-year survival rate of 5.2%2. Identification of molecular alterations in gastric tumorigenesis is a critical step for improving diagnostic and therapeutic options for gastric cancer.

microRNAs (miRNAs), short non-coding RNAs with a length of 18-22nt, downregulate protein levels through blocking mRNA translation or inducing mRNA degradation3. miRNA expression and function are tissue and context specific4. In cancer, miRNAs are classified as oncogenic or suppressive based on their molecular targets and biological functions5. The fact that miRNAs are more stable than mRNA and proteins, makes them ideal candidates for biomarkers as they can easily be detected and quantified in frozen and formalin-fixed, paraffin-embedded (FFPE) tissues as well as in body fluids such as serum, plasma, saliva and urine 6, 7. Although several studies have reported alterations of miRNAs’ expression in gastric cancer, such as overexpression of MIR21 and downregulation of MIR148A downregulation8, 9, identification of critical changes in early stages of gastric tumorigenesis have been hampered, not only by the lack of early-stage samples, but also by the observed immense heterogeneity among patients.

TFF1, a family member of the mammalian trefoil factor family (TFF), is a secreted protein expressed predominantly in the foveolar epithelial surface cells of the gastric mucosa 10. Recent studies have shown that TFF1 is a tumor suppressor gene, silenced in more than two-thirds of gastric carcinomas11, 12. The TFF1-knockout (KO) mouse model exhibits a pro-inflammatory phenotype with age-dependent histological changes of gastric neoplasia occurring in the pyloro-antrum glandular region of the mouse stomach11, 13. Integrated comprehensive analysis of gene expression demonstrated conserved activation of common transcription networks in the TFF1-KO mouse and human gastric cancers that included activation of STAT3, β-catenin and MYC signaling14. This data supports the notion that the TFF1-KO mouse model develops gastric tumors that closely mimic the human disease at the molecular level.

Bromodomain containing 2 (BRD2) is a family member of the bromodomain and extra-terminal motif (BET) protein family that also contains BRD3, BRD4, and BRDT in mammals15. BRD2, a transcription regulator, associates with transcription complexes and acetylated chromatin during mitosis15, 16. BRD2 selectively binds to the acetylated lysine-12 residue of histone 4 (H4) via its two bromodomains. Recent studies have demonstrated that BRD2 inhibition decreases MYC expression level in several human maliganances17–19. Currently, there are a number of clinical trials targeting BET family proteins, such as FT-1011 in hematologic malignancies20 and CPI-0610 in progressive lymphoma21.

In this study, we performed comprehensive miRNA sequencing with integrated bioinformatics analysis and identified a unique conserved miRNA signature in mouse and human gastric cancers. The results were validated in three independent human cohorts of gastric cancer, followed by molecular and functional analysis of MIR143-3p. We report BRD2 as a direct target of MIR143-3p, where its downregulation leads to induction of BRD2 and c-MYC with subsequent resistance to conventional chemotherapeutics.

Materials and Methods

Materials and Methods are described briefly, below. Additional methods and details are given in the Supplementary Materials and Methods section.

Mouse and human gastric tissue samples

We used gastric tissue samples from TFF1-KO and wild-type (WT) mice, all from the same C57BL/6J/129/Svj background. Tissue samples from the glandular antrum region of the stomach were collected from 39 TFF1-KO and 22 WT mice of matching ages (Supplementary Table S1). All vertebrate animal studies were approved by the Institutional Animal Care and Use Committees at the University of Miami and Vanderbilt University Medical Center. Based on histological evaluation, we selected tissue samples that showed low-grade dysplasia (LGD) or adenocarcinoma from the TFF1-KO mice. Tissue samples from the WT mice showed normal gastric mucosa histology. The histology and age are shown in Supplementary Table S1. De-identified human tissue samples from 25 gastric cancer and 28 normal gastric tissue samples were collected from the National Cancer Institute Cooperative Human Tissue Network (CHTN) and the pathology archives at Vanderbilt University Medical Center (Nashville, TN). All tissue samples were collected, coded, and de-identified in accordance with the Institutional Review Board-approved protocols. The histology and age information are included in Supplementary Table S2. 27 normal gastric (NG) tissues from non-cancer patients, 165 tumor-adjacent normal gastric tissues (TANG, >5cm away from the edge of cancer, pathologically negative for cancer), and 206 gastric cancer (GC) tissue samples were collected from patients who underwent surgery in the First Affiliated Hospital of Nanjing Medical University. Pathology diagnoses and archives were also obtained from the First Affiliated Hospital of Nanjing Medical University. This study and all protocols used in human sample collection were approved by the Ethics Committee with the permission number 2015-SRFA-027. The histology and age information for all gastric tissue samples are included in Supplementary Table S3. In addition, 63 tissue samples (29 gastric cancer and 34 non-tumor tissues) were selected from the Instituto Chileno Japonés de Enfermedades Digestivas-Hospital Clínico San Borja Arriarán (ICHJED-HCSBA), Sotero del Rio Hospital and Arturo Lopez Perez Foundation (FALP), Santiago, Chile between 2012 to 2016, in accordance with ICHJED-HCSBA Institutional Review Board-approved protocols. The histology information is included in Supplementary Table S4. Tissue samples from individual cases were classified in accordance to Japanese Research Society for Gastric Cancer recommendations22. This study was approved by the Institutional Review Boards (IRB) of each participating institution. Written informed consents were obtained from each participant involved in the study.

