Purpose: Recent studies revealed that both disseminated tumor cells and noncancerous cells contributed to cancer progression cooperatively in the bone marrow. Here, RNA-seq analysis of bone marrow from gastric cancer patients was performed to identify prognostic markers for gastric cancer.

Experimental Design: Bone marrow samples from eight gastric cancer patients (stages I and IV: n = 4 each) were used for RNA-seq analysis. Results were validated through quantitative real-time PCR (qRT-PCR) analysis of HIST1H3D expression in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from gastric cancer patients. miR-760 expression was assayed using qRT-PCR in 105 bone marrow and 96 primary tumor samples. Luciferase reporter assays were performed to confirm whether histone mRNAs were direct targets of miR-760. miR-760 expression was also evaluated in noncancerous cells from gastric cancer patients.

Results: RNA-seq analysis of bone marrow samples from gastric cancer patients revealed higher expression of multiple histone mRNAs in stage IV patients. HIST1H3D expression in the bone marrow, peripheral blood, and primary tumor of stage IV patients was higher than that in stage I patients (P = 0.0284, 0.0243, and 0.0006, respectively). In contrast, miR-760 was downregulated in the bone marrow and primary tumor of stage IV patients compared with stage I patients (P = 0.0094 and 0.0018, respectively). Histone mRNA and miR-760 interacted directly. Furthermore, miR-760 was downregulated in noncancerous mucosa in stage IV gastric cancer patients.

Conclusion: Histone mRNA was upregulated, whereas miR-760 was downregulated in the bone marrow and primary tumor of advanced gastric cancer patients, suggesting that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer. Clin Cancer Res; 19(23); 6438–49. ©2013 AACR.

Translational Relevance

The presence of isolated tumor cells in the peripheral blood and bone marrow is an important factor contributing to the metastasis of solid cancers. Moreover, recent studies have demonstrated that various types of host cells are also involved in cancer development and metastasis. We performed RNA-seq analysis of the bone marrow from patients with gastric cancer to identify candidate prognostic markers using next-generation sequencing and demonstrated that histone cluster genes were overexpressed in the bone marrow and primary tumor samples from stage IV gastric cancer patients compared with those from stage I gastric cancer patients. Furthermore, we proposed the possibility that microRNA-760 (miR-760) was downregulated in order to degrade upregulated histone mRNA in response to an increase in S-phase cells in the bone marrow and primary tumors of advanced gastric cancer patients. Our data also suggested that the histone mRNA/miR-760 axis had a crucial role in the development of gastric cancer.

The occurrence of distant metastases is the main cause of death in cancer patients. The presence of isolated tumor cells (ITC) is an important factor contributing to the metastasis of solid cancers. In clinical practice, the circulating tumor cell (CTC) detection system (CellSearch System) was first approved by the Food and Drug Administration for metastatic breast cancer and has now been approved for the detection and monitoring of CTCs in the blood from patients with metastatic prostate and colorectal cancer. Moreover, recent studies have demonstrated that various types of host cells are also involved in cancer development and metastasis, including fibroblasts (carcinoma-associated fibroblasts or myofibroblasts), tumor-associated macrophages, mesenchymal stem cells, platelets, and hematopoietic progenitor cells (1–8).

We previously investigated the presence of ITCs in peripheral blood and bone marrow samples using quantitative real-time PCR (qRT-PCR) analysis of CEA, CK-7, and CK-19 in more than 800 cases of gastric cancer (9). We found that ITCs circulated in patients with a range of clinical stages of gastric cancer and demonstrated that the simultaneous expression of ITC-associated genes and VEGFR-1, which may originate from hematopoietic progenitor cells in peripheral blood and bone marrow, was significantly associated with hematogeneous metastases (9). Therefore, multiple markers are currently needed to predict distant metastasis and/or prognosis from bone marrow or peripheral blood samples in gastric cancer patients by PCR analysis. Disseminated tumor cells (DTC) in the bone marrow have been detected in all solid tumor types, suggesting that the bone marrow may be a preferred reservoir for blood–bone DTCs. The bone marrow environment may allow these cells to persist for a prolonged period and to disseminate into other organs (10). Many cancer-associated host cells are derived from the bone marrow. According to these findings, differences in the gene expression status of bone marrow cells may reflect different cancer stages or the possibility of distant metastases. Moreover, the bone marrow is a convenient organ to sample for analysis and is more easily accessible than other organs that are often sites of metastases, such as the lungs or liver. In this study, we performed RNA-seq analysis of the bone marrow from patients with gastric cancer in order to identify candidate prognostic markers. We demonstrated that multiple histone cluster genes showed higher expression in the bone marrow of stage IV patients than in that of stage I patients and evaluated the molecules that regulate these multiple histone mRNAs, revealing an interesting association between histone mRNA and microRNA (miRNA).

Patients and sample collection

In our previous study, bone marrow and peripheral blood samples were collected from Japanese gastric cancer patients who underwent surgery (9). Bone marrow samples from 8 patients with gastric cancer, including 4 stage I and 4 stage IV patients with liver metastasis, were used for RNA-seq analysis (Supplementary Table S1). Quantitative real-time PCR (qRT-PCR) was performed to analyze the expression of histone mRNA in 175 bone marrow and 92 peripheral blood samples and expression of miRNA in105 bone marrow samples. For qRT-PCR analysis of primary tumors, another 127 gastric cancer and corresponding normal tissue samples were used. For miRNA microarray analysis of 3 fractions separated from the bone marrow, bone marrow samples were collected from another 4 gastric cancer patients. Detailed protocols for sample preparation are described in the Supplementary Data.

RNA-seq analysis of bone marrow samples from gastric cancer patients by massively parallel sequencing

Briefly, a total of 1 μg of extracted RNA was used as a template to construct RNA-seq libraries. In this step of sample preparation, starting with total RNA, the mRNA was poly-A selected, fragmented, and converted into single-stranded cDNA using random hexamer priming. Detailed protocols are described in the Supplementary Data. Fold enrichment of the RNA-seq tags between the samples was calculated for each mRNA using the assigned tag counts and was normalized to read per kilobase mRNA (RPKM; ref. 11).

