Identification of miRNAs progressively deregulated in the biological model of gastric carcinogenesis

doi:10.21203/rs.3.rs-1991665/v1

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Identification of miRNAs progressively deregulated in the biological model of gastric carcinogenesis

https://doi.org/10.21203/rs.3.rs-1991665/v1

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Gastric adenocarcinoma is the result of the progression of preneoplastic lesions in gastric mucosa. Helicobacter pylori (H. pylori) infection is the main environmental risk factor linked to the multistep progression from precancerous conditions to gastric cancer. Identification of early diagnosis biomarkers in preneoplastic lesions could prevent progression to cancer. microRNAs (miRNAs) are non-coding RNAs that have emerged as promising candidates with diagnostic and prognostic potential. miRNAs differentially expressed and functional enrichment analysis were identified in miRNomes of gastric preneoplastic samples available at the European Nucleotide Archive (ENA) and in gastric adenocarcinoma samples from TCGA databases by limma-voom linear model on the Galaxy Collections platform and R package. The prognostic value of miRNAs was evaluated by Kaplan-Meier assays. The expression level of miR-18a-5p was determined by RT-qPCR in preneoplastic lesion samples from Mexican patients positive to pathogenic H. pylori and in the H. pylori-AGS cells co-cultures. Fifteen miRNAs were progressively deregulated in the multistep gastric carcinogenesis model, and they were predictors of the outcome in gastric adenocarcinoma patients. Additionally, miR-18a-5p was significantly upregulated in gastric tumors compared to normal gastric epithelium samples and it was also associated with better overall survival in GC patients. The expression of miR-18a-5p was significantly inhibited in gastric preneoplastic lesions positive to pathogenic H. pylori. Further, miR-18a-5p was up-regulated in AGS cells infected with pathogenic H. pylori strain. In conclusion, miRNAs signature distinguished the gastric lesions through malignant transformation process, including miR-18a-5p, which was exclusively associated with H. pylori infection.

Preneoplastic lesions

gastric cancer

H. pylori

miRNAs

miR-18a-5p

Gastric cancer (GC) represents the fifth malignant tumor with 1,089,103 new cases diagnosed worldwide, and third cause of cancer-related mortality with 768,793 deaths reported for 2020. GC diffuse and intestinal subtypes are the most common histological types of the digestive systems since 95% of gastric tumors are classified as such [1–3]. Intestinal-type adenocarcinoma is the outcome of preneoplastic lesions, including chronic gastritis, atrophic gastritis, intestinal metaplasia and finally dysplasia that might evolve to GC [4–6]. The main risk factor for the development of preneoplastic lesions and GC is the infection with pathogenic strains of Helicobacter pylori (H. pylori), which is characterized by a 40 kb chromosomal segment called cag pathogenicity island (cytotoxin-associated genes; cagPAI). Among the genes contained at the cag pathogenicity island, the cagPAI gene encodes for the CagA protein (cytotoxin-associated gene A). All H. pylori strains contain the VacA gene (vacuolating cytotoxin), but only 40% of the strains produce a functional protein [7–10]. The structural and molecular changes in the gastric microenvironment caused by the chronic infection of pathogenic strains of H. pylori initiate a cascade of molecular events including the deregulation of microRNAs (miRNAs) involved in the development and maintenance of GC [11, 12]. Several studies have shown the role of miRNAs in the gastric carcinogenesis multistep model; therefore, miRNAs may be a potential tool to identify patients at early cancer stages [13–17].