Next generation MIRsequencing (MIRseq) analysis

22 RNA samples from mouse stomachs (7 wild-type normal, 8 TFF1-KO low grade dysplasia, and 7 TFF1-KO gastric adenocarcinoma tissues) and 64 RNA samples from human stomachs (9 normal from non-cancer patients, 16 tumor adjacent normal gastric (TANG), and 20 Stage I and 19 Stage III intestinal-type gastric adenocarcinoma tissue samples) were extracted using the miRNeasy Mini Kit (Qiagen) for MIRseq. An adjusted p value less than 0.05 was considered significant for MIRseq data analysis23. Detailed sample information can be found in Supplementary Table S1 and S3. Unsupervised cluster analysis and heat maps were generated using R package heatmap3 24. Spearman’s correlation coefficients were computed as distances between pair-wise samples and plotted as the edges in the dendrogram. Pathway analysis using Ingenuity Pathway Analysis software (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/) was performed in both human and mouse gastric tissue samples. miRNA changes with significant p-values (adjusted p < 0.05) were selected for pathway analysis. A detailed protocol for MIRseq is included in the Supplementary Materials and Methods.

Cell culture and reagents

Human gastric adenocarcinoma cell lines AGS, STKM2, MKN28, MKN45 and SNU-1 were maintained in culture using Ham’s F-12 Nutrient Mixture (AGS, MKN28 and STKM2) (GIBCO, Carlsbad, CA) or Dulbecco’s modified Eagle’s medium (MKN45 and SNU-1) supplemented with 5% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA) and 1% penicillin/streptomycin (GIBCO).

Quantitative real-time PCR (qRT-PCR) validation of miRNA alterations in mice and human gastric tissues

This analysis was performed using independent tissue samples from mice and humans. Mouse glandular stomach tissue samples included 16 TFF1-WT, 10 TFF1-KO LGD, and 13 TFF1-KO adenocarcinomas. The mouse age and gastric histology information are provided in Supplementary Table S1. Deidentified human stomach tissue samples included 28 normal and 25 GC for Cohort 1 (Supplementary Table S2), 34 normal gastric and 29 gastric adenocarcinoma tissue samples for cohort 2 (Supplementary Table S4), 27 NG, 165 TANG, and 167 GC for cohort 3 (Supplementary Table S3). miRNA expression levels were normalized to MIR101–3p and MIR140–3p combination expression levels in both human and mouse gastric tissue samples or cell lines based on a recent study of identification of reference miRNA in gastric cancer25.

Immunofluorescence and immunohistochemistry

Immunofluorescence and immunohistochemistry were performed as previously described26. Detailed antibody information is included in the Supplementary Materials and Methods.

Gastric cancer patients’ overall survival data analysis

BRD2 mRNA expression and gastric cancer patients’ overall survival data analysis of 320 gastric cancers was obtained from online database at http://kmplot.com/analysis/ 27. Clinical, survival, and MIR143-3p expression data of 60 gastric cancer patients was obtained from the First Affiliated Hospital of Nanjing Medical University. Kaplan-Meier survival analysis was performed using IBM SPSS statistics 24.

Statistical analyses

Data is demonstrated as mean ± standard deviation of 3 independent experiments. Statistical significance was determined by using Student’s t test, analysis of variance, Mann-Whitney U test, One-way ANOVA, liner regression or non-liner regression analysis. Differences with P values ≤0.05 were considered significant.