Evaluation of HIST1H3D and miR-760 expression in clinical samples

mRNA and miRNA levels were quantified using a LightCycler 480 Probes Master Kit (Roche Applied Science) following the manufacturer's protocol. HIST1H3D mRNA expression levels were measured in 175 bone marrow, 92 peripheral blood, and 115 primary tumor samples from patients with gastric cancer and corresponding noncancerous gastric mucosa samples. miR-760 expression was also assayed by qRT-PCR in 105 bone marrow, 96 primary tumor, and 84 corresponding normal gastric mucosa samples from gastric cancer patients. Detailed protocols are described in the Supplementary Data.

Cell lines and cell culture

Seven human gastric cancer cell lines (NUGC3, NUGC4, MKN74, AGS, KATOIII, MKN45, and KE39) were provided by the Cell Resource Center of Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University and the Riken Bioresource Center. Cells were maintained in RPMI 1640 containing 10% FBS or in serum-free conditions and cultured in a humidified 5% CO2 incubator at 37°C.

Evaluation of HIST1H3D and miR-760 expression in gastric cancer cells

For RNA analysis, cells were seeded at 1.0 to 4.0 × 105 cells per well in a volume of 2 mL in 6-well flat-bottomed microtiter plates. Total RNA from these cell lines was isolated using a miRNeasy Mini Kit (Qiagen) following 3 to 72 hours of incubation. Detailed protocols for qRT-PCR are described in the Supplementary Data.

Protein expression analysis

Western blotting was used to confirm HIST1H3D expression in gastric cancer cells. Primary antibodies targeting pan actin (NeoMarkers) and HIST1H3D (Abcam) were used. Detailed protocols are described in the Supplementary Data.

Construction of reporter plasmids and the luciferase reporter assay

To construct the luciferase reporter plasmid, full-length HSIT1H3D or HIST1H2AD was subcloned into the pmirGlo Dual-Luciferase miRNA Target Expression Vector (Promega) at a location 5′ to the firefly luciferase. Furthermore, to confirm the direct interaction between miR-760 and its binding sites on HIST1H3D and HIST1H2AD 3′ untranslated regions (UTR), we constructed luciferase reporter plasmids in which the miR-760 binding sites were mutated. To construct these mutants, positions 45 to 52 of the HIST1H3D 3′ UTR and 56 to 63 of the HIST1H2AD 3′ UTR (the sequences were common: CAGAGCCA) were mutated to the sequence CTGTGTCA. A detailed protocol of the luciferase reporter assay is described in the Supplementary Data.

Transfection of miR-760 precursor (Pre-miR-760)

Either Pre-miR-760 or a Pre-miR negative control (Ambion miRNA Inhibitors; Applied Biosystems) was transfected into gastric cancer cells at 30 nmol/L (final concentration) using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer's instructions.

miRNA microarray of bone marrow fractions from gastric cancer patients

In 4 patients with stage IV gastric cancer, bone marrow cells were separated into 3 fractions using a 3-step automagnetic-activated cell separation system (MACS) by MACS Cell Separators. CD45+, CD14+, and CD45/EpCAM+ cell fractions were collected using CD45, CD14, and EpCAM (CD326) microbeads according to the manufacturer's instructions (Miltenyi Biotec). RNA was extracted from each bone marrow fraction separated by the Auto MACS system, and miRNA microarrays were performed using the miRCURY LNA Array System. A detailed protocol is given in the Supplementary Data. The miRNA arrays have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database with accession code GSE40325.

Statistical analysis

Data from qRT-PCR analyses and in vitro transfected cell assays were analyzed with JMP 5 software (SAS, Inc.). Overall survival rates were calculated actuarially according to the Kaplan–Meier method and were measured from the day of surgery. Differences between groups were estimated using the χ2 test, Student t test, repeated-measures ANOVA, and log-rank test. Variables with a P-value of less than 0.05 in univariate analysis were used in subsequent multivariate analysis based on the Cox proportional hazards model for survival. All differences were considered statistically significant at the level of P < 0.05.

RNA-seq analysis of bone marrow from gastric cancer patients revealed increased histone mRNA expression in advanced cases

Hierarchical clustering of genes evaluated by RNA-seq resulted in 2 main groups, one consisting of 2 cases from stage I and the other consisting of 2 cases from stage I and 4 cases from stage IV (Supplementary Fig. S1A, left). This result indicated that some cases of stage I gastric cancer have characteristics similar to those of stage IV gastric cancer in the bone marrow microenvironment. Gene set enrichment analysis demonstrated that gene sets related to pathways of the immune response to cancer cells were significantly enriched in stage IV gastric cancer patients (Supplementary Table S3). Upregulation of these gene sets seemed to occur in immune competent cells in the bone marrow, and the immune response to cancer was more activated in stage IV gastric cancer patients than in stage I gastric cancer patients. RNA-seq analysis also revealed differential expression of many genes. Twenty-eight genes showed over a 5-fold significant increase in expression in the bone marrow from stage IV patients compared with that from stage I patients and had an RPKM value of at least 2.0 (Supplementary Table S4). These genes included 4 histone genes, namely, HIST1H1D, HIST1H3F, HIST1H2AD, and HIST1H2AL. Interestingly, many other histone mRNAs also showed higher expression in the bone marrow from stage IV patients. Thirty-seven histone genes were highly expressed (at least a 3-fold increase in expression) in stage IV patients compared with stage I patients (schematized in Fig. 1A). Hierarchical cluster analysis of histone cluster genes revealed that 3 of 4 cases of stage IV gastric cancer were clustered in one group with histone upregulation, and all cases of stage I and 1 of 4 cases of stage IV (gastric cancer 57) were clustered in the other group without histone upregulation (Supplementary Fig. S1A, right). This result indicated that some stage IV gastric cancer patients had few S-phase cells in the bone marrow and that bone marrow cells in these patients may be maintained in a dormant state of cell growth.

Figure 1.

Histone gene expression in gastric cancer (GC) patients. A, expression of histone cluster genes in bone marrow samples from GC patients. RPKM values of histone cluster genes were schematized. RPKM, reads per kilobase per million mapped reads. B–D, HIST1H3D expression in GC patients. HIST1H3D expression was analyzed in the bone marrow, peripheral blood (B), and primary tumors (C) of GC patients. D, Kaplan–Meier overall survival curves for GC patients based on the level of HIST1H3D expression from primary tumor samples.