In GC, the role of miRNAs in the interaction of pathogenic strains of H. pylori with the gastric epithelial cells that culminates in the establishment of GC is still poorly understood. A recent study using a co-culture model of H. pylori strain 26699 (cagPAI positive) and epithelial gastric cells identified some miRNAs associated with H. pylori infection (miR-196a, miR-127-5p, miR-206, miR-216, miR-488, miR-195, miR-34a, miR-141, miR-17-3p, miR-103, and let-7i), involved in chronic inflammatory processes [18]. In GC, the deregulation of miR-135-5p, miR-196a-5p, miR-18a-5p and miR-145-5p has been reported from which miR-30a, miR-143, miR-145, and miR-133b are associated with longer survival in GC [15]. Interestingly, only miR-18a-5p was altered in H. pylori-positive patients and the levels of the above-mentioned miRNAs have not been reported in H. pylori positive patients. Hence, we aimed identify miRNAs progressively deregulated in premalignant lesions and GC by bioinformatic approaches. Highlighting the expression analysis of miR-18a-5p in premalignant lesion of patients positive to pathogenic H. pylori and studying the effects of H. pylori infection in an in vitro model.

Analysis of RNA-seq and microarray data

The miRNAs microarray data of chronic non-active gastritis (n=16), chronic active gastritis (n=28), chronic atrophic gastritis (n=14), and metaplasia (n=35) were obtained from the data set E-MTAB-8890 of the European Nucleotide Archive (ENA) database (https://www.ebi.ac.uk/ena/browser/home). The miRNAs miRNA-seq data of 446 GC tumors and 45 normal gastric tissues were obtained from the TCGA-STAD data set of the Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov). The miRNA-seq and microarrays data were independently normalized and then, differential expression miRNAs analysis were performed using the limma-voom linear model on the Galaxy Collections platform (https://usegalaxy.org/) as follows: (I) chronic non-active gastritis vs. normal gastric tissues; (II) chronic active gastritis vs. chronic non-active gastritis; (III) chronic atrophic gastritis vs. chronic active gastritis; (IV) intestinal metaplasia vs. chronic atrophic gastritis; (V) GC tumors vs. intestinal metaplasia. Values of p.adj <0.05 and |log2FC| >= 1.5 were used to identify differentially expressed miRNAs (DEMs) in gastric preneoplastic lesions and GC. Identified DEMs were divided into two groups according to the expression levels: up-regulated (log2FC>1.5) and down-regulated (log2FC< -1.5). Additionally, miRNAs expression profile was obtained in GC tumors compared to normal gastric tissues from TCGA data.

Kaplan–Meier overall survival and ROC curves analysis

The prognostic value of miRNAs in GC was evaluated by Kaplan-Meier curves (www.kmplot.com). The prognostic value of each miRNA was evaluated according to high or low expression relative to the median expression of single miRNAs in GC patients. Log-rank tests were used to analyze the differences between the curves. The diagnostic performance was evaluated using receiver operating characteristic (ROC) curves. The clinic accuracy was defined as follow: AUC ≤ 6 without diagnostic value; AUC > 6 < 8 medium diagnostic value; AUC ≥ 8 < 9.5 high diagnostic value; AUC≥ 9.5 perfect diagnostic value. A p value <0.05 was considered statistically significant.

Functional enrichment analysis

The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and hallmarks of cancer from MSigDB collection enrichment analyses were performed with the “fgsa” R package (version 1.14.0). DIANA-miRPath v.3 (https://dianalab.e-ce.uth.gr/html/mirpathv3/index.php?r=mirpath) was used to probe the signaling pathways in both groups of DEMs by using default settings. The top terms with a p < 0.05 were extracted.

Clinical specimens

The study included 37 patients newly diagnosed with gastric disease referred to the Endoscopy Service of the Digestive Diseases Clinic of the Angel del Mar Hospital in Puerto Escondido, Oaxaca, Mexico from December 2019 to December 2021. Study was reviewed and approved by the Institutional Review Board. All patients agreed and signed the informed consent. The endoscopic analysis and biopsies collection were performed in accordance with the Sydney protocol [19] using the GIF-V2/H180 gastroscope, CV-145/CV-180 video processor (Olympus Corporation of the Americas, Unites States), and biopsy forceps (Boston Scientific, Massachusetts, Unites States). Two separate biopsies from the body and gastric antrum were collected in 10% buffered formaldehyde or RNAlater (Ambion, Austin, Texas), and stored at -70°C until further RNA extraction. The endoscopic evaluation and protocol biopsies collection were performed by two expert gastroenterologists. The study was carried out in accordance with the provisions in the Helsinki Declaration and the Ethics Committee of the Medical and Research Group of the Angel del Mar granted approval for this research.

