The diagnostic potential of miR-196a-1 in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a common malignancy worldwide. MicroRNAs (miRNAs) are important epigenetic alterations that notably impact various physiological and pathological processes by acting as negative regulators of gene expression. Furthermore, they have a vital function in different types of cancers, including CRC. In this research, we evaluated, for the very first time, the expression levels of miR-196a-1 in the tissue and plasma of patients with CRC and also homeobox D8 (HOXD8) as the target gene. MATERIALS AND METHODS: This study included a collection of 220 plasma and tissue samples from 55 patients diagnosed with CRC, as well as 55 healthy individuals matched by age and sex. Total RNA was extracted from plasma and tissue samples, and then polyadenylation and cDNA synthesis were performed. The expression levels of miR-196a-1 and HOXD8 as target gene was evaluated by quantitative RT-PCR (qRT-PCR) assay. We compared the diagnostic value of plasma miR-196a-1 with that of the circulating tumor markers CA19-9 and CEA using a Receiver Operating Characteristics (ROC) analysis. The association of miR-196a-1 with clinicopathological characteristics was assessed in tissue and plasma samples from patients with CRC. RESULTS: Our data demonstrated that the expression levels of miR-196a-1 in the tissue and plasma samples of CRC patients were 11.426- and 11.655-fold higher, respectively than those in adjacent normal tissue and plasma samples from normal subjects (p < 0.001). Through ROC curve analysis, it was identified that the sensitivity and specificity of miR-196a-1 for tissue samples, with an AUC of 0.925, were 89% and 98%, respectively. In addition, the sensitivity and specificity for plasma samples with an AUC of 0.801 were 70% and 98%, respectively. These findings reveal that miR-196a-1 is a useful biomarker for discriminating cases from controls. Furthermore, the expression of HOXD8 was not significantly altered in tumor tissue samples compared to adjacent normal tissues (P > 0.05). CONCLUSIONS: These results show that miR-196a-1 has an oncogenic impact and plays a significant role in CRC development. The results also indicate that miR-196a-1 could serve as a novel noninvasive biomarker for the detection of CRC.

Activities and polymorphisms of MMP-2 and MMP-9, smoking, diabetes and risk of prostate cancer.

Matrix metalloproteinases (MMPs) are a group of zinc dependent enzymes that are involved in tumor cell invasion and metastasis. The role of MMP-2 and -9 genetic polymorphism in different malignancies has been the subject of numerous studies. The present research has attempted to discover any positive correlation between MMP-2 and MMP-9 SNPs and prostate cancer (PCa) in patients with a history of either diabetes or smoking habits. 112 PCa-patients and 150 unrelated healthy-controls that matched for age and sex were selected for present case-control study. MMP-2 -1575G/A and MMP-9 -1562 C/T polymorphisms detected by PCR-RFLP, serum tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), testosterone, prostate-specific antigen (PSA), free-prostate-specific-antigen (fPSA), and fPSA/PSA levels were detected by ELISA and enzyme assay, respectively. MMP-2 and MMP-9 activities were measured by gelatin-zymography. Covariates were considered as age, status of cigarette smoking, and a possible history of diabetes mellitus (DM). The frequency of -1575 MMP-2 A/A + A/G and -1562 MMP-9 C/T + T/T genotypes were higher in PCa-patients with DM (74.3%,p = 0.003) and with smoking habits (72.5%,p = 0.005). These genotypes were associated with the increased risk of prostate cancer in smokers (3.52-folds) and in individuals with history of DM (4.34-folds). A significant positive association was found between level of TIMPs (TIMP -1 and TIMP-2) and BMI in PCa-patients and also between testosterone levels and MMP-9 activity in healthy control subjects. For the first time, this study demonstrated that activities of MMP-2 -1575G/A and MMP-9 -1562C/T variants in association with smoking and diabetes are considered significant risk factors for PCa.

The study of HMOX1 DNA methylation and gene expression and the diagnostic potential of miR-153-3p in preeclampsia.

Background: The objective was to elucidate the potential epigenetic regulatory mechanism in HMOX1 expression in preeclampsia. Materials & methods: HMOX1 promoter DNA methylation was evaluated in the placental tissue and blood of preeclamptic and normotensive pregnant women. HMOX1 and miR-153-3p gene expression were assessed in placental tissue and peripheral blood mononuclear cells (PBMCs). Related microarray datasets in the Gene Expression Omnibus database were also analyzed. Results: In placental tissue, despite HMOX1 expression downregulation, there was no significant change in HMOX1 methylation. In PBMCs, there was no significant alteration in HMOX1 expression, while hypomethylation was observed in blood. The miR-153-3p expression increased in the placental tissue and in the PBMCs of preeclampsia. Conclusion: DNA methylation does not affect HMOX1 expression, while miR-153-3p might be a biomarker for preeclampsia.