Results

Identification of a conserved miRNA signature in human and mouse gastric neoplasms

We hypothesized that an integrated analysis approach of mouse and human gastric tumors can identify a conserved miRNA signature and overcome the observed heterogeneity issues in studies of human samples. This approach allowed us to focus on molecular changes that are more likely drivers of the tumorigenic process. MIRseq analysis was utilized for analysis of gastric tissue samples from human (NG, TANG and stage I and II intestinal-type GC) and mouse [normal (WT) and GC (TFF1-KO)]. Within 2203 miRNA readings, we detected 795 significantly overexpressed and 62 down-regulated miRNAs in human GCs, as compared to normal gastric (NG) tissues from non-cancer patients (Figure 1A, P<0.05). By using tumor-adjacent normal gastric (TANG) tissues, that are histologically normal, we found that TANG tissues have 831 significantly up-regulated and 68 down-regulated miRNAs, as compared with NG tissues from non-cancer patients (Figure 1B, P<0.05). Of note, this signature overlapped with the signature that we obtained when gastric cancer tissues were compared to normal gastric tissues from non-cancer patients; similarities included 623 overexpressed (Figure 1C) and 42 down-regulated miRNAs (Figure 1D). These findings suggest that histologically normal tumor-adjacent normal gastric tissues display an aberrant molecular signature that may reflect early tumorigenic changes. Using clustering analysis, the majority of NG tissues clustered separate from GC and TANG tissues that showed similar patterns (Figure 1E). Analysis of MIRseq data from mouse tissues indicated that among the 1386 miRNA readings, 158 miRNAs were significantly up-regulated whereas 33 were down-regulated in mouse gastric cancer (mGC) tissues from the TFF1-KO mice, as compared with mouse normal gastric (mNG) tissues (Figure 1F, P<0.05). Clustering analysis of mouse data demonstrated a perfect separation of signatures from neoplastic lesions versus normal (Figure 1G). By comparing human and mouse data, we detected consistent and conserved alterations that included 58 up-regulated and 5 down-regulated miRNAs (Figure 1H). The top significantly de-regulated miRNAs in both mouse and human gastric cancers are listed in Supplementary Figure 1. Using integrated pathway analysis of the predicted target genes, we discovered 18 signaling pathways that are significantly regulated by the identified miRNA signatures in mouse and human gastric cancers (Figure 1I).

Figure 1. miRNA signature in mouse and human gastric cancer.

Figure 1.

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A) The number of significantly de-regulated miRNAs in Stage I or II human gastric cancer (GC), as compared with normal gastric tissues from non-cancer patients (NG) (P<0.05). B) The number of significantly de-regulated miRNAs in human tumor-adjacent normal gastric tissue (TANG) samples, as compared with NG (P<0.05). C) A Venn diagram analysis of panels A and B depicts 676 miRNAs that are similarly up-regulated in GC and TANG, as compared to NG (P<0.05). D) A Venn diagram analysis of panels A and B depicts 46 miRNAs that are similarly down-regulated in GC and TANG, as compared to NG (P<0.05). E) Hierarchical cluster analysis of miRNA expression in GC (stages I and II), normal gastric (NG), and tumor-adjacent normal gastric (TANG) tissues. F) The number of significantly de-regulated miRNAs in mouse gastric cancers (mGC) from the TFF1-KO mice 0 model, as compared with mouse normal gastric (mNG) tissues (P<0.05). G) Hierarchical cluster analysis of miRNA expression in mouse tissues (mGC and mNG). H) A Venn diagram analysis of significantly de-regulated miRNA in human gastric cancer and mouse gastric cancer samples demonstrates consistent and conserved up-regulation of 58 miRNAs and down-regulation of 5 miRNAs in both mouse and human (P<0.05). Upper panel for up-regulated miRNAs. Lower panel for down-regulated miRNAs. I) Circular plot from Ingenuity pathway analysis of sequencing data identified 18 significantly de-regulated signaling pathways (inner circle) in human (middle circle) and mouse (outer circle) gastric cancer samples, the number of miRNAs regulating each pathway is denoted.

Validation of miRNA signature in mouse and human gastric cancer tissue samples

We validated our results by using qRT-PCR for analysis of six representative miRNAs in human and mouse samples. We have screened the 10 up- and down-regulated miRNAs in a pilot study of 20 paired samples. These 6 miRNAs demonstrated the best P values and were selected for additional screening using a larger sample size from three independent cohorts. The human samples included three independent gastric cancer cohorts (see Materials and Methods section for details). Overexpression of MIR135B-5p, MIR196B-5p and MIR92A-5p was validated in both mouse and human samples. MIR135B-5p was significantly up-regulated in mouse gastric adenocarcinoma samples, as compared with normal gastric tissues (Figure 2A, P<0.01). MIR135B-5p expression was significantly overexpressed in GC or TANG tissue samples from three distinct cohorts (Figure 2B to D, P<0.01). Similar findings were noted for MIR196B-5p (Figure 2E to H) and MIR92A-5p (Figure 2I to L). Of note, MIR92A-5p expression level was also significantly higher in TANG tissues as compared to normal gastric tissues from non-cancer patients (Figure 2 H&L, P<0.01), suggesting that it may be an early molecular alteration in gastric tumorigenesis.