Histopathology analysis

Formalin fixed paraffin embedded (FFPE) tissue sections from the resection specimens were stained by hematoxylin and eosin (H&E). Slides were examined by pathologist that reported grading of alterations according to Sydney protocol [20, 21]. All samples were analyzed and examined under Zeiss Axion optical microscope (Carl Zeiss, Germany). Photomicrographs were taken with 20x, 40x, and 100x magnification.

Total RNA extraction and purification

Total RNA of all specimens and AGS cells was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the instruction manual. RNA quality and concentration were measured by UV absorbance at 260 and 280 nm (260/280 nm) spectrophotometry (A260nm/A280nm) on a NanoDrop lite (Thermo Fisher Scientific, USA).

Identification and genotyping of Helicobacter pylori

Identification and genotyping of H. pylori in biopsies of body and antrum gastric was carried out using the methodology described by Atherton et al. (1995) for the alleles of the VacA (s1 and s2). CagA expression was determined by the methodology described by Morales-Espinosa et al. (1999). cagL, virB10, and virD4 genes was carried out by using primers designed at Bacterial Genetics Laboratory of the Faculty of Medina of the National Autonomous University of Mexico by Morales-Espinoza et al. (data not shown, unpublished data).

Co-culture of Helicobacter pylori and AGS cells

AGS human gastric adenocarcinoma cell line (ATCC CRL-1739) was routinely cultured in Dulbecco´s Modified Eagle Medium (DMEM, Life Technologies), supplemented with 5% fetal bovine serum (FBS, Corning Costar) and penicillin and streptomycin (Gibco) at 37°C in a 5% CO₂ atmosphere. H. pylori strain 26695 (ATCC 700392) was maintained in blood agar plates (BAP) supplemented with 5% fetal bovine serum in microaerobic conditions for 48 h. Co-culture was carried out according to Morales-Espinosa et al. (2020). Briefly, 3.2x10⁵AGS cells were co-cultured with 3.2x10⁷ UFC of H. pylori for 6 and 12 h. After infection, cells were carefully washed with PBS and subsequently total RNA of AGS cell co-cultured with bacterial was obtained. H. pylori vapD gene expression was used as positive control of infection [23].

Quantification of miR-18a-5p levels

Total RNA was obtained from the tissue samples of each histology type (non-active gastritis, follicular gastritis, active gastritis, atrophic gastritis, and metaplasia) and then pooled. Pool of RNA from tissue samples (200 ng) or total RNA from AGS cells and AGS co-cultured with H. pylori (100 ng) were retro-transcribed using the Stem-loop RT microRNA assay (Applied Biosystem) in accordance with the manufacturer’s protocol. Retro-transcription was performed in the Applied biosystems 2720 Thermal. cDNA was used for qPCR analysis using probes of miR-18a-5p and TaqMan Universal PCR Master Mix (Applied Biosystems). qPCR was performed in 7500 Real-Time PCR System (Applied Biosystems). The relative expression of miR-18a-5p was determined using the comparative C_t (2^-^ΔΔ^Ct) method. RNU44 was used as a control for normalization of data.

Statistical analysis

All results were derived from three independent experiments which were plotted as the mean ± standard deviation (SD) using GraphPad Prism software ver. 9.3.1. The comparison between groups was performed using ANOVA test. Unpaired t-test was used to determine the difference between H. pylori-positive and negative samples in each lesion. Data are expressed as the mean SEM. Overall survival (OS) was evaluated using the database from Kaplan-Meier plotter (www.kmplot.com). Log-rank tests were used to analyze the differences between the curves. A P value <0.05 was considered statistically significant.