The influence of Nrf2 gene promoter methylation on gene expression and oxidative stress parameters in preeclampsia.

BACKGROUND AND AIMS: Preeclampsia (PE) is a serious medical condition that usually causes high blood pressure and affects multiple organs. Considering the adverse effect of oxidative stress on the process of PE in pregnant women and regarding the role of the Nrf2 gene in placental oxidative pathways, this study was conducted to investigate the DNA methylation status of Nrf2 in PE and healthy pregnant women. MATERIALS AND METHODS: The present case-control study consisted of 70 PE and 70 healthy pregnant women. Blood and placenta samples were taken from all subjects, and the percentage of the Nrf2 gene methylation in the samples was assessed by the Methyl Light PCR method. Also, the Nrf2 gene expression was evaluated by real-time PCR. The total antioxidant capacity (TAC) and total oxidative status (TOS) were measured by the colorimetric method. RESULTS: In PE women, there was a significant increase in blood pressure, term of pregnancy, and BMI. In addition, there were enhanced Nrf2 DNA methylation percentage in placenta tissue and increased TOS levels in placenta tissue and blood compared to healthy pregnant women (P < 0.05). Also, in the PE group, there was a significant decrease in Nrf2 gene expression and TAC level in placenta tissue compared to the control group (P < 0.05). CONCLUSION: The Nrf2 gene undergoes epigenetic modifications of DNA hypermethylation in the PE placenta. Decreased expression of this gene and the changes in the level of oxidative parameters (TAC, TOS) confirm it.

Diagnostic value of FOXF1 gene promoter-methylated DNA in the plasma samples of patients with colorectal cancer.

BACKGROUND: Epigenetic modifications such as DNA methylation in the CpG islands of genes occur at a high rate. In this study, we measured the methylation level of the promoter region of the FOXF1 gene as a new blood biomarker for the detection of colorectal cancer in the early stages. METHODS: The methylation level of the promoter region of the FOXF1 gene was measured in the plasma samples of 50 colorectal cancer patients and 50 normal individuals. DNA was extracted after exposure to sodium bisulfite by the MethyLight polymerase chain reaction (PCR) method. The percentage of promoter region was measured in all samples, and statistical analysis was done using SPSS v24 software. RESULTS: The average promoter region between the plasma samples of colorectal cancer patients and healthy individuals had a significant difference (P < 0.001). The average promoter region of the FOXF1 gene in tumor plasma samples was 7.1 and in the control samples was 0.48. The sensitivity and specificity of the sample plasma levels were 78% and 89.5%, respectively. CONCLUSION: The promoter region value of the FOXF1 gene in plasma samples using the MethyLight PCR method had high sensitivity and specificity as a non-invasive method for colorectal cancer diagnosis. This research is the first report that has been presented regarding the investigation of FOXF1 gene methylation in plasma samples in colorectal cancer. Therefore, it is necessary to conduct more studies with larger size samples to evaluate the efficiency of the gene under investigation.

Theobromine supplementation in combination with a low-calorie diet improves cardiovascular risk factors in overweight and obese subjects with metabolic syndrome: a randomized controlled trial.

Background & aims: The beneficial effects of theobromine (TB) on obesity and features of metabolic syndrome (MetS) have been reported in several studies. However, the findings are equivocal. The present study aimed to investigate the effects of 12 week pure TB supplementation (450 mg day-1) combined with a low-calorie diet on the anthropometric and metabolic syndrome indices in overweight and obese adults with MetS. Methods: In a randomized double-blind parallel controlled trial, 80 participants aged 40-55 years were randomly assigned to take 450 mg day-1 TB or placebo along with a low-calorie diet for 12 weeks. Dietary intake, anthropometric indices, blood pressure, lipid profile and glycemic indices were assessed at the start and end of the intervention. Results: Seventy-two participants completed the study. After 12 weeks, TB supplementation significantly decreased the waist circumference (WC) (-0.86 cm; P = 0.045), LDL-c/HDL-c (-0.26; P = 0.008), TG/HDL-c (-0.41; P = 0.001), TC/HDL-c (-0.38; P = 0.006) and increased HDL-c (1.72 mg dl-1; P = 0.036) compared to the placebo group. There were no significant differences regarding body weight, BMI, hip circumference (HC), hip-to-waist circumference ratio (WHR), systolic and diastolic blood pressure, fasting levels of total cholesterol (TC), triacylglycerol (TAG), low-density lipoprotein cholesterol (LDL-c), fasting blood glucose, insulin, homoeostatic model assessment for insulin resistance (HOMA-IR) and homeostasis model assessment of ß-cell function (HOMA-ß) between the two groups (p > 0.05). Conclusion: The results of the current study revealed that TB supplementation along with a low-calorie diet had favorable effects on WC, LDL-c/HDL-c, TG/HDL-c, TC/HDL-c, and serum level of HDL-c in overweight and obese subjects with MetS. Trial registration number: IRCT20091114002709N59. Registration date: 5 March 2022.

