Effects of alkaline water intake on gastritis and miRNA expression (miR-7, miR-155, miR-135b and miR-29c)

Background : It is known that abnormal expression of miRNAs in the gastric cancer (GC) contributes to its carcinogenesis. Therefore, ingestion of commercial (usual) water on a daily basis may be a contributing factor for the occurrence of alterations in the gastric mucosal. In this study, it was evaluated the expression of the miRNAs miR-29c, miR-7, miR-155, and miR-135b in the gastric tissue of patients with gastritis before and after the consumption of alkaline water (pH range from 8.0 to 10.0), as well as the clinic pathological characteristics. Methods : 50 subjects from the Amazon region, diagnosed with gastritis that routinely used commercial (usual) water with a lower than were to the to a to 10.0 for Results : Endoscopic ndings of gastritis were such different (less severe disease), p = 0.024; in 43% diagnosed with moderate gastritis upfront esophagogastroduodenoscopy (EGD) presented mild gastritis after the consumption of alkaline water, according to study methods; there were no worsening gastritis and there were a signicant increase in the expression of miR-135b (p = 0.039) and miR-29c (p = 0.039). Conclusions : Modied pH range water (from 8.0 to 10.0) ingested for 5 months was able lead to a less severe gastritis according to the Sidney classication system, suggesting that this lifestyle change represented a clinical benet in patients with gastritis on the Amazon region. In addition, higher expression of miR-135b and miR-29c was observed after the consumption of alkaline water for 5 months.


Abstract
Background : It is known that abnormal expression of miRNAs in the gastric cancer (GC) contributes to its carcinogenesis. Therefore, ingestion of commercial (usual) water on a daily basis may be a contributing factor for the occurrence of alterations in the gastric mucosal. In this study, it was evaluated the expression of the miRNAs miR-29c, miR-7, miR-155, and miR-135b in the gastric tissue of patients with gastritis before and after the consumption of alkaline water (pH range from 8.0 to 10.0), as well as the clinic pathological characteristics.
Methods : 50 subjects from the Amazon region, diagnosed with gastritis that routinely used commercial (usual) water with a pH lower than 5.0, were enrolled to change the consume water to a pH of 8.5 to 10.0 for 5 months.
Results : Endoscopic ndings of gastritis were such different (less severe disease), p = 0.024; in 43% diagnosed with moderate gastritis upfront esophagogastroduodenoscopy (EGD) presented mild gastritis after the consumption of alkaline water, according to study methods; there were no worsening gastritis and there were a signi cant increase in the expression of miR-135b (p = 0.039) and miR-29c (p = 0.039).
Conclusions : Modi ed pH range water (from 8.0 to 10.0) ingested for 5 months was able lead to a less severe gastritis according to the Sidney classi cation system, suggesting that this lifestyle change represented a clinical bene t in patients with gastritis on the Amazon region. In addition, higher expression of miR-135b and miR-29c was observed after the consumption of alkaline water for 5 months.