Identification of miRNAs differentially expressed during gastric malignant transformation process

To identify differentially expressed miRNAs through the progression from precancerous lesions to GC we analyzed the miRNomes available in ENA and TCGA databases. Fifteen miRNAs were differentially expressed through the gastric malignant transformation process (Table 1). Eight miRNAs (miR-873, miR-141-3p, miR-429-3p, miR-204-5p, miR-200a-3p, miR-592-5P, miR-146a-5p and miR-363-3p) were up-regulated in non-active chronic gastritis compared to normal gastric tissue; however, the expression of these miRNAs significantly decreased as the gastric carcinogenesis model progresses (Fig. 1a). In contrast, seven miRNAs (miR-122-5p, miR-196b-5p, miR-20b-5p, miR378a-3p, miR-99a-5p, miR-194-5p ad miR-101-2-3p) were down-regulated in non-active chronic gastritis compared to normal tissue, but as the degree of lesion progresses, the expression of this set of miRNAs increased significantly (Fig. 2a). These results show a set of miRNAs whose differential expression was associated with the progression of preneoplastic lesions towards tumor establishment. These miRNAs group could constitute biomarkers with diagnostic potential.

Differentially expressed miRNAs during gastric malignant transformation are associated to clinical outcome of GC patients.

To investigate the clinical significance of miRNAs on the outcome of GC patients, we evaluated OS of patients according to the expression levels of each miRNA by Kaplan-Meier assays. Results showed that low expression of miR-429-3p, miR-592-5p, miR-200a-3p, miR-146a-5p, and miR-141-3p whose expression decreases along the lesion grades was associated to poor OS in GC patients, while low levels of miR-363-3p was associated to improved OS (Fig. 1b). On the other hand, high levels of those miRNAs whose expression increased along the malignant progression (miR-194-5p, miR-196b-5p, and miR-378-3p) were associated with better OS (Fig. 2b). Contrariwise, higher expression of miR-99a-5p, miR20b-5p, and miR-122-5p were associated with poor OS of GC patients (Fig. 2b). miR-101-2-3p was not associated to OS in CG patients. Collectively, data showed that the expression levels of miRNAs could act as a predictive biomarker of the long-term outcome of GC patients. To evaluate the diagnostic power of miRNAs, ROC curves were constructed. Result indicated that combined AUC for the miRNAs significantly associated to OS was AUC = 0.68 to progressively downregulated miRNAs (Fig. 1c), while to those miRNAs progressively upregulated was AUC = 0.75 (Fig. 2c), suggesting that the combined expression of miRNAs might useful as potential tool for diagnosis of GC patients.

miRNAs differentially expressed during gastric malignant transformation regulate biological processes and signaling pathways associated to cancer

Gene ontology and biological pathways analysis showed that down-regulated miRNAs during progression of gastric lesions were significantly associated with biological pathways with relevance in cancer as adherent junction and metabolic processes including lysine degradation or fatty acid elongation, among others (Fig. S1a). Interestingly, several oncogenes as HDGF, TAZ, ZEB1/2, STAT4, KEAP1, Gli1, FSCN1, BCL2, ZEB1, L1CAM, MMP-9, ROCK1 and FBW7 have been reported as targets of this set of miRNAs in GC (Fig. S1b). On the other hand, up-regulated miRNAs were significantly associated to Hippo, p53 and TGF- β signaling pathways, ECM-receptor interaction and metabolic process related to proteoglycans in cancer as well as fatty acid biosynthesis (Fig. S1c). Reported target genes of these group of miRNAs include c-Myc, BCL2, PTEN, MAPK1, IGF1R, SOCS2 and FOXA1 (Fig. S1d). Taken together, these results suggest that progressive deregulation of these group of miRNAs could produce a deregulation of oncogenes or tumor suppressor genes associated to GC development. Remarkably, both up- and down-regulated miRNAs and their target genes have not been previously studied in preneoplastic lesions, neither their regulation by pathogenic strains of H. pylori plays through the carcinogenic process.

miRNAs expression profiles identify an overexpression of miR-18a-5p in gastric cancer