A novel epigenetic biomarker, plasma miR-138-5p gene promoter-methylated DNA, for colorectal cancer diagnosis.

Aim: The miR-138-5p promoter-methylated DNA level, miR-138-5p and PDL1 expression were investigated in colorectal cancer (CRC) patients. Materials & methods: miR-138-5p promoter methylation status and miR-138-5p expression were investigated using the MethyLight and qPCR method, respectively. For measuring PDL-1, we applied the Bioassay Technology Elisa kit. Results: The percentage of methylated reference values of plasma and tissue samples from patients was higher than control groups. The area under curve presented a sensitivity of 55% and a specificity of 82.5% for plasma samples. Compared with the control groups, lower expression of miR-138-5p and higher concentration of PDL1 protein were observed in the patients group. Conclusion: CRC may be detected early by identifying miR-138-5p methylated DNA in plasma as a diagnostic biomarker.

Unfortunately, most patients with colorectal cancer (CRC) are diagnosed late in advanced stages. Genetic alterations due to a person's behavior or environment, such as miRNA dysregulation and methylation, occur in the initial phases of tumorigenesis. This study investigated the disease causing role of methylation of the miR-138-5p gene and its target protein, PDL1, in CRC as well as their potential ability to be used in the early diagnosis of this cancer. Using molecular techniques, higher methylated miR-138-5p as well as a higher PDL1 concentration were found in patients. This suggested a link between PDL1 protein and hyper methylation of the miR-138-5p gene. On the other hand, the hypermethylation of miR-138-5p happened before the increase of PDL1 protein, which resulted in decreased miR-138-5p and as a result decreased PDL1 protein. Compared with other biomarkers, miR-138-5p methylation and PDL1 had high diagnostic accuracy (acceptable sensitivity and specificity). Thus, the methylated miR-138-5p and PDL1 are proposed as diagnostic biomarkers for CRC.

Quantitative detection of SRY-Box 21 (SOX21) gene promoter methylation as a stool-based noninvasive biomarker for early diagnosis of colorectal cancer by MethyLight method.

BACKGROUND: In recent years, the study of potential epigenetic biomarkers in feces has been an attractive research approach for the noninvasive diagnosis of colorectal cancer (CRC). The aim of this study was to evaluate the stool-based DNA methylation potential of SRY-Box 21 (SOX21) gene promoter as an appropriate candidate biomarker for differentiating CRC patients and healthy individuals for the first time. METHODS: The MethyLight method was performed to analyze the methylation status of SOX21 gene promoter in fecal samples from 40 patients with CRC and 40 healthy controls. In addition, the diagnostic efficiency of measuring the hypermethylated SOX21 gene in the feces to the fecal occult blood test (FOBT) was compared. RESULTS: The percentage of methylated reference (PMR) values in the stool of CRC patients (median 1.44) was higher than those of healthy individuals (median 0.00) (P < 0.001). A sensitivity of 72.5% and specificity of 100% were obtained for SOX21 gene promoter methylation status and 29 of the patients were considered as positive in methylation status. There was no significant association between PMR values and demographic/clinicopathological features (P > 0.05). CONCLUSION: The results of the present study demonstrated that the stool-based assay of SOX21 gene promoter methylation has a relatively high sensitivity and specificity and it may serve as a noninvasive biomarker for early detection of CRC. However, more studies with a wide range of samples are required to further confirm the role of hypermethylation of SOX21 in the early CRC diagnosis.

Study on SARS-CoV-2 strains in Iran reveals potential contribution of co-infection with and recombination between different strains to the emergence of new strains.

We aimed to describe SARS-CoV-2 strains in Iranians from nine distributed cities infected during two months expanding late 2020 and early 2021 by genotyping known informative single nucleotide in five PCR amplicons. Two variants associated with haplotype H1 (clade G) and nine additional variants associated with other haplotypes were genotyped, respectively, in RNA isolates of 244 and 85 individuals. The variants associated with the H1a (GR) and H1b (GH) haplotypes were most prevalent, indicating a significant change in infection pattern with passage of time. The most important findings were that recombinant genomes and co-infection, respectively, were surmised in 44.7% and 12.9% of the samples extensively genotyped. Partners of many of the recombinations were relatively common strains. Co-existing viruses were among those currently circulating in Iran. In addition to random mutations, co-infection with different existing strains and recombination between their genomes may significantly contribute to the emergence of new SARS-CoV-2 strains.

Effects of Breastfeeding and Formula Feeding on the Expression Level of FTO, CPT1A and PPAR-α Genes in Healthy Infants.