Figure 2. Quantitative real-time RT-PCR (qRT-PCR) validation of expression levels of up-regulated miRNAs in human and mouse gastric tumors.

Figure 2.

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A) Expression analysis of MIR135B-5p analysis in TFF1-KO gastric low-grade dysplasia (LGD) and cancer, as compared to normal gastric tissues. (B) Expression analysis of MIR135B-5p in human gastric cancer (GC) as compared to tumor-adjacent normal gastric (TANG) tissues, cohort 1 (United States). (C) Expression analysis of MIR135B-5p in GC as compared to normal gastric tissues from non-cancer patients (NG), 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<0.05, **P<0.01, ***P<0.001, Mann Whitney Test.

In the meantime, down-regulation of MIR143-3p, MIR204-5p and MIR133-3p was validated in both mouse and human samples. MIR143-3p was significantly down-regulated in mouse LGD and gastric cancer samples, as compared with normal gastric tissues (Figure 3A, P<0.01).

Figure 3. Quantitative real-time RT-PCR validation of expression levels of down-regulated miRNAs in human and mouse gastric tumors.

Figure 3.

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A) Expression analysis of MIR143-3p analysis in TFF1-KO gastric low-grade dysplasia (LGD) and cancer, as compared to normal gastric tissues. (B) Expression analysis of MIR143-3p in human gastric cancer (GC) as compared to tumor-adjacent normal gastric (TANG) tissues, cohort 1 (United States). (C) Expression analysis of MIR143-3p in GC as compared to normal gastric 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<0.05, **P<0.01, ***P<0.001, Mann Whitney Test.

MIR143-3p expression was significantly down-regulated in human gastric cancer tissues samples from three different cohorts (Figure 3B to D, P<0.05). Furthermore, MIR143-3p expression level was also significantly down-regulated in TANG tissues as compared with NG tissue samples (Figure 3D, P<0.05), suggesting that down-regulation of MIR143-3p may be an early molecular change in gastric tumorigenesis. Similar results were found for MIR204-5p (Figure 3E to H) and MIR133–3p (Figure 3I to L).

Of note, our qRT-PCR data indicated that there were no significant differences of miRNA (MIR135B-5p, MIR92A-5p, MIR196B-5p, MIR143-3p, MIR133–3p, and MIR204-5p) expression levels between early stage (stages I & II) and late stage (stages III & IV) gastric cancer tissue samples (Supplementary Figure 2). In summary, miRNA dysregulation from our data was validated in gastric cancer tissue samples from three independent cohorts, suggesting that integrated combined analysis of mouse and human tissue samples is a promising approach for discovering molecular driver alterations in gastric tumorigenesis.

MIR143-3p reconstitution suppresses cellular proliferation in gastric cancer cells and tissue organoids

For functional analysis of the role of miRNAs in gastric tumorigenesis, we selected MIR143-3p that was validated in human and mouse gastric tumors, with little known information regarding its molecular function role in gastric tumorigenesis. We also found that this miRNA was downregulated in TANG in human and LGD lesions in mice, suggesting a possible role in the early stages of gastric tumorigenesis. Similar to human tissues, its levels were significantly lower in several gastric cancer cell lines, STKM2, MKN45, AGS, and MKN28, as compared to normal gastric tissues (Supplementary Figure 3, P<0.05). To establish its biological functions, MIR143-3p was stably reconstituted and confirmed in STKM2 cells (Figure 4A left panel, P<0.01). The ATP-glo cell viability assay demonstrated a significant reduction in cell viability following reconstitution of MIR143-3p in STKM2, as compared with control cells in day 5 (Figure 4A right panel, P<0.001). Similar findings were observed in MKN45, AGS and MKN28 cells (Figure 4 B to D, P<0.01), confirming the reproducibility of the results in multiple in vitro cell models. Using the EdU staining assays, as a measure of cellular proliferative capacity, we detected a significant reduction in the EdU positive cells in all in vitro cell models (Figure 4E to H, P<0.05).

Figure 4. MIR143-3p suppresses cellular proliferation and gastric organoids’ growth.

Figure 4.