Figure 1.

Histone gene expression in gastric cancer (GC) patients. A, expression of histone cluster genes in bone marrow samples from GC patients. RPKM values of histone cluster genes were schematized. RPKM, reads per kilobase per million mapped reads. B–D, HIST1H3D expression in GC patients. HIST1H3D expression was analyzed in the bone marrow, peripheral blood (B), and primary tumors (C) of GC patients. D, Kaplan–Meier overall survival curves for GC patients based on the level of HIST1H3D expression from primary tumor samples.

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HIST1H3D expression in gastric cancer patients

Intron spanning primers can prevent amplification of contaminating genomic DNA. The genes encoding canonical histones generally lack introns, whereas HIST1H3D, corresponding to the 27th histone in Fig. 1A, is unique in that it contains 1 intron; this gene showed elevated mRNA expression in the bone marrow from stage IV gastric cancer patients. We validated HIST1H3D mRNA expression in the bone marrow and peripheral blood of gastric cancer patients (175 and 92 cases, respectively) by qRT-PCR using intron-spanning primers (Supplementary Fig. S2A), and revealed that HIST1H3D expression was significantly higher in the bone marrow and peripheral blood of stage IV patients than in those of stage I patients (Fig. 1B). Furthermore, HIST1H3D expression in another set of 115 primary gastric cancer tissues was also evaluated. Primary tumor tissues exhibited higher HIST1H3D expression compared with corresponding normal tissues (P = 0.0127; Fig. 1C, left), and the average expression of HIST1H3D mRNA in stage I tumors was significantly lower than in other stages (Fig. 1C, right). We then classified these 115 gastric cancer cases into 2 groups according to average HIST1H3D mRNA expression; patients with an average HIST1H3D expression below 1.069 (normalized to GAPDH) were assigned to the low expression group (n = 77), whereas those with an average expression above 1.069 were assigned to the high expression group (n = 38). These 2 groups were then used to analyze clinicopathological factors in relation to HIST1H3D levels. Patients with high expression of HIST1H3D exhibited significantly more frequent tumor invasion, lymph node metastasis, peritoneal dissemination, and advanced-stage cancers than those with low HIST1H3D expression (χ2 test; Supplementary Table S2A). In terms of overall survival, patients in the HIST1H3D high-expression group had a significantly poorer prognosis than those in the HIST1H3D low-expression group (Fig. 1D). Univariate analysis of overall survival revealed that the level of HIST1H3D expression was a prognostic predictor; however, multivariate analysis showed that HIST1H3D expression was not an independent prognostic predictor of prognosis in gastric cancer patients (Supplementary Table S2B).

Expression of specific genes involved in histone mRNA metabolism in the bone marrow of gastric cancer patients

To identify candidate prognostic/metastatic markers in gastric cancer patients, we compared the expression status of genes that regulate multiple histone mRNAs between stage I and stage IV gastric cancer patients. Many specific genes are involved in histone mRNA transcription, cleavage, translation, and degradation (12). Interestingly, most of these genes showed about a 2-fold increase in bone marrow samples from stage IV patients compared with those from stage I patients by RNA-seq analysis (Supplementary Table S5). These results indicated enhanced histone mRNA metabolism in the bone marrow of advanced gastric cancer patients and were consistent with our previous results demonstrating increased histone mRNA expression in the bone marrow of patients with stage IV gastric cancer. Some histone metabolism-associated genes showed significantly higher expression in the bone marrow from stage IV patients, suggesting that these genes, which are involved in controlling the metabolism of dozens of histone mRNAs, could be prognostic markers for gastric cancer patients. However, these genes may regulate not only upregulated histones in stage IV patients (schematized in Fig. 1A) but also many other histones that showed no increased expression in stage IV patients.

In silico analysis predicted an association between histone mRNA and miRNA

Because one or several miRNAs often regulate multiple target genes in a specific pathway of cellular function, we investigated the possibility that miRNAs regulate multiple histone mRNAs using an in silico prediction algorithm (TargetScan v. 6.0). TargetScan predicts biological targets of miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA (13). Our analysis predicted that several miRNAs bind to the histone genes upregulated in bone marrow samples from stage IV gastric cancer patients (Table 1). Surprisingly, in this analysis, most of the histone genes upregulated in bone marrow samples from stage IV gastric cancer patients had predictive target sites for several common miRNAs in their 3′ UTR. In particular, 3 miRNA, namely, miR-760, miR-1276, and miR-4766-5p, were predicted to bind the 3′ UTRs of more than 20 histone genes (Table 1). Although histone H4 family genes had no binding sites for these 3 miRNAs, they had target sites for 2 other common miRNAs, miR-1291 and miR-4512 (Table 1). These results suggested that several miRNAs were involved in histone mRNA metabolism. Because the context score percentile of miR-760 for each of the histone mRNAs was higher than those of miR-1276 and miR-4766-5p, we examined miR-760 expression and function in gastric cancer in subsequent experiments.

Table 1.

The association between histone cluster genes and conserved miRNAs predicted by in silico analysis

