Prognostic biomarkers and molecular pathways mediating Helicobacter pylori–induced gastric cancer: a network-biology approach

Cancer of the stomach is the second most frequent cancer-related death worldwide. The survival rate of patients with gastric cancer (GC) remains fragile. There is a requirement to discover biomarkers for prognosis approaches. Helicobacter pylori in the stomach is closely associated with the progression of GC. We identified the genes associated with poor/favorable prognosis in H. pylori–induced GC. Multivariate statistical analysis was applied on the Gene Expression Omnibus (GEO) dataset GSE54397 to identify differentially expressed miRNAs (DEMs) in gastric tissues with H. pylori–induced cancer compared with the H. pylori–positive with non-cancerous tissue. A protein interaction map (PIM) was built and subjected to DEMs targets. The enriched pathways and biological processes within the PIM were identified based on substantial clusters. Thereafter, the most critical genes in the PIM were illustrated, and their prognostic impact in GC was investigated. Considering p-value less than 0.01 and |Log2 fold change| as >1, five microRNAs demonstrated significant changes among the two groups. Gene functional analysis revealed that the ubiquitination system, neddylation pathway, and ciliary process are primarily involved in H. pylori–induced GC. Survival analysis illustrated that the overexpression of DOCK4, GNAS, CTGF, TGF-b1, ESR1, SELE, TIMP3, SMARCE1, and TXNIP was associated with poor prognosis, while increased MRPS5 expression was related to a favorable prognosis in GC patients. DOCK4, GNAS, CTGF, TGF-b1, ESR1, SELE, TIMP3, SMARCE1, TXNIP, and MRPS5 may be considered prognostic biomarkers for H. pylori–induced GC. However, experimental validation is necessary in the future.

Designing a novel ELISA method based on CagA, NapA recombinant antigens to increase sensitivity and specificity of Helicobacter pylori whole cell antigen detection

Aim: In this research, we designed a direct Enzyme Linked Immunoassay method to detect Helicobacter pylori antigens in stool specimens

Background: Helicobacter pylori infection as the worldwide problem is related to many gastrointestinal disorders such as gastritis, gastric cancer, non-ulcer disease, peptic ulcer disease and duodenal ulcer

Methods: We produced and purified recombinant CagA and NapA antigens in Escherichia coli and extracted their antibodies from a panel of positive sera specimens. We designed a novel enzyme linked immunoassay direct method in combination with the whole cell for the qualitative and quantitative detection of Helicobacter pylori antigens in human stool. Assay performance was evaluated by histopathology staining and urease activity

Results: The sensitivity and specificity of assay was determined as 91.7 [95% confidence interval: 89.3-95.6%] and 93.1% [95% CI: 91.2-96.4%], respectively. Novel ELISA exhibits enhanced sensitivity and specificity of Helicobacter pylori detection in comparison with another commercially available kit

Conclusion: Combination of the recombinant antigens and whole cell of Helicobacter pylori in immunoassay designing is a new approach about early diagnosis, treatment and fallowing up of the Helicobacter pylori infected patients, especially in peptic cancer cases

Investigation of MicroRNA-21 expression levels in serum and stool as a potential non-invasive biomarker for diagnosis of colorectal cancer

Background: Most cancer studies focus on exploring non-invasive biomarkers for cancer detection. In the present study, we sought to investigate the expression level of microRNA-21 [miR-21], as a potential diagnostic marker, in serum and stool samples from 40 patients with colorectal cancer [CRC] and 40 healthy controls

Methods: Quantitative real-time RT-PCR was applied to determine the relative expression level of miR-21 in serum and stool. At the same time, the sensitivity and specificity of this marker was evaluated by receiver operating characteristic [ROC] curve analysis

Results: miR-21 expression levels of serum and stool were up-regulated 12.1 [P<0.05, 95% CI: 5.774-34.045] and 10.0 [P<0.05, 95% CI: 0.351-16.260] times in CRC patients, respectively, when compared to the control group. The sensitivity and specificity of miR-21 was found to be 86.05% and 72.97%, respectively [an area under the ROC curve [AUC] of 0.783]. The stool miR-21 level in CRC patients was much higher than that in the healthy controls, showing a sensitivity of 86.05% and a specificity of 81.08% [AUC: 0.829]. The expression level of miR-21 in stool was able to significantly distinguish CRC tumor, node, metastasis stages III-IV from stages I-II, with a sensitivity and specificity of 88.1% and 81.6%, respectively [AUC: 0.872]