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Reconstitution of MIR143-3p was established by using lenti-virus infection followed by puromycin selection (A-H). A) qRT-PCR analysis of MIR143-3p expression level following its stable reconstitution in STKM2 cells, as compared to control (left panel). ATP-glo cell viability assay analysis of MIR143-3p stably reconstituted STKM2 cells and control cells (right panel). Similar experiments, as in A, were performed in MKN45 (B), AGS (C), and MKN28 (D). E) EdU immunofluorescence staining in MIR143-3p stably reconstituted STKM2 cells and controls (left), quantification of data is shown on the right panel. Similar experiments, as in E, were performed in MKN45 (F), AGS (G), and MKN28 (H). Reconstitution of MIR143-3p in gastric organoids from mouse low-grade dysplasia (LGD) lesions in TFF1-KO mouse (I-N). I) Light field images of mouse gastric organoids (Day 1, 3, 5, and 7). J) Light field images of mouse gastric organoids, 3 days after MIR143-3p reconstitution using lenti-virus infection, or control lentivirus. K) Quantification data of organoids’ diameter from J, Mann Whitney Test. L) qRT-PCR analysis of MIR143-3p expression levels, following reconstitution of MIR143-3p in gastric organoids and controls. M) Ki-67 immunofluorescence staining in gastric organoids, 3 days after MIR143-3p reconstitution using lenti-virus infection, or control lentivirus. N) Quantification data of Ki67 positive cells in M. *, P<0.05, **P<0.01, ***P<0.001, Student t test.

Because we detected down-regulation of MIR143-3p in mouse LGD tissues, (Figure 3A, P<0.01), we reconstituted MIR143-3p into the TFF1-KO LGD gastric organoids by using lenti-virus particles to confirm in vitro functional data. Bright field images of LGD gastric organoids from day 1 to day 7 (passage 1) are shown in Figure 4I. By measuring the diameter of the organoids, data indicated that MIR143-3p reconstitution significantly decreased LGD gastric organoids diameter, as compared to control organoids (Figure 4J and K, P<0.001). The expression level of MIR143-3p in the organoids was confirmed by qRT-PCR analysis (Figure 4L, P<0.05). Immunofluorescence staining against Ki-67 indicated that MIR143-3p reconstitution significantly decreased the number of Ki-67 stained cells in the LGD organoids (Figure 4M&N, P<0.001). In summary, our data demonstrated that MIR143-3p reconstitution decreased cell viability and proliferative capacity of human gastric cancer cells and neoplastic mouse tissue organoids.

MIR143-3p downregulates BRD2 protein level leading to transcriptional repression of MYC

To identify downstream targets of MIR143-3p in gastric cancer cells, analysis of three miRNA database [miRDB (http://mirdb.org/), Target Scan (http://www.targetscan.org/vert_71/) and microRNA.org (http://34.236.212.39/microrna/microrna/home.do)] demonstrated BRD2 as a predicted target gene in the three databases for both human and mice (Figure 5A). Other predicted target genes of MIR143-3p with the score over 90 from three databases for both human and mice are listed in Supplementary Table S5. Additional analysis identified two MIR143-3p binding sites within its 3′UTR region of MIR143-3p (Figure 5B). We determined MIR143-3p expression and BRD2 protein levels in gastric cancer cells. qRT-PCR results indicated that STKM2, MKN45, MKN28, and AGS cells expressed low levels of MIR143-3p, as compared with normal gastric mucosa samples (Supplementary Figure 3, P<0.01). Western blot data showed that expression levels of BRD2 and MYC, a known direct target of BRD2, were remarkably higher in gastric cancer cells, as compared with normal gastric mucosa (Figure 5C). Following reconstitution of MIR143-3p using a mimic, Western blot data demonstrated that MIR143-3p reconstitution caused lower BRD2 protein expression levels without any notable change in the BRD4 protein level, confirming that BRD2 is a specific downstream target of MIR143- (Figure 5D). Western blot data in AGS, STKM2 and MKN45 cells showed that stable reconstitution of MIR143-3p decreased BRD2 and MYC protein expression levels, as compared with control cells (Figure 5E to G). Similarly, BRD2 and MYC protein expression levels were significantly higher in TFF1-KO mouse gastric cancer tissue samples, as compared with wild-type normal gastric samples (Supplementary figure 4 A&B, P<0.01). Meanwhile, MIR143-3p and Tff1 mRNA expression levels were significantly down-regulated in TFF1-KO mice gastric cancer tissue samples, as expected (Supplementary Figure 4C, P<0.01). In addition, following reconstitution of MIR143-3p in dysplastic gastric organoids from the TFF1-KO mouse, we detected a decrease in BRD2 and MYC protein levels, as compared with controls (Figure 5H).

Figure 5. BRD2 is a direct downstream target of MIR143-3p.

Figure 5.