Histone genesConserved miR (Context score percentile)
HIST1H2AK miR760 (98)  miR4766-5p (54)   
HIST1H2BM  miR1276 (75) miR4766-5p (39)   
HIST1H1B miR760 (98) miR1276 (78) miR4766-5p (43)   
HIST1H3B miR760 (99) miR1276 (84) miR4766-5p (51)   
HIST1H1D miR760 (99) miR1276 (74) miR4766-5p (48)   
HIST1H1E miR760 (98) miR1276 (80) miR4766-5p (44)   
HIST1H3F miR760 (98) miR1276 (82) miR4766-5p (47)   
HIST1H2AD miR760 (98) miR1276 (83) miR4766-5p (48)   
HIST1H2AH miR760 (99) miR1276 (83) miR4766-5p (55)   
HIST1H3C miR760 (98) miR1276 (81) miR4766-5p (46)   
HIST1H4I     miR4512 (98) 
HIST1H2BN miR760 (99) miR1276 (76) miR4766-5p (43)   
HIST1H4L    miR1291 (99) miR4512 (93) 
HIST1H4K     miR4512 (98) 
HIST1H4E    miR1291 (89) miR4512 (95) 
HIST1H2BJ miR760 (66) miR1276 (78)    
HIST1H3A miR760 (98) miR1276 (82) miR4766-5p (45)   
HIST2H2AB miR760 (98) miR1276 (78) miR4766-5p (47)   
HIST1H2BC miR760 (67) miR1276 (87)    
HIST1H2AL miR760 (99) miR1276 (80) miR4766-5p (51)   
HIST1H4B    miR1291 (89)  
HIST1H2AG  miR1276 (70)    
HIST2H2BF miR760 (99) miR1276 (82) miR4766-5p (52)   
HIST1H2BF miR760 (67) miR1276 (87)    
HIST1H2AI 
HIST1H2AC miR760 (86) miR1276 (79) miR4766-5p (42)   
HIST1H3D miR760 (99) miR1276 (84) miR4766-5p (50)   
HIST1H3G  miR1276 (88)    
HIST1H3H miR760 (99) miR1276 (82) miR4766-5p (47)   
HIST1H2BH miR760 (99)     
HIST1H4C    miR1291 (92) miR4512 (97) 
H3F3C 
HIST2H2BE miR760 (97) miR1276 (85) miR4766-5p (46)   
HIST1H4D    miR1291 (99)  
HIST1H3I miR760 (85) miR1276 (80) miR4766-5p (43)   
HIST1H2BO miR760 (98) miR1276 (84)    
HIST1H2BB miR760 (99) miR1276 (82)    
Average (%) 93.6 80.9 47.05 93.6 96.2 
Histone genesConserved miR (Context score percentile)
HIST1H2AK miR760 (98)  miR4766-5p (54)   
HIST1H2BM  miR1276 (75) miR4766-5p (39)   
HIST1H1B miR760 (98) miR1276 (78) miR4766-5p (43)   
HIST1H3B miR760 (99) miR1276 (84) miR4766-5p (51)   
HIST1H1D miR760 (99) miR1276 (74) miR4766-5p (48)   
HIST1H1E miR760 (98) miR1276 (80) miR4766-5p (44)   
HIST1H3F miR760 (98) miR1276 (82) miR4766-5p (47)   
HIST1H2AD miR760 (98) miR1276 (83) miR4766-5p (48)   
HIST1H2AH miR760 (99) miR1276 (83) miR4766-5p (55)   
HIST1H3C miR760 (98) miR1276 (81) miR4766-5p (46)   
HIST1H4I     miR4512 (98) 
HIST1H2BN miR760 (99) miR1276 (76) miR4766-5p (43)   
HIST1H4L    miR1291 (99) miR4512 (93) 
HIST1H4K     miR4512 (98) 
HIST1H4E    miR1291 (89) miR4512 (95) 
HIST1H2BJ miR760 (66) miR1276 (78)    
HIST1H3A miR760 (98) miR1276 (82) miR4766-5p (45)   
HIST2H2AB miR760 (98) miR1276 (78) miR4766-5p (47)   
HIST1H2BC miR760 (67) miR1276 (87)    
HIST1H2AL miR760 (99) miR1276 (80) miR4766-5p (51)   
HIST1H4B    miR1291 (89)  
HIST1H2AG  miR1276 (70)    
HIST2H2BF miR760 (99) miR1276 (82) miR4766-5p (52)   
HIST1H2BF miR760 (67) miR1276 (87)    
HIST1H2AI 
HIST1H2AC miR760 (86) miR1276 (79) miR4766-5p (42)   
HIST1H3D miR760 (99) miR1276 (84) miR4766-5p (50)   
HIST1H3G  miR1276 (88)    
HIST1H3H miR760 (99) miR1276 (82) miR4766-5p (47)   
HIST1H2BH miR760 (99)     
HIST1H4C    miR1291 (92) miR4512 (97) 
H3F3C 
HIST2H2BE miR760 (97) miR1276 (85) miR4766-5p (46)   
HIST1H4D    miR1291 (99)  
HIST1H3I miR760 (85) miR1276 (80) miR4766-5p (43)   
HIST1H2BO miR760 (98) miR1276 (84)    
HIST1H2BB miR760 (99) miR1276 (82)    
Average (%) 93.6 80.9 47.05 93.6 96.2 

miRNA-760 expression in gastric cancer patients

miR-760 expression in bone marrow samples from stage IV patients (n = 53) was lower than that of stage I patients (n = 52; P = 0.0094; Fig. 2A). Similarly, miR-760 expression in stage IV primary gastric cancer tissues was significantly lower than in early-stage primary gastric cancer tissues (Fig. 2B, left). These expression patterns for miR-760 were opposite to those of HIST1H3D in both the bone marrow and primary tumors from gastric cancer patients. We then classified 96 gastric cancer cases, which had been examined for miR-760 expression in primary tumor tissues, into 2 groups according to the average miR-760 mRNA expression. Patients with expression of miR-760 that was below an average value of 42.256 (normalized to RNU6B) were assigned to the low-expression group (n = 65), whereas those with expression values above an average of 42.256 were assigned to the high-expression group (n = 31). Clinicopathological factors were then analyzed in relation to miR-760 levels. Patients with low miR-760 expression exhibited significantly larger tumor sizes and more frequent tumor invasion, lymph node metastasis, peritoneal dissemination, and advanced stages than patients with high miR-760 expression (Table 2). In terms of overall survival, patients in the high miR-760 expression group had a significantly better prognosis than those in the low miR-760 expression group (Fig. 2B, right). Multivariate analysis of overall survival showed that the level of miR-760 expression was an independent prognostic predictor [relative risk (RR) = 1.67; 95% confidence interval (CI), 1.03–3.11; P = 0.0374 by Cox proportional hazards model; Table 3].

Figure 2.

miR-760 expression in gastric cancer patients. miR-760 expression was analyzed in the bone marrow (A) and primary tumors (B) of gastric cancer patients. Kaplan–Meier overall survival curves for gastric cancer patients based on the level of miR-760 expression from primary tumor samples (B, right). C, miR-760 expression in bone marrow fractions separated by the AutoMACS system using CD45, EpCAM, and CD14 microbeads from 4 gastric cancer patients. D, left, miR-760 expression in corresponding noncancerous gastric mucosa from gastric cancer patients. Right, Kaplan–Meier overall survival curves of gastric cancer patients based on the level of miR-760 expression in corresponding noncancerous gastric mucosa.