Background
Gastric cancer (GC) is the fth most common malignant neoplasm worldwide and accounts for 8.2% of all cancer deaths (1,2). In northern Brazil, it is the second most common cancer in men and fth in women and is a public health problem in the state of Pará (3).
Gastric carcinoma is the most common cancer type in the stomach, and the intestinal subtype is the most frequent (4). It develops in multiple stages: normal mucosa, chronic gastritis, gastric atrophy, intestinal metaplasia, dysplasia and malignant neoplasia (4). These conditions are usually sequential and occur over a period of many years as a result of the interaction between a variety of endogenous and exogenous factors (4).
The association between GC and diet is well established in the literature (5). The characteristic diet of Pará, e.g., high intake of salty foods (jerky, sh, and shrimp), low intake of greens and vegetables and high consumption of glucose ( our), has contributed to the high incidence of GC in the region (6).
The cellular transformation characteristics of gastric carcinogenesis may take time to present; however, epigenetic transformations, such as DNA methylation, histone changes and miRNA expression changes (7), can be identi ed early and are more likely to be in uenced by environmental factors (smoking, sun exposure, in ammation and pollutants, and diet) (8).
Previous studies have shown that the abnormal expression of several miRNAs contribute to the carcinogenesis and progression of neoplasms, including GC, and may also be useful as predictors of drug resistance and as prognostic factors (8)(9)(10). The epigenetic targets chosen in the present study (miR-29c, miR-7, miR-155, miR-135b) are dysregulated in GC and altered in tissues with in ammation, as occurs in gastritis, and are thus potential biomarkers for the carcinogenesis of GC (10)(11)(12)(13).
In the North region of Brazil, the waters sold have a low pH, ranging from 3.0 to 5.0, and are therefore outside the standards required by the World Health Organization and the Brazilian Ministry of Health (14,15). In addition, in this region, there is also a high incidence of gastritis and GC, above the national average (3). Thus, water pH may be a contributing factor for the occurrence of these gastric issues, and therefore, its evaluation is extremely important for understanding the high incidence rates of this cancer in the region. Mousa (16). Furthermore, it was observed that electrolyzed alkaline water has antiin ammatory properties because of its ability to reduce the expression of TNF-α in the gastric mucosaNaito (17).
In addition, a study showed the ability of water with a pH of 8.8 to inactivate pepsin. This irreversible inactivation of pepsin in this alkaline medium can generate a possible bene t of an alkalizing diet for the treatment of acid re ux because this enzyme is related to protein lysis and is activated when pepsinogen is in acidic medium (18).
Thus, the objective of this study was to evaluate the expression of the miRNAs miR-29c, miR-7, miR-155, and miR-135b in the gastric tissue of patients with gastritis before and after the consumption of alkaline water to understand how changes in consumption may affect the modulation of the expression of these microRNAs as well as the clinicopathological characteristics of the patients.

Methods
A total of 50 individuals, positive and negative for Helicobacter pylori, were recruited by nonprobabilistic and convenience sampling. This study was approved by a research ethics committee under protocol 2.033.180.
Individuals older than 35 years, diagnosed with gastritis by anatomopathological examination, were included. The patients excluded were those with a prior or familial history of GC, those who were using alkaline water and/or antacids, those who had endoscopic criteria for immediate eradication of H. pylori (past and present gastric and/or duodenal ulcers with or without complications), or those who had gastric MALT lymphoma or atrophic gastritis.
The individuals recruited routinely used water with a pH lower than 5.0 and began to consume water with a pH of 8.5 to 10.0 voluntarily for a median of 5 months. These lters provided pretreated, ltered, puri ed, ionized and alkaline water with negative potential renal acid load (PRAL), obtained using a water ionizer. Table 1 shows the composition of the alkaline water provided to the participants. Table 1 Composition and chemical characteristics of the alkaline water used in the study. After this period, all the individuals underwent a new esophagogastroduodenoscopy (EGD) during which the anatomopathological parameters and the proposed outcomes were again evaluated.
For all participants, miR-155, miR-7, miR-29c and miR-135b expression was evaluated before and after the study period (median of 5 months). Total RNA extraction was performed using an mirVana miRNA isolation kit (Ambion, Texas, USA) according to the manufacturer's instructions. RNA concentrations were determined using a SpectraMax i3 spectrophotometer (Molecular Devices, Sunnyvale, California, USA) with a concentration of 10 ng/µL as the standard, and RNA integrity was visualized in an agarose gel.
To detect miRNA expression, reverse transcription was performed using total RNA samples (TaqMan MicroRNA Reverse Transcription Kit, Life Technologies, Foster City, CA), followed by real-time PCR with commercially available primers and probes (TaqMan MicroRNA Assays, Life Technologies, Foster City, CA). The endogenous miRNAs used for standardization were RNU6B, miR-1403p and miR-101, which were selected after analysis of data available in the literature (19)(20)(21). After analyzing the results in NormFinder software (22), RNU6B and miR-1403p were the endogens selected to calculate differential expression.
All assays followed the manufacturer's recommendations and were performed in quadruplicate, according to the following thermocycling program (95 °C for 10' and 40 cycles of 95 °C for 15'' and 60 °C for 1'). Gene expression was evaluated using the comparative CT method (-2 ΔΔCT ).
Identi cation of H. pylori bacteria was performed both by histological analysis and by real-time PCR through ampli cation of the bacterial 16SRNA region (23,24). The sample was considered infected when positive in any of the methodologies.
For the statistical analysis, clinical-epidemiological qualitative variables are presented as absolute and relative frequencies. The quantitative variable "age" (Shapiro-Wilks < 0.05) is presented as the median and 25% -75% percentiles. Fisher's exact test was used to assess the correlation between categorical variables.
The difference in microRNA expression between the 2 groups (before and after alkaline water intake) was determined by the Wilcox test for all samples (Stata 11.0 and BioStat 5.3). Two-tailed p < 0.05 was considered statistically signi cant.

