miR-218-5p, miR-124-3p and miR-23b-3p act synergistically to modulate the expression of NACC1, proliferation, and apoptosis in C-33A and CaSki cells.

Background: In cervical cancer (CC), miR-218-5p, -124-3p, and -23b-3p act as tumor suppressors. These miRNAs have specific and common target genes that modulate apoptosis, proliferation, invasion, and migration; biological processes involved in cancer. Methods: miR-218-5p, -124-3p, and -23b-3p mimics were transfected into C-33A and CaSki cells, and RT-qPCR was used to quantify the level of each miRNA and NACC1. Proliferation was assessed by BrdU and apoptosis by Annexin V/PI. In the TCGA and The Human Protein Atlas databases, the level of NACC1 mRNA and protein (putative target of the three miRNAs) was analyzed in CC and normal tissue. The relationship of NACC1 with the overall survival in CC was analyzed in GEPIA2. NACC1 mRNA and protein levels were higher in CC tissues compared with cervical tissue without injury. Results: An increased expression of NACC1 was associated with lower overall survival in CC patients. The levels of miR-218-5p, -124-3p, and -23b-3p were lower, and NACC1 was higher in C-33A and CaSki cells compared to HaCaT cells. The increase of miR-218-5p, -124-3p, and -23b-3p induced a significant decrease in NACC1 mRNA. The transfection of the three miRNAs together caused more drastic changes in the level of NACC1, in the proliferation, and in the apoptosis with respect to the individual transfections of each miRNA. Conclusion: The results indicate that miR-218-5p, -124-3p, and -23b-3p act synergistically to decrease NACC1 expression and proliferation while promoting apoptosis in C-33A and CaSki cells. The levels of NACC1, miR-218-5p, -124-3p, and -23b-3p may be a potential prognostic indicator in CC.

Aryl Hydrocarbon Receptor Involvement in the Sodium-Dependent Glutamate/Aspartate Transporter Regulation in Cerebellar Bergmann Glia Cells.

Glutamate, the major excitatory neurotransmitter in the vertebrate brain, exerts its functions through the activation of specific plasma membrane receptors and transporters. Overstimulation of glutamate receptors results in neuronal cell death through a process known as excitotoxicity. A family of sodium-dependent glutamate plasma membrane transporters is responsible for the removal of glutamate from the synaptic cleft, preventing an excitotoxic insult. Glial glutamate transporters carry out more than 90% of the brain glutamate uptake activity and are responsible for glutamate recycling through the GABA/Glutamate/Glutamine shuttle. The aryl hydrocarbon receptor is a ligand-dependent transcription factor that integrates environmental clues through its ability to heterodimerize with different transcription factors. Taking into consideration the fundamental role of glial glutamate transporters in glutamatergic synapses and that these transporters are regulated at the transcriptional, translational, and localization levels in an activity-dependent fashion, in this contribution, we explored the involvement of the aryl hydrocarbon receptor, as a model of environmental integrator, in the regulation of the glial sodium-dependent glutamate/aspartate transporter. Using the model of chick cerebellar Bergmann glia cells, we report herein that the aryl hydrocarbon receptors exert a time-dependent decrease in the transporter mRNA levels and a diminution of its uptake activity. The nuclear factor kappa light chain enhancer of the activated B cell signaling pathway is involved in this regulation. Our results favor the notion of an environmentally dependent regulation of glutamate removal in glial cells and therefore strengthen the notion of the involvement of glial cells in xenobiotic neurotoxic effects.

Phenolic Compounds of Therapeutic Interest in Neuroprotection.

The number of elderly people is projected to double in the next 50 years worldwide, resulting in an increased prevalence of neurodegenerative diseases. Aging causes changes in brain tissue homeostasis, thus contributing to the development of neurodegenerative disorders. Current treatments are not entirely effective, so alternative treatments or adjuvant agents are being actively sought. Antioxidant properties of phenolic compounds are of particular interest for neurodegenerative diseases whose psychopathological mechanisms strongly rely on oxidative stress at the brain level. Moreover, phenolic compounds display other advantages such as the permeability of the blood-brain barrier (BBB) and the interesting molecular mechanisms that we reviewed in this work. We began by briefly outlining the physiopathology of neurodegenerative diseases to understand the mechanisms that result in irreversible brain damage, then we provided an overall classification of the phenolic compounds that would be addressed later. We reviewed in vitro and in vivo studies, as well as some clinical trials in which neuroprotective mechanisms were demonstrated in models of different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), ischemia, and traumatic brain injury (TBI).

LncRNA SOX9-AS1 triggers a transcriptional program involved in lipid metabolic reprogramming, cell migration and invasion in triple-negative breast cancer.