Figure 2.

miR-760 expression in gastric cancer patients. miR-760 expression was analyzed in the bone marrow (A) and primary tumors (B) of gastric cancer patients. Kaplan–Meier overall survival curves for gastric cancer patients based on the level of miR-760 expression from primary tumor samples (B, right). C, miR-760 expression in bone marrow fractions separated by the AutoMACS system using CD45, EpCAM, and CD14 microbeads from 4 gastric cancer patients. D, left, miR-760 expression in corresponding noncancerous gastric mucosa from gastric cancer patients. Right, Kaplan–Meier overall survival curves of gastric cancer patients based on the level of miR-760 expression in corresponding noncancerous gastric mucosa.

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Table 2.

miR-760 expression and clinicopathological factors

Low expressionHigh expression
(n = 65)(n = 31)
FactorsN (%)N (%)P value
Age (mean) 
 ≥ 65 35 (53.85) 15 (48.39) 0.6166 
 < 65 30 (46.15) 16 (51.61)  
Sex 
 Male 44 (67.69) 17 (54.84) 0.2211 
 Female 21 (32.31) 14 (45.16)  
Size 
 (Large) > 50 mm 40 (61.54) 10 (32.26) 0.0056a 
 (Small) < 50 mm 24 (36.92) 21 (67.74)  
Histologyb 
 Well and moderately 28 (43.08) 14 (45.16) 0.8967 
 Poorly and others 36 (55.38) 17 (54.84)  
Tumor stage 
 T1 6 (9.23) 14 (45.16) <0.0001a 
 T2–T4 59 (90.77) 17 (54.84)  
Lymph node metastasis 
 Absent 17 (26.15) 15 (48.39) 0.0307c 
 Present 48 (73.85) 16 (51.61)  
Lymphatic invasion 
 Absent 16 (24.62) 12 (38.71) 0.1694 
 Present 48 (73.85) 19 (61.29)  
Venous invasion 
 Absent 44 (67.69) 24 (77.42) 0.3797 
 Present 20 (30.77) 7 (22.58)  
Liver metastasis 
 Absent 62 (95.38) 30 (96.77) 0.75 
 Present 3 (4.62) 1 (3.23)  
Peritoneal dissemination 
 Absent 51 (78.46) 30 (96.77) 0.0209c 
 Present 14 (21.54) 1 (3.23)  
Stage 
 I 14 (21.54) 15 (48.39) 0.0075a 
 II 14 (21.54) 8 (25.81)  
 III 17 (26.15) 6 (19.35)  
 IV 20 (30.77) 2 (6.45)  
Low expressionHigh expression
(n = 65)(n = 31)
FactorsN (%)N (%)P value
Age (mean) 
 ≥ 65 35 (53.85) 15 (48.39) 0.6166 
 < 65 30 (46.15) 16 (51.61)  
Sex 
 Male 44 (67.69) 17 (54.84) 0.2211 
 Female 21 (32.31) 14 (45.16)  
Size 
 (Large) > 50 mm 40 (61.54) 10 (32.26) 0.0056a 
 (Small) < 50 mm 24 (36.92) 21 (67.74)  
Histologyb 
 Well and moderately 28 (43.08) 14 (45.16) 0.8967 
 Poorly and others 36 (55.38) 17 (54.84)  
Tumor stage 
 T1 6 (9.23) 14 (45.16) <0.0001a 
 T2–T4 59 (90.77) 17 (54.84)  
Lymph node metastasis 
 Absent 17 (26.15) 15 (48.39) 0.0307c 
 Present 48 (73.85) 16 (51.61)  
Lymphatic invasion 
 Absent 16 (24.62) 12 (38.71) 0.1694 
 Present 48 (73.85) 19 (61.29)  
Venous invasion 
 Absent 44 (67.69) 24 (77.42) 0.3797 
 Present 20 (30.77) 7 (22.58)  
Liver metastasis 
 Absent 62 (95.38) 30 (96.77) 0.75 
 Present 3 (4.62) 1 (3.23)  
Peritoneal dissemination 
 Absent 51 (78.46) 30 (96.77) 0.0209c 
 Present 14 (21.54) 1 (3.23)  
Stage 
 I 14 (21.54) 15 (48.39) 0.0075a 
 II 14 (21.54) 8 (25.81)  
 III 17 (26.15) 6 (19.35)  
 IV 20 (30.77) 2 (6.45)  

aP < 0.01.

bWell, moderately, and poorly: differentiated types of gastric adenocarcinoma.

cP < 0.05.

Table 3.

Results of univariate and multivariate analysis of clinicopathological factors for 5-year overall survival (Cox proportional hazard model)

Univariate analysisMultivariate analysis
FactorsRR (95% CI)P valueRR (95% CI)P value
Sex (female/male) 1.12 (0.81–1.60) 0.5054 – – 
Tumor size (<5 cm/>5 cm) 1.63 (1.19–2.27) 0.0021a 0.95 (0.62–1.48) 0.8316 
Histological differentiation (wellb, moderatelyc/othersd1.32 (0.97–1.83) 0.0749   
Tumor depth (T1/T2–T4) 3.31 (1.83–8.20) <0.0001a 1.13 (0.45–5.05) 0.8306 
Lymph node metastasis (negative/positive) 6.41 (2.98–27.02) <0.0001a 3.3 (1.45–14.24) 0.0016a 
Lymphatic invasion (negative/positive) 5.05 (2.35–21.30) <0.0001a 1.59 (0.64–7.13) 0.3623 
Venous invasion (negative/positive) 2.17 (1.58–2.98) <0.0001a 1.62 (1.04–2.56) 0.032e 
miR-760 expression (high/low) 1.96 (1.23–3.63) 0.0035a 1.67 (1.03–3.11) 0.0374e 
Univariate analysisMultivariate analysis
FactorsRR (95% CI)P valueRR (95% CI)P value
Sex (female/male) 1.12 (0.81–1.60) 0.5054 – – 
Tumor size (<5 cm/>5 cm) 1.63 (1.19–2.27) 0.0021a 0.95 (0.62–1.48) 0.8316 
Histological differentiation (wellb, moderatelyc/othersd1.32 (0.97–1.83) 0.0749   
Tumor depth (T1/T2–T4) 3.31 (1.83–8.20) <0.0001a 1.13 (0.45–5.05) 0.8306 
Lymph node metastasis (negative/positive) 6.41 (2.98–27.02) <0.0001a 3.3 (1.45–14.24) 0.0016a 
Lymphatic invasion (negative/positive) 5.05 (2.35–21.30) <0.0001a 1.59 (0.64–7.13) 0.3623 
Venous invasion (negative/positive) 2.17 (1.58–2.98) <0.0001a 1.62 (1.04–2.56) 0.032e 
miR-760 expression (high/low) 1.96 (1.23–3.63) 0.0035a 1.67 (1.03–3.11) 0.0374e 

aP < 0.01.

bWell, well-differentiated type.

cModerately, moderately differentiated type.

dOthers, poorly differentiated and scirrhous type.

eP < 0.05.