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A) BRD2 is a predicted downstream target of MIR143-3p, by using three 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, following transient reconstitution of MIR143-3p using a mimic (2.5–40 pmol). BRD2, not BRD4, is downregulated following reconstitution. Western blot analysis following reconstitution of MIR143-3p in AGS (E), STKM2 (F) MKN45 (G) and mouse LGD gastric organoids (H). Wild-type or mutant (missing both MIR143-3p bindings sites) BRD2 3′UTR luciferase reporter analysis in AGS cells (I) or MKN45 (J) following stable reconstitution of MIR143-3p or control. K) c-MYC promoter (4xTBE1 wt) luciferase reporter analysis in AGS cells following reconstitution of MIR143-3p or control, with and without BRD2 transient transfection.K) qRT-PCR analysis of c-MYC gene expression level in mouse LGD gastric organoids following reconstitution of MIR143-3p or control. *, P<0.05, **P<0.01, ***P<0.001, Student t test or one-way ANOVA.

To further validate that BRD2 is a direct downstream target of MIR143-3p, wild-type or mutant (lacks both MIR143-3p binding sites) BRD2 3′UTR reporters were generated and utilized in experiments using AGS and MKN45 cells. The mutation of BRD2 3′UTR MIR143-3p binding sites was confirmed by DNA sequencing (Supplementary Figure 5). Luciferase reporter analysis indicated that MIR143-3p reconstitution significantly decreased wild-type BRD2 3′UTR reporter luciferase value, but not the mutant BRD2 3′UTR reporter activity in both AGS and MKN45 cells, as compared with control cells (Figure 5I and J, P<0.001). To determine that down-regulation of MYC protein levels was caused by transcriptional regulation, c-MYC promoter (4xTBE1 wt) luciferase reporter was utilized, following reconstitution of MIR143-3p or control with or without BRD2 overexpression. Our data indicated that MIR143-3p reconstitution in AGS cells significantly decreased c-MYC promoter luciferase reporter activity (Figure 5K, P<0.001). As expected, BRD2 overexpression alone significantly increased c-MYC promoter luciferase reporter activity (Figure 5K, P<0.001). At the same time, BRD2 overexpression, following reconstitution of MIR143-3p, antagonized the inhibition of c-MYC promoter luciferase reporter activity that was mediated by MIR143-3p (Figure 5K, P<0.001). Using qRT-PCR, we did not detect significant differences in BRD2 mRNA expression levels, following reconstitution of MIR143-3p in STKM2 or AGS cells, as compared with control cells (Supplementary Figure 6A&B). On the other hand, c-MYC mRNA expression levels were significantly down-regulated following reconstitution of MIR143-3p (Supplementary Figure 6C&D, P<0.05). Furthermore, using qRT-PCR, we detected strong inverse correlation between MIR143-3p and c-MYC mRNA expression levels in human gastric tissue samples (Supplementary figure 7, P<0.05). Similarly, the mRNA expression level of c-MYC gene in TFF1-KO LGD organoids was significantly decreased with reconstitution of MIR143-3p (Figure 5L, P<0.001). These data suggest that MIR143-3p directly binds to BRD2 leading to its decreased protein levels and subsequent downregulation of c-MYC transcription in gastric cancer.

MIR143-3p promotes cleavage of PARP and sensitizes gastric cancer cells to cisplatin

The use of several Bromodomain inhibitors that target the BRD family including BRD2, has become an investigational therapeutic approach in cancer, as a single agent or in combination with other chemotherapeutics18. Given the function of MIR143-3p in suppressing BRD2, we investigated whether its reconstitution may be therapeutically effective against gastric cancer cells by sensitizing to cisplatin (CDDP) treatment, a commonly used drug in chemotherapeutic regimens. Using ATP-glo assay, the results demonstrated that MIR143-3p reconstitution in several gastric cancer cell models (STKM2, MKN45, AGS and MKN28) led to reduction in CDDP IC50s, as compared with cisplatin treatment alone and controls (Figure 6 A to D). Clonogenic cell survival assay demonstrated that MIR143-3p reconstitution significantly decreased AGS cell survival with CDDP treatment, as compared to CDDP treatment alone (Figure 6E to G, P<0.001). We next investigated the signaling outcome of MIR143-3p reconstitution, as compared to JQ-1 (BRD2 inhibitor) alone or in combination with CDDP. Western blot analysis demonstrated that MIR143-3p reconstitution decreased BRD2 and c-MYC protein levels, as expected, with a notable increase in cleaved PARP protein expression levels with CDDP alone or JQ-1 alone treatment (Figure 6H). Similarly, the combination of CDDP and JQ-1 induced higher levels of cleaved PARP, as compared with CDDP or JQ-1 treatment alone, in both control and MIR143-3p reconstituted MKN45 cells (Figure 6H).

Figure 6. MIR143-3p sensitizes gastric cancer cells to cisplatin treatment.

Figure 6.