To determine whether miRNAs associated to GC multistep might also distinguish between GC and normal gastric tissue, we evaluate miRNAs expression profiles in GC tissues compared to normal tissues. We identified 159 down-regulated and 339 up-regulated miRNAs (Fig. 3a, Table S1). Among deregulated miRNAs, we detected the up-regulation of miR-196b, miR-200a, miR-141, miR-429 and miR-592, while miR-204 was down-regulated (Fig. 3b). The ten most deregulated miRNAs in GC were miR-139 (down-regulated) and miR-21, miR-196a-1, miR-196a-2, miR-196b, miR-135b, miR-146b, miR-501, and miR-18a (up-regulation) (Fig. 3c). Of these, up-regulation of miR-18a has previously been reported in GC patients positive to H. pylori-infection [24]; however, miR-18a has not been evaluated in gastric premalignant lesions. Hence, we compared the expression of miR-18a-5p in normal gastric tissues and GC tissues finding that miR-18a was overexpressed in GC (Fig. 3d). Moreover, higher expression of miR-18a was associated to better OS in GC patients (Fig. 3e) and ROC curves analysis showed AUC = 0.89 (Fig. 3f) demonstrated that the expression of miR-18a might useful as tool for diagnosis and prognosis of GC patients. These results indicate a potential role of miR-18a in gastric carcinogenic process, hence we aimed to evaluate miR-18a in gastric lesions.

miR-18a-5p is down-regulated in preneoplastic lesions infected with H. pylori

The expression of miR-18a was evaluated in premalignant gastric lesions collected from gastric antrum and body from 37 patients with gastric disease by endoscopic analysis (Fig. 4a-c). Seventy-four biopsies were examined (37 samples of gastric antrum and 37 samples gastric body). All samples were evaluated by hematoxylin and eosin (Fig. 4d-i) for histopathology diagnosis and to detect the presence of Helicobacter-like microorganisms. Genotyping of H. pylori was carried out in all samples (Table 2) by evaluating the presence of VacA (s1 and s2 alleles) gene, and cagPAI genes (cagA, cagL, virB10 and virD4), which distinguish pathogenic or non-pathogenic strains of H. pylori (Fig. S2a). The identification of cagPAI genes and the VacA s1 allele indicates an infection by pathogenic H. pylori strain (Fig. S2b), while the expression of VacA s2 allele and the absence of the expression of cagPAI genes indicated the infection by non-pathogenic H. pylori strain (Fig. S2 c, d). Samples with negative gene expression of bacterial genes were considered as negative to the infection by H. pylori.

Histopathological and genotyping examination of biopsies showed 82.4% of non-active gastritis (71.6% H. pylori positive), 12.2% follicular gastritis (77.7% H. pylori positive), 17.6% active gastritis (100 % H. pylori positive), 27.0% atrophic gastritis (85.0% H. pylori positive) and 5.4% intestinal metaplasia (75% H. pylori positive). Clinicopathologic characteristic of patients are shown in table 2. All samples positive to H. pylori were positive for pathogenic strains, except one sample from gastric body with diagnosis of non-active gastritis, that was positive to a non-pathogenic strain.

When evaluating the relative expression of miR-18a-5p in the different gastric lesions a down-regulation of miR-18a-5p was evidenced during malignancy progression (Fig. 5a). However, in metaplasia samples we observed an up-regulation of miR-18a. These results suggest a loss of expression of miR-18a at the initial premalignant gastric lesion, nevertheless, in advanced malignancy lesions the miR-18a expression might be reestablished to contribute with carcinogenesis. Afterwards, we evaluated the impact of the H. pylori infection on the expression of miR-18a hence we compared its expression among H. pylori-positive and -negative premalignant lesions (Fig. 5b). We observed diminished expression of miR-18a in non-active and follicular gastritis samples positive to H. pylori, however in atrophic gastritis the expression of miR-18b was unchanged. Interestingly, in H. pylori positive metaplasia, the expression of miR-18a was lower than those samples negative for bacterial presence. Moreover, miR-18a was down-regulated in infected tissues compared with non-active gastric tissues uninfected (Fig. 5c) however, the change of expression in infected tissues was unclear (Fig. 5d). These results suggested that miR-18a may differentially regulated in absence or presence of H. pylori.