Direct interaction between miR-760 and histone mRNA in gastric cancer cells

To confirm whether histone mRNAs were direct targets of miR-760, we generated HIST1H3D- and HIST1H2AD-luciferase constructs. Interestingly, among the histones upregulated in stage IV bone marrow in Fig. 1A, HIST1H2AD shared a common genomic region with HIST1H3D in a region of the 5′ sides of transcripts (Supplementary Fig. S2A). Therefore, we evaluated the differences in the binding abilities between these 2 histone variants. Cotransfectants expressing both miR-760 and HIST1H3D or HIST1H2AD showed significant reductions in luciferase activity compared with controls in the gastric cancer cell line, NUGC3 (P < 0.01 and P < 0.001, respectively; Fig. 3A). This reduction in luciferase activity was more pronounced in cells transfected with the HIST1H2AD construct than with the HIST1H3D construct. The activities of both reporter constructs harboring point mutations in miR-760 binding sites were unaffected by simultaneous transfection with Pre-miR-760 (Fig. 3A).

Figure 3.

The association between histone mRNA and miR-760 in gastric cancer cells. A, luciferase analysis in NUGC3 cells. HIST1H3D (left) or HIST1H2AD (right) luciferase vector + miR-760 transfectants showed lower luciferase activities than control cells. The luciferase activities of both reporter constructs harboring point mutations in miR-760 binding sites were unaffected by simultaneous transfection with Pre-miR-760. Pre-miR n.c., Pre-miR negative control; WT, wild type. B, HIST1H3D and miR-760 mRNA expression in gastric cancer cell lines. Top, HIST1H3D and miR-760 expression in normal culture conditions. Middle and bottom, changes in HIST1H3D and miR-760 expression in gastric cancer cells. Cells were cultured for 72 hours in serum-free media and were restimulated with serum after 24 hours of serum starvation. Expression of HIST1H3D and miR-760 was measured at 24 and 72 hours after starvation and at 48 hours after restimulation. C, miR-760 and HIST1H3D mRNA and protein expression after treatment with negative control or Pre-miR-760 for 3 hours (mRNA) and 48 hours (protein) in gastric cancer cell lines. Top, miR-760; middle, HIST1H3D mRNA; bottom, Western blotting analysis of HIST1H3D and pan-actin. Bottom, graphical representation of gel images in the top panels. D, miRNA binding site in the 3′ UTR of histone mRNA. Gray region indicates the binding site for miR-760 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 3.

The association between histone mRNA and miR-760 in gastric cancer cells. A, luciferase analysis in NUGC3 cells. HIST1H3D (left) or HIST1H2AD (right) luciferase vector + miR-760 transfectants showed lower luciferase activities than control cells. The luciferase activities of both reporter constructs harboring point mutations in miR-760 binding sites were unaffected by simultaneous transfection with Pre-miR-760. Pre-miR n.c., Pre-miR negative control; WT, wild type. B, HIST1H3D and miR-760 mRNA expression in gastric cancer cell lines. Top, HIST1H3D and miR-760 expression in normal culture conditions. Middle and bottom, changes in HIST1H3D and miR-760 expression in gastric cancer cells. Cells were cultured for 72 hours in serum-free media and were restimulated with serum after 24 hours of serum starvation. Expression of HIST1H3D and miR-760 was measured at 24 and 72 hours after starvation and at 48 hours after restimulation. C, miR-760 and HIST1H3D mRNA and protein expression after treatment with negative control or Pre-miR-760 for 3 hours (mRNA) and 48 hours (protein) in gastric cancer cell lines. Top, miR-760; middle, HIST1H3D mRNA; bottom, Western blotting analysis of HIST1H3D and pan-actin. Bottom, graphical representation of gel images in the top panels. D, miRNA binding site in the 3′ UTR of histone mRNA. Gray region indicates the binding site for miR-760 (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

HIST1H3D expression was downregulated in gastric cancer cell lines after Pre-miR-760 transfection

In terms of mRNA expression, both HIST1H3D transcripts and miR-760 were expressed at low levels in AGS cells, and KE39 cells showed high HIST1H3D and low miR-760 expression among the 7 gastric cancer cell lines investigated (Supplementary Fig. S2B). HIST1H3D protein levels did not necessarily correspond to HIST1H3D mRNA levels in each cell line (Supplementary Fig. S2C), indicating that the proportions of S-phase cells differed between cell lines and that redundant untranslated histone mRNAs may be present in some types of gastric cancer cells. Three cell lines (NUGC3, KATOIII, and KE39) were used in the following Pre-miR transfection experiments because of their high transfection efficiency for miRNAs. In these cell lines, contrasting expression patterns of HIST1H3D and miR-760 were observed in terms of mRNA levels (Fig. 3B, top). The expression of HIST1H3D mRNA was downregulated at 3 hours after Pre-miR-760 transfection (Fig. 3C, middle), but upregulated at 24 to 48 hours after transfection in these 3 cell lines. A time course of HIST1H3D expression data after Pre-miR-760 transfection in NUGC3 cells is shown in Supplementary Fig. S2D. Although HIST1H3D protein was upregulated at 3 hours after Pre-miR-760 transfection in all 3 cell lines (Supplementary Fig. S2E), downregulation of HIST1H3D protein was observed in NUGC3 and KE39 cells at 48 hours (Fig. 3C, bottom). In KATOIII cells, further upregulation of HIST1H3D was observed at 48 hours (Fig. 3C, bottom). Notably, only in NUGC3 cells, overexpression of miR-760 induced morphological changes at 24 to 48 hours after transfection (Supplementary Fig. S2F).