Conclusion: The results of this study indicated that miR-21 expression levels in serum and stool can be considered as a potential diagnostic biomarker for the diagnosis of CRC patients. However, more studies are required to confirm the validity of miR-21 as a valuable non-invasive diagnostic tool for CRC

MicroRNA-183 Family in Inner Ear: Hair Cell Development and Deafness

miRNAs are essential factors of an extensively conserved post-transcriptional process controlling gene expression at mRNA level. Varoius biological processes such as growth and differentiation are regulated by miRNAs. Web of Science and PubMed databases were searched using the Endnote software for the publications about the role miRNA-183 family in inner ear: hair cell development and deafness published from 2000 to 2016. A triplet of these miRNAs particularly the miR-183 family is highly expressed in vertebrate hair cells, as with some of the peripheral neurosensory cells. Point mutations in one member of this family, miR-96, underlie DFNA50 autosomal deafness in humans and lead to abnormal hair cell development and survival in mice. In zebrafish, overexpression of the miR-183 family induces extra and ectopic hair cells, while knockdown decreases the number of hair cell. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cell in the eye, nose and inner ear. In the inner ear, mechanosensory hair cells have a robust expression level. Despite much similarity of these miRs sequences, small differences lead to distinct targeting of messenger RNAs targets. In the near future, miRNAs are likely to be explored as potential therapeutic agents to repair or regenerate hair cells, cell reprogramming and regenerative medicine applications in animal models because they can simultaneously down-regulate dozens or even hundreds of transcripts.

Peroxisome Proliferator-Activated Receptor-γGene Expression and Its Association with Oxidative Stress in Patients with Metabolic Syndrome / 전남의대학술지

Regulation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene plays an important role in controlling the metabolism of lipids and inflammatory processes. Therefore, it can be associated with the pathogenesis of metabolic syndrome (MetS). The purpose of this study was to determine the expression of this gene in peripheral blood mononuclear cells (PBMC) in patients with metabolic syndrome. Using real-time polymerase chain reaction (PCR), mRNA expression of PPAR-γ was found in PBMC from 37 subjects with MetS and 30 healthy controls. Serum levels of glucose and lipid profiles were measured. The total antioxidant capacity (TAC) was measured using the ferric reducing ability of plasma (FRAP) test. Malondialdehyde (MDA) was determined using a fluorimetric method. Total oxidant status (TOS) in serum was assayed according to oxidation of ferric to ferrous in the presence of methyl orange. Super oxide dismutase (SOD) activity was measured using a Randox kit. Expression of PPAR-γ gene was significantly increased in patients with MetS compared to the control subjects (p=0.002). There was no difference in serum levels of TAC, MDA and SOD between the two study groups, but a significant difference was observed in the TOS (p=0.03). Serum levels of triglycerides and glucose were significantly higher in subjects with MetS. According to the results of our study, an increase in the expression of PPAR-γ in subjects with MetS indicated a possible role of PPAR-γ in the pathogenesis of this disease.

Peroxisome Proliferator-Activated Receptor-γGene Expression and Its Association with Oxidative Stress in Patients with Metabolic Syndrome / 전남의대학술지

Regulation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) gene plays an important role in controlling the metabolism of lipids and inflammatory processes. Therefore, it can be associated with the pathogenesis of metabolic syndrome (MetS). The purpose of this study was to determine the expression of this gene in peripheral blood mononuclear cells (PBMC) in patients with metabolic syndrome. Using real-time polymerase chain reaction (PCR), mRNA expression of PPAR-γ was found in PBMC from 37 subjects with MetS and 30 healthy controls. Serum levels of glucose and lipid profiles were measured. The total antioxidant capacity (TAC) was measured using the ferric reducing ability of plasma (FRAP) test. Malondialdehyde (MDA) was determined using a fluorimetric method. Total oxidant status (TOS) in serum was assayed according to oxidation of ferric to ferrous in the presence of methyl orange. Super oxide dismutase (SOD) activity was measured using a Randox kit. Expression of PPAR-γ gene was significantly increased in patients with MetS compared to the control subjects (p=0.002). There was no difference in serum levels of TAC, MDA and SOD between the two study groups, but a significant difference was observed in the TOS (p=0.03). Serum levels of triglycerides and glucose were significantly higher in subjects with MetS. According to the results of our study, an increase in the expression of PPAR-γ in subjects with MetS indicated a possible role of PPAR-γ in the pathogenesis of this disease.