H. pylori is up-regulating miR-18a in infected AGS cells

To explore the role of H. pylori on the expression of miR-18a-5p we performed co-cultures of H. pylori-AGS cells (Fig. 6a). Infection of AGS cells were evaluated at 6 and 12 hours after co-culturing by quantifying VapD gene expression. We demonstrate that VapD gene was overexpressed in co-cultures indicating that infection was established (Fig. 6b). Surprisingly, the expression of miR-18a-5p was significantly elevated in AGS cells at 12 hours of infection (Fig. 6c). These results suggesting that H. pylori favor the expression of miR-18a in cancer cells.

Gastric carcinogenesis is the result of progressive malignancy of preneoplastic lesions. Multiple factors including H. pylori infection, diet, genetic and epigenetic factors produce the required molecular changes along the multistep gastric carcinogenesis. Nonetheless, the complete mechanisms underlying of phenotypic preneoplastic lesions from gastric epithelia to adenocarcinoma are still unknown. Moreover, the molecular characterization of this multistep gastric carcinogenesis model provides a source for biomarker identification that may predict the risk for GC development. In this regard, miRNAs comply with qualities as biomarkers [25]

Deregulation of some miRNAs occur at the initial stages of cancer development influenced by intrinsic and extrinsic factors [26]. However, the expression and function of miRNAs during progressive malignant transformation from preneoplastic lesions to GC is limited [27–29]. In this work we evaluated the miRNomes derived from preneoplastic lesions encompassing non-active gastritis to GC. We identify eight miRNAs diminished, and seven miRNAs increased progressively along the multistep gastric carcinogenesis. Firstly, we performed an initial identification of miRNAs altered in non-active gastritis compared to normal gastric tissues. Secondly, we evaluated the expression of miRNAs in consecutive lesions including chronic active gastritis, chronic atrophic gastritis, metaplasia, and GC. This approach allowed the identification of miRNAs progressively deregulated from the earliest gastric alterations, which has not been previously reported. Furthermore, identified miRNAs were associated to GC patient’s outcome some of them providing a clear protection of dead. Therefore, identified miRNAs represent a feasible set of diagnostic and prognostic biomarkers.

miRNAs identified in this study have been extensively reported as tumor suppressor miRNAs in GC regulating EMT, metastasis, proliferation, cell cycle, apoptosis and chemoresistance genes including ZEB1, HDGF, Gli1, BCL2, MMP-9 and ROCK1 among others (Fig. 2) [30–46]. All these results suggest that this set of miRNAs may favor gastric carcinogenesis by differentially regulate oncogenes that contribute with hallmarks of cancer.

miRNome analyzed data made not possible to evaluate the positivity to H. pylori, but some of the DEMs have been reported associated to H. pylori infection in gastric premalignant lesions. For instance, miR-146a is overexpressed in H. pylori-induced chronic gastritis and counteract the proinflammatory response of infected cells by targeting IRAK1 and TRAF6 genes [47]. We also detected an overexpression of miR-146a in non-active chronic gastritis which agrees with result reported by Liu et al. (2010). In addition, miR-101 was reported to be significantly downregulated in H. pylori positive gastric tissues, including superficial gastritis, atrophic gastritis, metaplasia, and GC [35]. Interestingly, we detected that miR-101 was also downregulated in non-active gastric, and its expression was substantially downregulated in metaplasia and GC.

When comparing the miRNome of GC to normal epithelial tissues we identified 498 miRNAs deregulated in GC (Table S1), fifteen of them were associated to the progression of gastric lesions. Moreover, five overexpressed miRNAs and one under-expressed miRNA in GC tissues were predictors of OS in GC patients. Additionally, some miRNAs previously reported in GC were identified but not in premalignant lesions indicating a dynamic regulation of DEMs gastric carcinogenesis.