miR-760 expression was altered in response to histone mRNA expression in gastric cancer cells

It is possible that miR-760 expression changes in response to histone mRNA expression. We evaluated changes in miR-760 expression in gastric cancer cells under conditions of serum starvation and restimulation in which histone mRNA expression may be altered depending on cell-cycle progression. Gastric cancer cells were cultured in serum-free media for 72 hours and then restimulated with serum for 24 hours. The expression of HIST1H3D transcripts and miR-760 was measured at 24 and 72 hours after starvation and at 48 hours after restimulation. Although HIST1H3D and miR-760 behaviors differed in each cell line, contrasting expression patterns of these genes were observed under conditions of serum starvation and restimulation (Fig. 3B, middle and bottom). These results indicated that the expression levels of histone mRNA and miR-760 were altered in opposite directions in response to culture conditions in 3 gastric cancer cell lines.

Contrasting expression of histone mRNA and miR-760 was also observed in noncancerous cells in gastric cancer patients

Next, we examined which cells in the bone marrow exhibited altered miR-760 expression in another set of 4 gastric cancer patients. Because all of these 4 patients had stage IV gastric cancer, the expression of miR-760 was expected to be low. However, differential expression of miR-760 was observed in each fraction. In bone marrow samples from all 4 patients, the CD45/EpCAM+ fraction showed highest miR-760 expression among all fractions (Fig. 2C). Tumor cells may be enriched in the CD45/EpCAM+ fraction compared with other fractions. Interestingly, the CD14 fraction exhibited the same degree of miR-760 expression (Fig. 2C).

In corresponding noncancerous tissues from primary gastric cancer tumors, miR-760 expression was lower in stage IV patients than in stage I patients (P = 0.0243; Fig. 2D, left). We then classified 84 gastric cancer patients, whose miR-760 expression could be evaluated in corresponding noncancerous gastric mucosa, into 2 groups based on the median expression of miR-760 (17.57 normalized to RNU6B). In terms of overall survival, patients in the low miR-760 expression group (n = 42) had a significantly poorer prognosis than those in the high miR-760 expression group (n = 42; Fig. 2D, right). These results suggested that changes in miR-760 expression in host noncancerous cells were also associated with gastric cancer progression.

Histone protein synthesis is restricted to the S-phase of the cell cycle, and regulation of histone protein synthesis is accomplished by regulation of histone mRNA levels. Histone mRNAs are tightly regulated during the cell cycle, allowing the synthesis of histone proteins to occur coordinately with the replication of DNA. In malignant tumors, upregulation of histone mRNA indicates proliferative activity of tumor cells, and many studies have demonstrated that histone mRNA accumulates in tumors (14–16). In most previous studies, histone levels have been measured by only considering H3 core histone levels. Histone gene clusters in mammals are heterogeneously organized and contain 1 or more copies of the 5 histone subtypes, that is, core (H2A, H2B, H3, H4, and variants thereof) and linker (H1) histone genes (17, 18). The vast majority of the 74 human histone genes can be found within the major and minor clusters located on chromosomes 6p21 and 1q21, respectively. Recent studies have demonstrated that these histone clusters genes are overexpressed in several types of malignancies. Sadikovic and colleagues performed integrative whole-genome analysis of DNA copy numbers, promoter methylation, and gene expression using 10 osteosarcomas and identified significant changes, including the hypomethylation, gain, and overexpression of histone cluster 2 genes on chromosome 1q21.1–q21.3 (19). Moreover, Perez-Margan and colleagues identified candidate genes related to meningioma recurrence by differential gene expression profiling analyses of 33 original and 7 recurrent meningiomas. They demonstrated that 16 histone cluster 1 genes, composed of 3 H1 linker and 13 H2 core histones, were overexpressed in recurrent meningiomas (20). Our current study presents the first evidence for overexpression of histone cluster genes, mainly histone cluster 1, in bone marrow and primary tumor samples from stage IV gastric cancer patients compared with those from stage I gastric cancer patients. Because histone mRNAs are tightly regulated and increase or decrease simultaneously during the cell cycle, we speculate that genes regulating multiple histone mRNAs may be useful prognostic/metastatic markers for gastric cancer patients. Unlike most RNA polymerase II-transcribed mRNAs, histone mRNAs are not polyadenylated, but instead end in a conserved 30-bp stem-loop structure, which is recognized and cleaved by the stem-loop binding protein (SLBP; ref. 12). SLBP binds to the 3′ end of histone mRNA and participates in many aspects of histone mRNA metabolism. SLBP is a cell cycle–regulated protein, accumulating just before entry into S-phase and then rapidly degraded by the proteasome at the end of S-phase, similar to the timing of degradation of histone mRNAs (21). A sequence in the amino-terminal domain of SLBP is necessary for the rapid degradation of SLBP at the end of S-phase. This region contains consensus cyclin phosphorylation and binding sites. Mutation of either of these sequences stabilizes SLBP. Despite the fact that SLBP is stabilized, histone mRNA is still degraded at the appropriate time (22). Thus, it has been suggested that histone mRNA is degraded at the end of S-phase through a separate mechanism. We proposed the possibility that several miRNAs were involved in histone mRNA degradation and uncovered contrasting expression patterns of miR-760 and histone mRNAs in gastric cancer patients and gastric cancer cells subjected to serum starvation or restimulation. Furthermore, Pre-miR-760 transfection experiments also indicated that the interaction between HIST1H3D and miR-760 occurred in living gastric cancer cells (Fig. 3C). HIST1H3D mRNA and corresponding protein expression changes induced by miR-760 overexpression were somewhat complex. Just after Pre-miR-760 transfection, HIST1H3D mRNA was downregulated as expected, whereas HIST1H3D protein expression was upregulated in 3 cell lines (Fig. 3C and Supplementary Fig. S2E). At 48 hours after Pre-miR-760 transfection, HIST1H3D protein was downregulated in NUGC3 and KE39 cells (Fig. 3C, bottom); however, the expression of HIST1H3D mRNA was significantly upregulated in all cell lines (Supplementary Fig. S2D). Recently, 4 miRNAs, including miR-760, have been reported to cooperatively induce cellular senescence by targeting a subunit of protein kinase CKII in human colorectal cancer cells (23). Our in silico analysis also indicated that several miRNAs, including miR-760, targeted histone mRNAs cooperatively. Furthermore, an interaction between the histone 3′ UTR and miRNA is only one degradation pathway for histone mRNAs. Therefore, the expression of HIST1H3D mRNA and its corresponding protein is likely maintained by compensatory and/or feedback mechanisms, although exogenous miR-760 caused temporary destabilization of histone mRNA by targeting its 3′ UTR. Both mRNA and protein expression of HIST1H3D were continuously elevated only in KATOIII cells. Because KATOIII was a floating cell line, different from the other 2 cell lines, the effects of miR-760 may vary between cell types. Interestingly, morphological changes were observed only in NUGC3 cells by overexpression of miR-760. Pre-miR-760-transfected NUGC cells did not show spindle formation, which was observed in control cells (Supplementary Fig. S2F). There were no differences in proliferation rates between Pre-miR-760-transfected cells and controls, whereas p21 expression was upregulated in transfected cells (Supplementary Fig. S2G and S2H). These results suggested that miR-760 may prevent spindle formation and direct senescence in specific cells. The predictive miR-760-binding site in histone 3′ UTRs was located in the stem-loop end (Fig. 3D). All of the predicted binding sites for other miRNAs in histone 3′ UTRs shown in Table 1 were also in the stem loop regions. Under normal conditions, miRNAs are not likely to bind to these histone mRNA stem-loop structures. However, loss of normal histone pre-mRNA processing has been shown to result in the production of polyadenylated mRNAs from histone genes (24, 25). Levels of these polyadenylated histone mRNAs are very low in proliferating cells (24, 26), but may increase during terminal differentiation (27, 28) or tumorigenesis (29–31). The histone mRNAs detected in our RNA-seq analysis may also be polyadenylated because polyA-containing RNA was selected before first-strand cDNA synthesis (see Materials and Methods). Therefore, it is possible that upregulation of these polyadenylated histone mRNAs and downregulation of miR-760, which can bind to polyadenylated histone 3′ UTRs, are also involved in gastric cancer progression.