Circulating Betatrophin Levels Are Associated with the Lipid Profile in Type 2 Diabetes / 전남의대학술지

Betatrophin is a newly characterized circulating hormone that is produced in tissues such as adipose tissue and liver and stimulates pancreatic beta-cell proliferation. The purpose of the current study was to examine circulating betatrophin levels in Iranian patients with type 2 diabetes mellitus (T2DM) and in normal controls. Seventy-five subjects were enrolled in this case-control study in the following two groups: T2DM patients (n=40) and a group of age-, sex-, and BMI-matched normal control subjects (n=35). Circulating betatrophin concentrations as well as the blood lipid profile, body mass index (BMI), fasting blood sugar (FBS), glycated hemoglobin (HbA1c), and insulin resistance were determined. Circulating betatrophin levels were significantly higher in patients with T2DM than in the normal subjects (4.79+/-1.53 ng/mL vs. 2.79+/-1.11 ng/mL respectively; p=0.001). Serum triacylglycerol and total cholesterol were also significantly higher in patients with T2DM than in the control group. In the patients with T2DM, serum betatrophin was positively correlated with age, FBS, TG, total cholesterol, and HbA1c. The results of this initial study in Iran have shown that circulating betatrophin levels are significantly increased in Iranian patients with T2DM compared with a control group. Additionally, it is postulated that betatrophin as a novel hormone may be involved in the generation of an atherogenic lipid profile.

Circulating Betatrophin Levels Are Associated with the Lipid Profile in Type 2 Diabetes / 전남의대학술지

Betatrophin is a newly characterized circulating hormone that is produced in tissues such as adipose tissue and liver and stimulates pancreatic beta-cell proliferation. The purpose of the current study was to examine circulating betatrophin levels in Iranian patients with type 2 diabetes mellitus (T2DM) and in normal controls. Seventy-five subjects were enrolled in this case-control study in the following two groups: T2DM patients (n=40) and a group of age-, sex-, and BMI-matched normal control subjects (n=35). Circulating betatrophin concentrations as well as the blood lipid profile, body mass index (BMI), fasting blood sugar (FBS), glycated hemoglobin (HbA1c), and insulin resistance were determined. Circulating betatrophin levels were significantly higher in patients with T2DM than in the normal subjects (4.79+/-1.53 ng/mL vs. 2.79+/-1.11 ng/mL respectively; p=0.001). Serum triacylglycerol and total cholesterol were also significantly higher in patients with T2DM than in the control group. In the patients with T2DM, serum betatrophin was positively correlated with age, FBS, TG, total cholesterol, and HbA1c. The results of this initial study in Iran have shown that circulating betatrophin levels are significantly increased in Iranian patients with T2DM compared with a control group. Additionally, it is postulated that betatrophin as a novel hormone may be involved in the generation of an atherogenic lipid profile.

Expression of two basic mRNA biomarkers in peripheral blood of patients with non-small cell lung cancer detected by real-time RT-PCR,individually and simultaneously

Although extensive research has been conducted on lung cancer markers, a singular clinically applicable marker has not been found yet. The objective of this study was to evaluate the sensitivity and the specificity of carcinoembryonic antigen [CEA] mRNA and lung-specific X protein [LUNX] mRNA biomarkers in peripheral blood to detect lung cancer individually and simultaneously.Thirty patients affected by lung cancer and 30 healthy individuals were studied in this research. Three vials of cDNA were made from each sample after taking peripheral blood samples and extracting total RNA. Each sample was examined by the real-time RTPCR technique. The result from each vial was then compared with the sensitivity of overall marker. The CEA mRNA was positive in 24 out of 30 lung cancer patients. Hence, its sensitivity was determined at 80%,differing significantly from that observed in healthy individuals, where 11 positive cases were seen. The overall sensitivity of this marker was significantly associated with positivity in vials 2 and 3 but not in vial 1 The LUNX mRNA was positive in 21 out of 30 patients, indicating 70% sensitivity. This finding significantly differed from that in healthy individuals. The overall sensitivity of this marker was significantly associated with positivity in vials 1 and 3, but not in vial 2. In 93.3% of the patients, at least one positive marker was observed.The mentioned mRNA could be suggested as sensitive and specific markers in peripheral blood for primary diagnosis of lung cancer.