Results
A total of 50 individuals, positive and negative for H. pylori, were recruited between May 2017 and February 2018 after con rmation of gastritis by EGD to evaluate the effects of changing the type of water consumed from water with an acidic pH (pH less than 5) to water with an alkaline pH (pH between 8.5 and 10). Of these, 28 completed all steps of the study with the use of alkaline water for the period established and then performed a new EGD.
The absolute and relative frequencies of qualitative variables for the data collected before and after the consumption of alkaline water are shown in Tables 2 (provided in the Supplemental section) and 3, respectively.   Figure 1 shows a box plot comparing the expression levels of miR-135b (p = 0.039) and miR-29c (p = 0.039) before and after the median period of 5 months of alkaline water consumption. The expression levels were higher after the consumption of alkaline water. There was no signi cant increase in miR-155 and mir-7 expression (Fig. 1).
The FoldChange analysis of the differences between 2 −ΔΔCT , before and after the consumption of alkaline water for a median of 5 months, of the target microRNAs showed values greater than 1 for every miRNA (Fig. 2).

Discussion
Currently, there is a wide discussion about the effects of the pH of drinking water and its consequences on human health, such as its relationship with in ammation and cancer (25). However, there is little scienti c evidence supporting this association.
In the present study, we found a signi cant increase in the expression of 2 microRNAs, miR-29c and miR-135b, after the consumption of alkaline water for 5 consecutive months, as well as an improvement in gastritis as evaluated by a second EGD; i.e., 42% of the patients who had moderate gastritis in the rst EGD had mild gastritis in the second EGD, according to the Sydney classi cation system. It is also worth noting that the patients did not undergo pharmacological treatment for gastritis during the intervention period and were not instructed to change their diet during the period.
Gastritis is considered a preneoplastic condition, as it participates in gastric carcinogenesis in the early stages of the process, followed by gastric atrophy, ulcerations, intestinal metaplasia, dysplasia and, nally, malignant neoplasia (4,26,27). Thus, the improvement in gastritis observed in the present study with the use of alkaline water may represent a protective factor of in ammation in the gastric mucosa.
Thus, this simple lifestyle change may act positively in the early stages of the carcinogenesis cascade of GC.
Proton pump inhibitors (PPIs) are among the most commonly prescribed drugs in the world; however, although they are generally considered safe, they have several adverse effects associated with prolonged use (28,29). These medications are associated with an increased risk of the development of premalignant lesions (fundic gland polyps, worsening of gastric atrophy and metaplasia) and GC, particularly in individuals infected with H. pylori, especially when PPIs are used for over one year (28)(29)(30). The use of alkaline water does not compare with the mechanism of action of PPIs because there is no inhibition of acid secretion but, rather, a reduction in exposure to acidic substances of a mucosa that is damaged by gastritis (reference). miR-29c is commonly described as a tumor suppressor because it inhibits the proliferation, invasion and migration of malignant cells. Decreased expression of this microRNA has been reported in several human malignancies, such as pancreatic cancer (31), breast cancer (32), prostate cancer (33), hepatocellular cancer (34), nasopharyngeal cancer (35) and GC (10,36).
The miR-29c expression levels were signi cantly decreased (p < 0.001) in GC tissues compared to normal tissue (37). In addition, it has been demonstrated by in vitro and in vivo assays that the increased expression of this miRNA suppressed tumor growth through the downregulation of ITGB1 (β1 integrin, also known as CD29) (37).