PURPOSE: The study aimed to investigate the effect of breastfeeding, formula feeding and mix feeding (breastfed plus formula-fed) on the expression level of obesity-predisposing genes including fat mass and obesity-associated (FTO), carnitine palmitoyltransferase 1A (CPT1A), and peroxisome proliferator-activated receptor-α (PPAR-α) in 5- to 6-month-old infants. PATIENTS AND METHODS: A total of 150 infants participated in this case-control study. All subjects were healthy infants aged 5-6 months that divided into 3 groups: breastfed, formula-fed, and mix-fed. The expression level of FTO, CPT1A, and PPAR-α genes in peripheral blood mononuclear cells (PBMC) was evaluated in each group using reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Our findings showed that the current weight, height, and head circumference of infants in the formula feeding and mix feeding groups were significantly higher than those in the exclusive breastfeeding group. The expression level of FTO and CPT1A genes in formula-fed and mix-fed infants was significantly higher (p<0.001) than that in breastfed infants, while the expression level of PPAR-α gene was significantly lower (p<0.05). CONCLUSION: Breastfeeding showed modulatory effects on the expression level of obesity-predisposing genes and can protect against obesity and subsequent non-communicable diseases. However, more investigations are required to explain the epigenetic effects of breast milk.

High potential of SOX21 gene promoter methylation as an epigenetic biomarker for early detection of colorectal cancer.

BACKGROUND: Despite the advances in screening during the past decades, colorectal cancer (CRC) still is a leading cause of cancer deaths worldwide. Therefore, the development of new diagnostic methods is necessary. AIM: The aim of this study was to compare methylation changes of SRY-Box 21 (SOX21) gene promoter in tumor tissues and their normal adjacent mucosa in patients with CRC and to examine the relationship between the methylation levels and demographic/clinicopathological factors. MATERIALS AND METHODS: A total of 41 CRC patients participated in the present study. After the extraction of DNA and bisulfite treatment of the samples, the methylation levels were determined by using the MethyLight method. STATISTICAL ANALYSIS: Two-sided Mann-Whitney U test was used to compare the median level of methylation in tumor tissues and their adjacent normal mucosa. RESULTS: The methylation rates in tumor tissue samples were significantly higher compared to their adjacent normal mucosa (P < 0.0001). No association between demographic/clinicopathological factors and methylation status observed in tumor tissues. A receiver operating characteristics curve was constructed and tissue samples exhibited a sensitivity of 80.5% and specificity of 97.6% for SOX21 promoter methylation. CONCLUSION: The results of this study indicated the high potential of SOX21 gene promoter methylation as a candidate noninvasive diagnostic biomarker in stool and plasma of colorectal cancer patients. However, further studies with larger sample sizes are required to evaluate the specific role of SOX21 methylation as a biomarker for early detection of CRC.

Detection of SPG20 gene promoter-methylated DNA, as a novel epigenetic biomarker, in plasma for colorectal cancer diagnosis using the MethyLight method.

Aberrant promoter methylation of genes is a common epigenetic alteration in colorectal cancer (CRC). In the present study, spastic paraplegia 20 (SPG20) promoter-methylated DNA, as a potential diagnostic biomarker, was investigated in plasma and tumor tissue samples from patients with CRC. To the best of our knowledge, the quantification of SPG20 promoter-methylated DNA in plasma samples remains unreported. SPG20 promoter methylation was investigated in 32 paired tumor and healthy adjacent tissues, 37 plasma samples from patients with CRC, and in 37 plasma samples from a healthy control group, using the MethyLight method. The percentage of methylated reference (PMR) values was determined for each sample, and the sensitivity and specificity of this unique biomarker were evaluated. PMR values were significantly higher in plasma samples from patients with CRC compared with in those from the control group (P<0.05). Plasma specimens from patients and healthy controls exhibited median PMR values of 7.7 (95% CI, 4.15-15.28) and 0.59 (95% CI, 0.14-1.12), respectively. Notably, the median PMR values were identified as 42.39 (95% CI, 27.69-72.26) and 3.61 (95% CI, 1.07-5.29) in tumor and adjacent healthy tissues, respectively. Using receiver-operating characteristics curve analysis, the area under curve (AUC) was demonstrated to be 0.984 for plasma samples, exhibiting a sensitivity of 81.1% and a specificity of 96.9%. Furthermore, the AUC was 0.996 for tissue samples, revealing a sensitivity of 93.8% and specificity of 99.96%. Results from the present study indicate that the identification of SPG20 promoter-methylated DNA in plasma is a potential diagnostic biomarker for the detection of CRC. Furthermore, the results demonstrate a satisfactory sensitivity and specificity, indicating the importance of SPG20 methylation as a novel noninvasive biomarker.