In the case of miR-21, no statistically significant differences between Barrett's esophagus and normal epithelia were acquired (see Figure ). Refering to miR-192, there is a statistically significant upregulation of miR-192 expression in any grade of the disease with the p < 0,00001 (see Figure ). miR-196a expression shows the upregulation as well with p < 0,05 (see Figure ). On the other hand, miR-203 is definitely downregulated in the impaired tissue with the p < 0,00001 (see Figure ). In the case of miR-196a, there is a statistically significant (p < 0,005) trend in relative expression dependent on the disease grading (see Figure ). The results acquired from the analysis of disease grading did not confirm the dependency on sex, hiatal hernia, obesity, smoking, alcohol abuse, family anamnesis of any cancer, each of this risk factor being estimated separately (Pearsons Chi-Quadrat Test with Monte Carlo Simulations). Kruskal-Wallis Chi-Quadrat Test did not confirm the correlation of the grading versus age of patients as well as it did not confirm the correlation of the grading versus alcohol and smoking (every risk factor considered separately). However, correlation of grading versus hiatal hernia, obesity, smoking, alcohol abuse, family anamnesis (all parameters considered together) has confirmed that there are statistically significant parameters for alcohol (p < 0,046) and smoking (p < 0,065). Further, the correlation of grading to all the previous parameters plus sex and age shows statistically significant values of the influence of sex (p < 0,008), age (p < 0,065), smoking (p < 0,02), and alcohol abuse (p < 0,0045).
miRNA-21 expression in normal healthy esophagus vs Barrett's metaplasia. No statistically significant differences in miR-21 expression were observed.
miRNA-192 statistically significant upregulation in BE patients.
miRNA-196a upregulation in BE patients is statistically significant.
Statistically significant downregulation of miR-203 in BE patients correlated to normal healthy esophageal tissue.
Rising miRNA-196a upregulation in BE patients in NBI detected BE(1), BE(2), low-grade dysplasia(3), high-grade dysplasia/adenocarcinoma(4).
It is supposed that Barrett's metaplasia arises on the basis of chronic inflammation [34
] which originates as a reaction to the gastroesophageal reflux disease (GERD). GERD occurs when the acid content of the stomach returns to the distal part of the esophagus. The continuous irritation of the esophageal squamous stratified nonkeratinized epithelium induces its replacement by the columnar one which is less mechanically resistant but it resists to the low pH of the esophageal microenvironment. Connection between BE and GERD was well established in the 1970s [6
]. However, when there is GERD in the esophagus, BE must not be necessary present. GERD is not the only risk factor for development of BE. It is the presence of hiatal hernia, obesity (visceral fat), smoking or alcohol abuse as well. Moreover, men are three times more in danger than women. O'Riordan et al. [34
] demonstrated that in the inflammation - BE - EAC sequence the levels of proinflammatory cytokines IL-8 and IL-1β increase. Thus, interleukins seem to be one possible target of the therapeutic intervention in the future. Another possible target of BE therapy could be the microRNAs - short single-stranded molecules with high potential of gene regulation.
It is well known that many microRNAs are deregulated in the connection with malignant progression. Changes in microRNA-196a expression are cited in connection with both pancreatic adenocarcinoma and breast carcinoma as well as in Barrett's esophagus. In combination with another miRNA (miR-217), miR-196a level can help distinguish between malignant and benign pancreatic tissue [37
]. Moreover, in long-term survival adenocarcinoma patients (24 months), the levels of miR-196a are conversely correlated with survival [38
]. As well as Mathé et al. [21
], we also revealed that microRNA-196a is definitely overexpressed in Barrett's esophagus of any grade compared to normal healthy esophageal tissue, even the statistical analysis shows that the trend of the rising expression of this microRNA during the BE progression does exist. Moreover, compared to [20
] (11 FFPE patients), we demonstrated this fact in our study with 71 patients in 4 gradings of BE.
In the case of miR-21, it was published, that six solid tumor tissues (lung, breast, stomach, prostate, colon, pancreas) where miR-21 is the only overexpressed microRNA do exist [39
]. Feber et al. [19
] describes the overexpresson of miR-21 and downexpression of miR-203 in his experiments with Barrett's esophagus as well as with esophageal adenocarcinoma. In the paper from Saini et al. [40
], microRNA-203 is described as a anti-metastatic small RNA with the possible therapeutic intervention. Our results suggest the unequivocal miR-203 downregulation in BE, but non-unequivocal miR-21 expression. Compared to our study, Feber performed his experiments for 35 frozen specimens without the microdissection step. The same conclusion (for miR-21 and miR-203) can be find in the paper from Mathé et al. [22
] who compared miRNA expression profiles of esophageal adenocarcinoma and squamous cell carcinoma to the adjacent noncancerous tissue pairs. In addition, Mathé et al. [22
] describe the elevation of levels of miR-192 in esophageal adenocarcinoma. In our set of patients, we have come to the same conclusion.
Dealing with the possibility of getting the tissue samples through all stages of BE progression in one individuality brings controversial situation. We succeed in obtaining of tissue samples from all stages of BE progression in one patient's case, however we have to point out that the NBI technique, which we are using in the endoscopic examination helps to vizualize the architecture of the BE lesion and gives us the most precise picture of the stage of potential dysplasia and if there are any indications of proliferation to higher stage of BE dysplasia, it is therapeutically intervened. Our aim is not to let the patient to escalate to HGD/EAC stage which is very difficult to cure.
Most of the gene expression studies cited deal with relative expression data, but not all of them deal with the endogenous controls. Nonn et al. [28
] use the normalization of the results to two chosen small nucleolar RNA RNU44 and RNU48, Maru et al. [21
] use miRNA-16 as the reference gene. In his Application Note (Applied Biosystems/Life Technologies; Carlsbad, CA, USA) Wong et al., 2007 describe all the applicable endogenous controls suitable for microRNA data normalization, however with the necessity of verification. That's why in this paper six possible endogenous controls were chosen and the most stable couple of them was chosen for relative expression data calculation.