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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, 50 μmol/L). B-D) Similar experiments in MKN45 (B), AGS (C), and MKN28 (D) cells. IC50s were calculated using the Prism 5 software. E) Representative clonogenic survival assay in AGS cells with stable reconstitution of MIR143-3p, or controls, with or without 2.5μM of CDDP treatment, quantification of surviving colonies is showing in F. G) IC50 analysis based on clonogenic assay similar to E (CDDP: 10, 5, 2.5, 1.25, 0.625 μmol/L) for 24h 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, following treatment with CDDP or JQ-1 (BRD2 inhibitor) alone or in combination. ***P<0.001, Student t test.

To find out if BRD2 levels have a prognostic significance in gastric cancer, we analyzed the overall survival data from 320 gastric cancer patients (KMplot database)28. The results demonstrated that patients with high levels of BRD2 had a poor overall survival outcome, as compared to patients with low levels (lower than the average of BRD2 expression level) (Supplementary Figure 8, P<0.001). Survival data from 60 gastric cancer patients of the First Affiliated Hospital of Nanjing Medical University indicated that higher MIR143-3p expression (higher than the average of MIR143-3p expression in 60 gastric cancer samples) may predict better survival in gastric cancer; although this was not significant (P=0.26) due to the limited sample size and relatively short follow-up time (Supplementary Figure 9).

Reconstitution of MIR143-3p suppresses tumor xenografts growth and sensitizes to CDDP treatment in vivo

To confirm the findings from in vitro cell models and gastric organoids, AGS control and MIR143-3p stably reconstituted cells were tested in tumor xenograft experiments with or without CDDP treatment. Representative tumor xenograft images of AGS control or MIR143-3p stably reconstituted cells with or without CDDP treatment are shown in Figure 7A. The expression levels of MIR143-3p in all test groups are shown in Figure 7B. Tumor xenograft growth curves’ data demonstrated that MIR143-3p reconstitution significantly suppressed tumor growth, as compared with the control group (P<0.01) (Figure 7C). In the meantime, CDDP treatment alone displayed a weak effect on tumor growth inhibition. While the combination of MIR143-3p reconstitution and CDDP treatment not only suppressed tumor growth, but also led to regression of tumor volume, as compared with the control group (Figure 7C, P<0.001). Western blot analysis showed a decrease in BRD2 and c-MYC protein levels following MIR143-3p reconstitution (Figure 7D). While CDDP treatment alone showed minimal effects on BRD2, c-MYC, and cleaved-PARP protein levels, there was a notable reduction of BRD2 and c-MYC, and an increase of cleaved-PARP in tumor xenografts of combined MIR143-3p reconstitution and CDDP treatment (Figure 7D). Immunohistochemistry results indicated that CDDP treatment alone decreased Ki-67 positive cell percentage (Figure 7E, P<0.05). However, MIR143-3p reconstitution alone or with CDDP treatment had significantly more profound effects on the percentage of Ki-67 positive cells, as compared with control groups (Figure 7E, P<0.001). Similarly, MIR143-3p reconstitution and CDDP combination treatment significantly induced cleaved-caspase 3 (Figure 7E, P<0.001). These data indicate that MIR143-3p reconstitution decreases tumor xenografts’ growth and sensitizes to CDDP treatment.

Figure 7. MIR143-3p inhibits gastric cancer tumor xenograft growth and sensitizes to cisplatin treatment in vivo.

Figure 7.

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A) Representative images of gastric cancer cells (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 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<0.05, **P<0.01, ***P<0.001, Student t test or one-way ANOVA.

Discussion

Gastric cancer is the third leading cause of cancer-related deaths worldwide1. The overall 5-year survival rate is 30.6% for all patients which plunges to 5.2% for patients with advanced stages2. Helicobacter pylori (H. pylori) is a type I carcinogen and the main risk factor for gastric cancer29. Although the H. pylori infection affects more than half of the world’s population, only a small proportion of infected subjects develop gastric adenocarcinoma30, suggesting the existence of complex molecular and environmental factors that drive gastric tumorigenesis.

In this study, we utilized an integrated bioinformatics approach for the analysis of human and mouse miRNA alterations. miRNAs are highly conserved among species and known for their stability in various body tissues and fluids, making them ideal molecular markers for the diagnosis and prognosis of human cancers31, 32. Because loss of TFF1 expression is a frequent finding in human gastric cancer11 and in most gastric cancer mouse models33–35, we utilized the TFF1-KO mouse model to overcome limitations of acquisition of tissues from early stages of human gastric cancer and to eliminate the heterogeneity factors observed in humans. The results demonstrated a miRNA signature that was conserved across the species. Since these miRNA alterations were conserved in mouse and human tumors, it is plausible to conclude that these changes are more likely related to the tumorigenic cascade. Earlier studies have noted similarities in molecular pathways and signaling networks between the TFF1-KO gastric cancer and human intestinal-type gastric cancer 11, 13. Of note, we validated a representative set of miRNAs using more than 400 human samples from three independent cohorts from three independent cohorts. We have observed miRNA changes in tumor-adjacent histologically normal human tissues that were similar to changes in cancer tissues. These changes were also detected in premalignant lesions of the TFF1-KO gastric mucosa, suggesting that indeed they may be early molecular events in gastric tumorigenesis. Nevertheless, we also acknowledge that changes in tumor-adjacent histologically normal tissues may also reflect field defects due to other confounding factors associated with carcinogenesis such as changes in metabolic activities or changes in immune cell infiltration.