We also evaluated the association between HIST1H3D or miR-760 expression and the status of DTC markers in the bone marrow. Significant differences were not observed between DTC-positive and -negative cases in both HIST1H3D and miR-760 expression (Supplementary Fig. S1B), indicating that contrasting expression of HIST1H3D and miR760 did not occur only in DTCs of gastric cancer patients. To investigate whether host cells were involved in cancer development and metastasis, we evaluated the miR-760 expression status of noncancerous cells in gastric cancer patients. Fractionated bone marrow from gastric cancer patients was subjected to miRNA microarray; this analysis revealed that miR-760 was highly expressed in both the CD14+ and CD45/EpCAM+ fractions (Fig. 2C). Generally, it is thought that several types of host cells, such as macrophages and myofibroblasts, are enriched in CD14+ fractions, whereas tumor cells are enriched in CD45/EpCAM+ fractions. Consistent with this, in corresponding noncancerous gastric mucosa, miR-760 expression was downregulated in tissues from stage IV patients; moreover, low expression of miR-760 was associated with a poorer prognosis than high expression of this miRNA (Fig. 2D). Kaplan and colleagues found that bone marrow–derived hematopoietic progenitor cells expressing VEGFR-1 are attracted to tumor-specific, premetastatic sites and form cellular clusters before the arrival of tumor cells, functioning as a cancer niche to facilitate metastasis (7). Furthermore, bone marrow–derived myofibroblasts have been reported to contribute to cancer-induced stromal reactions in the later stages of tumor development (32). From studies in mouse models of inflammation-induced gastric cancer, Quante and colleagues also demonstrated that carcinoma-associated fibroblasts were derived from mesenchymal stem cells in the bone marrow and that myofibroblasts expressing alpha-smooth muscle actin increased markedly during cancer progression (33). Therefore, our results indicate that downregulation of miR-760 expression not only in gastric cancer cells, but also in specific host cells in the bone marrow and gastric tissue may affect gastric cancer progression.

Area under the curve (AUC) values of both miR-760 and HIST1H3D could not be used as sufficient predictors in receiver operating characteristic (ROC) curve analysis for discriminating patients with stage I or stage IV gastric cancer from bone marrow samples (AUC = 0.680 for miR-760; AUC = 0.585 for HIST1H3D; Supplementary data and Supplementary Fig. S1C). Thus, further studies are warranted to develop prediction approaches for the prognosis of gastric cancer patients using these markers.

Histone mRNAs seemed to be upregulated in response to the increased proportion of S-phase cells in advanced gastric cancer tissue and the bone marrow. Because redundant untranslated histone mRNAs are degraded rapidly at the end of S-phase, a large amount of specific molecules involved in the degradation of histone mRNA, including miR-760, may be required at this period in advanced gastric cancer. Our present study revealed an interaction between miR-760 and histone mRNA. Although it is unclear whether low levels of miR-760 are the result or the cause of histone mRNA degradation in advanced gastric cancer, the histone mRNA/miR-760 axis may have a crucial role in the development of gastric cancer and may become a new therapeutic target in the treatment of advanced gastric cancer.

No potential conflicts of interest were disclosed.

Conception and design: T. Iwaya, T. Fukagawa, F. Tanaka, M. Mori, M. Sasako, K. Mimori

Development of methodology: T. Iwaya, J. Kurashige

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Iwaya, T. Fukagawa, Y. Takahashi, G. Sawada, M. Ishibashi, J. Kurashige, F. Endo, K. Ishida, K. Kume, M. Sasako

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Iwaya, Y. Suzuki, J. Kurashige, F. Endo, K. Ishida, K. Kume, M. Mori

Writing, review, and/or revision of the manuscript: T. Iwaya, F. Endo, K. Ishida, K. Kume

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Iwaya, T. Sudo, H. Katagiri, S. Nishizuka, H. Iinuma

Study supervision: T. Fukagawa, K. Shibata, G. Wakabayashi, M. Mori, M. Sasako, K. Mimori

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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