Simultaneous analysis of multidrug resistance 1[MDR1] C3435T, G2677T/A, and C1236T genotypes in Hamadan city population, west of Iran

One of the limitations in the treatment of common diseases such as cancer chemotherapy is development of multidrug resistance [MDR]. Polymorphisms could alter the expression level of MDR1 gene, which plays an important role in MDR. In this research, the frequency of C3435T, C1236T, and G2677T/A polymorphisms of MDR1 gene was investigated in a large group of population from Hamadan city to provide a sample data resource.Peripheral blood [2 ml] was taken, and DNA extraction was carried out. Multiplexed mutagenically separated PCR, which was followed by polyacrylamide gel electrophoresis and silver staining, was applied to detect the mentioned polymorphisms in 935 individuals. Sequencing performed for confirmation of gel electrophoresis resulted in 10 random cases. In total, alleles and genotypes of 933 persons [776 women and 157 men] were determined. The most frequent alleles of the polymorphisms were: 3435T, C1236, and G2677. The most frequent genotypes were: 3435C/T, 1236C/T, and 2677G/A, and their concurrent presence was also found as the most frequent simultaneous genotypes. There was not any meaningful difference among the prevalence of these genotypes in groups of men and women. Our results were close to those of other studies performed in Iran and compared to the other ethnic groups, which showed more similarity to Asian peoples than Europeans. As an aspect of personalized medicine, it could be used by chemotherapists to improve the routine methods of cancer treatment.

Expression analysis of mir-21 and mir-221 in cancerous tissues from Iranian patients with gastric cancer

Early detection is a key to survival for gastric cancer. Molecular markers such as miRNA [microRNA] can have great importance in the early diagnosis of gastric cancer. Expression of miR-21 and miR-221 are deregulated in many types of human cancers. This study aimed to investigate the differences in miRNA expression patterns within the Iranian population. Total RNA was extracted from gastric cancer tissues and adjacent non-cancerous tissues from 32 patients. Expression levels of miR-21 and miR-221 were detected by Real time RT-PCR using a specific primer, with 5s rRNA as the internal reference gene. Our data showed that the expression levels of miR-21 and miR-221 in gastric cancer samples were significantly higher than in paired non-cancerous samples [P < 0.05]. The receiver operating characteristic [ROC] analyses yielded the area under the curve [AUC] values of 80.30 for miR-21 and 93.30 for miR-221, and combined ROC analysis revealed the highest AUC value of 96.90 in discriminating GC patients from healthy controls. It seems that miR-21 and miR-221 expression pattern in Iranian patients with gastric cancer are similar to any other population. Considering the increased expression level of two miRNAs in cancerous tissue compared to normal tissue as well as the area under ROC curve, miR-21 and miR-221 can be used for early detection of gastric cancer

Effect of myomectomy on endometrial glutathione peroxidase 3 [GPx3] and glycodelin mRNA expression at the time of the implantation window

In fertile women, glycodelin and glutathione peroxidase 3 [GPx3] genes expression rises during the luteal phase, with a peak occurring during the implantation window. The expression of these genes decreases in women with myomas. To determine whether myomectomy would reverse glycodelin and GPx3 expression, we evaluated the transcript levels of these genes in the endometrium of patients before and after myomectomy. Expression of glycodelin and GPx3 genes were examined prospectively during the midluteal phase in the endometrium obtained from infertile women with myoma [n = 12] before and three months after myomectomy. Endometrial expression of these genes was evaluated using quantitative real-time RT-PCR. Endometrial glycodelin mRNA expression levels [normalized to 18S rRNA expression] were increased significantly in endometrium of patients after myomectomy [P = 0.02]. GPx3 mRNA expression was increased insignificantly after myomectomy [P = 0.43]. The results showed that myomectomy increased endometrial glycodelin [significantly] and GPx3 [not significantly] gene expression after 3 months. Study at different times and detecting expression of these genes can reveal more details

Improved real-time RT-PCR assays of two colorectal cancer peripheral blood mRNA biomarkers: a pilot study

Background: efficient screening for detection of colorectal cancer [CRC] at earlier stages reduces its mortality. The purpose of this study was to investigate expression of carcinoembryonic antigen [CEA] and human telomerase reverse transcriptase [hTERT] mRNA in peripheral blood of CRC patients and to present strategies for early detection screen test

Methods: twenty seven patients in non-metastatic stage and 27 healthy individuals were studied. Expression of CEA, hTERT mRNA and 18srRNA [18s subunit of ribosomal RNA, as reference gene] were determined based on real-time RT-PCR on 3 [micro]g of total RNA from blood in 3 separate vials [1 [micro]g per vial]

Results: positive expression rate of CEA mRNA [78%] and hTERT mRNA [81%] were higher in patient group [P<0.001]. These rates were meaningfully higher than the results of individual vials containing only 1 [micro]g of total RNA. Difference between Ct values of markers with 18srRNA [[DELTA]Ct] was higher in healthy group than patient one. Therefore, a [DELTA]Ct cut-off value was determined for distinguishing between true- and false-positive results. Concurrent expression of both markers was found in 67% of the patients, which was higher than healthy cases [11%]. Combination of concurrent marker expression with cut-off point strategy increased specificity to 100%