The expression levels of miRNAs play important roles in regulating signaling and biological processes in health and disease through their ability to modify the expression of a large set of target genes in tissue specific and context dependent manners36. Alterations in miRNAs expression have been reported in almost every human malignancy, regulating cancer cell proliferation, angiogenesis, metastasis, and drug resistance37, 38. We performed an integrated, comprehensive bioinformatics analysis to predict the impact of miRNA changes on signaling pathways and networks in gastric tumorigenesis in mouse and human. This analysis predicted the involvement of several signaling pathways that play important roles in gastric tumorigenesis such as IGF-1, WNT/β-catenin, ERBB, NF-kB, and STAT3 39, 40. These pathways were similarly present in both mouse and human data. Taken together, our results further support the relevance of the TFF1-KO mouse to study gastric cancer.

We verified significant low expression levels of MIR143-3p in human and mouse gastric tumors. Of note, the expression of MIR143-3p was also lower in dysplastic lesions in mouse and in tumor adjacent normal tissues in human. Although deregulation of MIR143-5p was reported in some cancer types, its functional role in gastric carcinogenesis is understudied41, 42,43. Earlier studies suggested that MIR143-3p can inhibit cell migration and epithelial-mesenchymal transition44, 45. We report that MIR143-3p reconstitution not only inhibits gastric cancer cell proliferation and gastric organoid growth, but also sensitizes cancer cells to CDDP treatment. Our results demonstrate BRD2 as a novel direct downstream target of MIR143-3p. Our choice to focus on BRD2 was based not only on its ranking in three databases but also its biological, translational and clinical relevance in cancer. BRD2 mediates highly specific histone acetylation by tethering transcriptional HATs to specific chromosomal sites, or to the activity of multi-protein complexes in chromatin remodeling46. BRD2 overexpression in human cancers promotes tumor proliferation and drug resistance47, 48. Of note, Bromodomain inhibitors such as OTX015 and JQ-1 are in several clinical trials for treatment of cancer 49, 50. We have shown that loss of MIR143-3p led to an increase in BRD2 protein levels leading to transcription induction of its downstream target c-MYC 51, 52. Reconstitution of MIR143-3p reduced the protein levels of BRD2 and c-MYC resulting in restoration of sensitivity to CDDP in vitro and in vivo. Concordant with these results, high expression levels of BRD2 were associated with poor clinical outcome and reduced overall survival in gastric cancer patients. However, as miRNAs can regulate several targets, it is possible that the effects of MIR143-3p are not limited to BRD2 and c-MYC but also include several additional targets. There are a number of recent advances in approaches utilizing miRNAs for treatment of cancer53, 54, including early efforts for developing edible plants that express miRNAs for treatment of human diseases55. In this context, approaches that reconstitute MIR143-3p could have an impact on prevention and or treatment of gastric cancer

In summary, this first integrated, comprehensive analysis of human and mouse miRNA signatures in gastric cancer successfully unveiled consistent and conserved miRNAs that are more likely related to gastric tumorigenesis. The results demonstrated complex and concerted roles of miRNAs in regulating signaling pathways and networks that are important for gastric tumorigenesis. The impact of MIR143-3p loss on BRD2 expression and CDDP resistance, suggests that approaches to restore MIR143-3p may aid in prevention and treatment of gastric cancers.

Supplementary Material

1

Acknowledgement

This study was supported by the NIH (R01CA93999 and R01CA177372), Research Career Scientist Award (1IK6BX003787), and a merit award (I01BX001179) from the U.S. Department of Veterans Affairs (to W. El-Rifai). This work was also supported by the National Natural Science Foundation Project of International Cooperation (NSFC-NIH, 81361120398); the National Natural Science Foundation of China (81572362); Jiangsu Key Medical Discipline (ZDXKA2016005) and CONICYT-FONDAP 15130011 and Fondecyt 1151411 from de Government of Chile. In addition, this study was supported by Bioinformatics and Biostatistics and Oncogenomics Shared Resources at Sylvester Comprehensive Cancer Center, University of Miami.

Footnotes

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Conflict of interest statement: The authors declare no conflicts of interest.

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