It is worth mentioning that miR-18a-5p was significantly overexpressed in GC patients, and it was associated to better overall survival, a result consistent with that reported by Yuan et al [48]; Furthermore, recently miR-18a-5p was identified in exosomes of serum samples obtained from GC patients [49]. Our results were consistent with other previous reports, therefore miR-18a-5p representing as possible non-invasive biomarker in GC patients. However, we did not identify miR-18a-5p in the preneoplastic lesions analysis, probably due to the quality of the samples or other factors specific to the analyzed population. In this sense, we evaluated the expression of miR-18a-5p in a cohort of Mexican patients diagnosed with non-active, follicular, active, and atrophic gastritis, and metaplasia. Interestingly, the expression of miR-18a diminished in gastritis lesions but increased in metaplasia, suggesting that miR18a could be overregulated in carcinogenic lesions to perform oncogenic functions [14, 48]. Regarding the presence of H. pylori, it has been reported that Mexican population might be infected by different genotypes. In this study, H. pylori positive samples corresponded to the pathogenic genotype strain. The expression of miR-18a-5p was significantly downregulated in premalignant lesions infected with H. pylori suggesting that H. pylori play a key role in the deregulation of miR-18a-5p in preneoplastic lesions and GC [24]. To the best of our knowledge, this is the first study analyzing the role of the infection of pathogenic H. pylori in preneoplastic gastric lesions on the deregulation of miR-18a-5p. Surprisingly, the expression of miR-18a-5p in the co-culture of H. pylori-AGS cells was significantly increased, suggesting that the genetic background of carcinogenic AGS cell could contribute with the regulation of miR-18a-5p. During progressive malignancy, the overexpression of miR-18a could be a biomarker of early gastric carcinogenesis and it could be evaluated as non-invasiveness biomarker in the biological model of GC.

The regulation of miR-18a-5p because of H. pylori infection suggests that H. pylori could induce different signaling pathways in gastric premalignant lesions and gastric malignant cells [14]. In fact, miR-18a-5p has is up-regulated in patients with H. pylori-positive GC [24] supporting the idea that DEMs regulate different carcinogenic processes along gastric transformation.

We identified a set of candidate miRNAs that may be used as diagnostic biomarkers in the progression of gastric carcinogenesis and as prognostic biomarkers in GC patients. H. pylori regulates miR-18a-5p along the different gastric lesions and GC. The mechanistic function of H. pylori on the expression of miR-18a-5p must be further explored.

Acknowledgments

Sergio Ayala-Díaz is recipient of CONACyT fellowship (#758874).

Funding

This work was partially supported by Programa Especial de Apoyo a la Investigación-Rectoría Universidad Autónoma Metropolitana [Number: 47301023].

Competing Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author contributions

Conception and design: SAD, JMM, RME, EAO

Development of methodology: SAD, MFF, JMM, RME, JAPO, ARGN, LSLS, GD, EAO

Acquisition of data (provided animals, acquired, and managed patients, provided facilities, etc.): SAD, JMM, RME, MFF, JAPO, ARGN, LSLS, GD, AGR, DGV, EAO

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): SAD, JMM, RME, MFF, GD, EAO

Writing, review, and/or revision of the manuscript: SAD, JMM, RME, MFF, JAPO, ARGN, LSLS, GD, AGR, DGV, LFO, RHG, EAO

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): SAD, JMM, RME, MFF, JAPO, ARGN, LSLS, GD, EAO

Study supervision: SAD, JMM, RME, EAO

Data Availability

Not applicable.

Ethics approval

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (Hospital Ángel del Mar, Mexico) and with the Helsinki Declaration of 1964 and later versions.

Consent to participate

Informed consent to be included in the study, or the equivalent, was obtained from all patients.

Consent for publication

Informed consent was obtained from each one of the patients for the development of this project, the publication of the article, and the use of clinical images.

Availability of supporting data

The datasets generated during and/or analysed during the current study are not publicly available due to involve personal data from but are available from the corresponding author on reasonable request.