As previously described, miR-29c expression levels gradually decrease as gastric carcinogenesis progresses at different stages of mucosal aggression, demonstrating a direct correlation between the loss of mir-29c expression and the tumorigenesis of this cancer (10). Thus, in our study, the increased expression of this miRNA after the consumption of alkaline water may suggest that its frequent use may function as a modifying factor of the epigenetic environment of these tissues, providing an environment with lower chances of carcinogenic progression. Transfection of the miR-7 precursor into AZ521 and Kato III GC cells signi cantly inhibited cell proliferation capacity (13).
miR-135b has been described in the literature as an oncomiR in most tumor tissues, such as colon cancer (38), lung cancer (39), oral cancer (40) and breast cancer (41), but few studies have evaluated its role in gastric tissue. Vidal, Cruz (10) observed that miR-135b expression was increased in gastric lesions when compared with normal gastric mucosa but was decreased when gastritis tissue was compared with cancer tissue; thus, its role in the various stages of gastric carcinogenesis remains uncertain.
miR-135b has been described as a tumor suppressor in osteosarcoma, and the MYC gene, which is related to the progression of several tumors, is its direct target (42). That role is similar to that observed for prostate cancer, for which Wang, Tao (43) observed a decrease in miR-135b expression in prostate cancer tissues compared to normal tissues, acting as a tumor suppressor by inactivating the STAT6 pathway, known as oncogenic in several tumors. Thus, as the signaling pathways and activity of this miRNA are not yet certain, we cannot a rm or suggest protective or deleterious effects generated by increased miR-135b expression after alkaline water consumption.

Conclusions
The present study evaluated the effect of a patient lifestyle intervention on the rst abnormal stage of the gastric carcinogenesis cascade, gastritis, and it was observed that the pH change in the water ingested for 5 months was able to improve gastritis according to the Sidney classi cation system, suggesting that this lifestyle change represented a clinical bene t in patients with gastritis. In addition, higher expression of miR-135b (p = 0.039) and miR-29c (p = 0.039) was observed after the consumption of alkaline water for 5 months.
Thus, the present study generates preliminary hypotheses and provides initial information about the ideal pH for regularly consumed beverages, such as water, and can serve as a basis for larger, randomized studies.

Abbreviations
Gastric cancer (GC) Negative potential renal acid load (PRAL) Declarations Ethics approval and consent to participate: This study was approved by a research ethics committee under protocol 2.033.180, at the ethics committee of Federal University of Pará. The informed consent of the study participants obtained was written (the model of the document is attached, as supplementary material 2).

Consent for publication: Not applicable
Availability of data and materials: All data generated or analysed during this study are included in this published article [and its supplementary information les] Competing interests: The authors declare that they have no competing interests Funding: We declare this study was sponsored by the company Acqualive, that provides water alkalization lters.
Authors' contributions: JRC idealized the theme, wrote the project, collected the data and wrote the nal results of the work. CRTS, AACM, FCM and EBT helped in data analysis and nal writing of the work. JSS helped in the writing of the project, in the data analysis and in the nal writing of the work. TSRA helped in the initial writing of the project, data collection and tabulation, in the statistical analysis and writing of the nal results of the work. TMTA and ASK helped in the initial design of the project, corrections, statistical analysis and nal writing of the project. All authors read and approved the nal manuscript. Figure 1 Expression of miRNAs miR-155, miR135, miR29c and miR7 before and after the consumption of alkaline water for a median of 5 months ( rst and second EGD).