Conclusion: these results showed that concurrent evaluation of marker expression and performing the test on 3 [micro]g of samples in 3 separate vials may increase specificity and sensitivity of real-time RT-PCR for early detection of non-metastatic CRC. However, more investigations with larger numbers of samples are needed to verify these results. Iran. Biomed. J. 17 [1]: 15-21, 2013

High prevalence of multidrug resistance and metallo-beta-lactamase [MbetaL] producing acinetobacter baumannii isolated from patients in ICU wards, Hamadan, Iran

Acinetobacter baumannii is gram- negative opportunistic coccobacilli, the most important agent in nosocomial infections with high mortality rate. Multidrug resistance in strains isolated from nosocomial infections, making it difficult to treat and sometimes impossible. The aim of the present study was to investigate antibiotic resistance in A. baumannii isolates from Iranian patients in Hamadan, west of Iran. In this cross sectional study 100 A. baumannii isolated from trachea, blood, urine, sputum and wound samples of patients bedridden in Intensive care unit [ICU] wards of three educational hospitals during June 2011 to October 2012 was included. Isolates confirmed at species level using biochemical tests and tracing bla[OXA-51] gene using Polymerase chain reaction [PCR] and preserved frozen at -70 °C until examination. Their susceptibility to 17 antibiotics was performed using Kirby-Bauer disc diffusion method. Determination of minimum inhibitory concentration and Metallo-beta-lactamase production was carried out using E-test method. Resistance rate of isolates were 94%, 85%, 84%, 97%, 95% and 98% against meropenem, imipenem, amikacin, ciprofloxacin, piperacillin/tazobactam and cefotaxime, respectively. No resistant isolate was observed against tigecycline and also no sensitive isolate seen against aztreonam and cefotaxime. Results of E-test illustrated that 99% of all isolates were Metallo-beta-lactamase [MbetaL] producing, which were resistance to imipenem; also 85% of them were resistance to meropenem. MIC50 and MIC90 of the isolates were >/= 256 and >/= 32 micro/ml for imipenem and meropenem, respectively. The antibiotic resistance against most of the antibiotics, especially carbapenems is very high in Hamadan region. In addition colistin sulfate and tigecycline were most effective antibiotics and to be used in A. baumannii infections

Effect of intramural myomectomy on endometrial HOXA10 and HOXA11 mRNA expression at the time of implantation window

HOXA11 and HOXA10 are expressed in endometrium throughout the menstrual cycle and show a dramatic increase during the mid-luteal phase at the time of implantation. The expression of these genes is decreased in women with myomas. To determine whether myomectomy would reverse HOXA11 and HOXA10 expression, we evaluated the transcript levels of these genes in the endometria of patients before and after myomectomy. Expression of HOXA11 and HOXA10 were examined prospectively during the midluteal phase in endometrium obtained from infertile women [n=12] with myoma before and three months after myomectomy. Endometrial HOXA11 and HOXA10 expression were evaluated using quantitative real-time reverse transcriptase-polymerase chain reaction [RT-PCR]. Endometrial HOXA11 and HOXA10 mRNAs expression levels [normalized to 18SrRNA] were increased insignificantly in endometrium of patients after myomectomy [p=0.7 and p=0.15 respectively]. The results suggest that the alteration in expression pattern of these genes could not account for some aspects of fertility after myomectomy

Semi-quantitative analysis of HOXA11, leukemia inhibitory factor and basic transcriptional element binding protein 1 mRNA expression in the mid-secretory endometrium of patients with endometriosis

HOXA11 and leukemia inhibitory factor [LIF] and basic transcriptional element binding protein1 [BTEB1] are expressed in endometrium throughout the menstrual cycle and show a dramatic increase during the mid-luteal phase at the time of implantation. In this case-control study, the expression pattern of these mRNA was evaluated in patients with endometriosis at the time of implantation. We also describe a semi-quantitative RT-PCR protocol optimized in our laboratory. Eight patients with endometriosis were considered as our case and 8 fertile women as control group. Expression levels of HOXA11, LIF and BTEB1 mRNA were measured in endometrium during the mid-secretory phase using semi-quantitative RT-PCR. We describe the detailed procedure for the analysis of HOXA11, LIF and BTEB1 mRNA levels. Endometrial HOXA11 and LIF mRNA expression levels [normalized to beta -actin expression] were significantly decreased in endometrium of infertile patients with endometriosis compared with healthy fertile controls at the time of implantation [P<0.05]. A similar trend was seen in BTEB1 mRNA expression. The results suggest that the alteration in expression pattern of the some genes could account for some aspects of infertility in endometriosis