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Table 1 Differentially expressed miRNAs in gastric carcinogenesis multistep from non-active gastritis to gastric adenocarcinoma.

miRNA	LogFC						p value
miRNA	Non-active gastritis		Active gastritis	Atrophy	Metaplasia	Adenocarcinoma	p value
miR-141-3p	Down-regulated	6.37	-0.18	-0.43	-0.51	-2.86	0.042*
miR-873-5p		6.10	-0.47	-0.51	-2.20	-4.92	0.063
miR-429-3p		5.50	-0.14	-0.17	-1.03	-2.55	0.0055*
miR-204-5p		4.87	-0.04	-0.66	-2.13	-6.24	0.093
miR-200a-3p		4.35	-0.38	-0.46	-1.33	-1.87	0.017*
miR-592-5p		3.86	-1.91	-0.57	-0.28	-0.59	0.015*
miR-146a-5p		2.65	-0.94	0.33	1.78	-4.11	0.023*
miR-363-3p		2.19	-0.76	-0.46	-1.92	-2.32	0.00065*
miR-122-5p	Up-regulated	0.89	-0.16	-0.48	-1.69	-0.13	0.028*
miR-196b-5p		-0.83	0.94	0.45	1.73	2.88	0.013*
miR-20b-5p		-1.63	-0.56	-0.59	-1.65	1.62	0.012*
miR-378a-3p		-3.24	-0.58	-0.02	0.19	1.97	0.041*
miR-99a-5p		-3.64	0.19	-0.82	-0.68	3.27	0.0047*
miR-194-5p		-5.03	1.78	1.64	3.81	3.24	0.001*
miR-101-2-3p		-5.58	0.01	-0.17	-1.09	-5.34	0.070

Table 2 Clinicopathologic features of patients

Gender		n	%
	Male	14	37.8
	Female	23	62.2
Mean age	48 years
Age range	14-75 years
Histologic diagnosis		Biopsies (n)		%	H. pylori*
		Body	Antrum		n	%
	Non-active gastritis	22	18	54.1	28	71.6
	Active gastritis	3	5	12.2	8	100
	Active follicular gastritis	1	1	2.7	1	50
	Non-active follicular gastritis	2	5	9.5	6	85.7
	Atrophic gastritis	8	5	16.2	12	92.3
	Atrophic gastritis with metaplasia	1	3	5.4	3	75
Histopathological findings
	Lymphoplasmacytic infiltrate	23	31.1
	Edema in the submucosa	20	27
	Submucosal hemorrhage	19	25.7
	Vacuolization of the epithelium	3	4.1
	No apparent pathological changes	2	2.7
	H. pylori-like microorganism	18	24.3

* VacA s1 genotype

Keymessages.docx
FigureS1.jpg
Fig. S1Gene ontology, biological pathways, and target genes of miRNAs in the progression of premalignant gastric lesions. a Heatmap of KEGG pathways modulated by down-regulated miRNAs, the dendrogram shows the cancer and non-cancer pathways. b Target genes and biological processes regulated by the down-regulated miRNAs in GC. c Heatmap of KEGG pathways modulated by up-regulated miRNAs, the dendrogram shows the cancer and non-cancer pathways. d Target genes and biological processes regulated by the down-regulated miRNAs in GC. Intensity of colors represent the significant levels (p=value). P ≤ 0.05 was considered statistically significant.
FigureS2.jpg
Fig. S2Genotyping of H. pylori of premalignant gastric lesions. a Schematic representation of VacA gene and cagPAI genes in pathogenic H. pylori strain 26695. bRepresentative electrophoretic gel of RT-PCR assays of the non-pathogenic H. pylori genotype s1 from a patient with active gastritis in gastric antrum. cRepresentative electrophoretic gel of RT-PCR assays of the pathogenic H. pylori genotype s2 from a patient with non-active gastritis in gastric antrum. d Representative electrophoretic gel of RT-PCR assays of positive controls. lines: molecular weight (DNA Ladder 100bp, Axygen); VacA s2 gene 286 bp (DNA from H. pylori strain Tx30a); VacA s1 gene 259 bp; CagA gene 153 bp; cagL gene 210 bp; virB10gene 211 bp; virD4 gene 271 bp (DNA from H. pylori strain 26695). Gels were stained with SYBR Green (6X GelRed, Biotium).
TableS1.csv

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Identification of miRNAs progressively deregulated in the biological model of gastric carcinogenesis

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