At the molecular level, triple-negative breast cancer (TNBC) is frequently categorized as PAM50 basal-like subtype, but despite the advances in molecular analyses, the clinical outcome for these subtypes is uncertain. Long non-coding RNAs (lncRNAs) are master regulators of genes involved in hallmarks of cancer, which makes them suitable biomarkers for breast cancer (BRCA) diagnosis and prognosis. Here, we evaluated the regulatory role of lncRNA SOX9-AS1 in these subtypes. Using the BRCA-TCGA cohort, we observed that SOX9-AS1 was significantly overexpressed in basal-like and TNBC in comparison with other BRCA subtypes. Survival analyzes showed that SOX9-AS1 overexpression was associated with a favorable prognosis in TNBC and basal-like patients. To study the functions of SOX9-AS1, we determined the expression levels in a panel of nine BRCA cell lines finding increased levels in MDA-MB-468 and HCC1187 TNBC. Using subcellular fractionation in these cell lines, we ascertained that SOX9-AS1 was located in the cytoplasmic compartment. In addition, we performed SOX9-AS1 gene silencing using two short-harping constructs, which were transfected in both cell models and performed a genome-wide RNA-seq analysis. Data showed that 351 lncRNAs and 740 mRNAs were differentially expressed in MDA-MB-468 while 56 lncRNAs and 100 mRNAs were modulated in HCC1187 cells (Log2FC < - 1.5 and > 1.5, p.adj value < 0.05). Pathway analysis revealed that the protein-encoding genes potentially regulate lipid metabolic reprogramming, and epithelial-mesenchymal transition (EMT). Expression of lipid metabolic-related genes LIPE, REEP6, GABRE, FBP1, SCD1, UGT2B11, APOC1 was confirmed by RT-qPCR. Functional analysis demonstrated that the knockdown of SOX9-AS1 increases the triglyceride synthesis, cell migration and invasion in both two TNBC cell lines. In conclusion, high SOX9-AS1 expression predicts an improved clinical course in patients, while the loss of SOX9-AS1 expression enhances the aggressiveness of TNBC cells.

RNA-Binding Proteins: A Role in Neurotoxicity?

Despite sustained efforts to treat neurodegenerative diseases, little is known at the molecular level to understand and generate novel therapeutic approaches for these malignancies. Therefore, it is not surprising that neurogenerative diseases are among the leading causes of death in the aged population. Neurons require sophisticated cellular mechanisms to maintain proper protein homeostasis. These cells are generally sensitive to loss of gene expression control at the post-transcriptional level. Post-translational control responds to signals that can arise from intracellular processes or environmental factors that can be regulated through RNA-binding proteins. These proteins recognize RNA through one or more RNA-binding domains and form ribonucleoproteins that are critically involved in the regulation of post-transcriptional processes from splicing to the regulation of association of the translation machinery allowing a relatively rapid and precise modulation of the transcriptome. Neurotoxicity is the result of the biological, chemical, or physical interaction of agents with an adverse effect on the structure and function of the central nervous system. The disruption of the proper levels or function of RBPs in neurons and glial cells triggers neurotoxic events that are linked to neurodegenerative diseases such as spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and frontotemporal dementia (FTD) among many others. The connection between RBPs and neurodegenerative diseases opens a new landscape for potentially novel therapeutic targets for the intervention of these neurodegenerative pathologies. In this contribution, a summary of the recent findings of the molecular mechanisms involved in the plausible role of RBPs in RNA processing in neurodegenerative disease is discussed.

DNMT3B overexpression downregulates genes with CpG islands, common motifs, and transcription factor binding sites that interact with DNMT3B.

DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.

Utility of in silico-identified-peptides in spike-S1 domain and nucleocapsid of SARS-CoV-2 for antibody detection in COVID-19 patients and antibody production.

SARS-CoV-2 contains four structural proteins, two of which, the spike and nucleocapsid, are commonly used for the standardization of novel methods for antibody detection; however, some limitations in their use have been observed due to the homology of this virus with other phylogenetically-related viruses. We performed in silico analysis to search for novel immunogenic and antigenic peptides. A total of twenty-five peptides were preliminarily selected, located in the 3D structure of both proteins. Finally, eight peptides were selected: one located in the N protein and seven in the S1 domain of the spike protein. Additionally, the localization of selected peptides in 2D structures and possible changes in the sequences of these peptides in SARS-CoV-2 variants of concern were analyzed. All peptides were synthetized in MAP8 format, and recombinant S (trimer and RBD) and N proteins were used as antigens to search for antibodies in serum samples derived from COVID-19 patients, and for antibody response in New Zealand rabbits. Results showed high recognition of the serum derived from COVID-19 patients to all selected peptides; however, only the RBD3 peptide induced antibody production. In conclusion, this work provides evidence for a new strategy in peptide selection and its use for antibody detection or antibody production in animals.

Variation in the Humoral Immune Response Induced by the Administration of the BNT162b2 Pfizer/BioNTech Vaccine: A Systematic Review.

The BNT162b2 Pfizer/BioNTech vaccine was the first emergency approved vaccine during the COVID-19 pandemic. The aim of this systematic review was to examine the variations in the humoral immune response induced by the administration of the BNT162b2 vaccine in patients with previous SARS-CoV-2 infection, the elderly, and those with comorbidities and immunosuppression states. Additionally, we analyzed the effect of generated neutralizing antibodies against the new variants of concern of SARS-CoV-2. Pubmed, Science Direct, Mendeley, and WorldWide Science were searched between 1 January 2020 and October 2021 using the keywords "BNT162b2", "serology", "comorbidity", "immunosuppression", and "variants of concern"dA total of 20 peer-reviewed publications were selected. The analysis showed that those individuals with previous infections have a considerably higher antibody response after the administration of BNT162b2 vaccine in contrast with seronegative individuals. With regard to variation in immune responses, elderly individuals, patients with cancer, or patients who had undergone a kidney transplant, dialysis, or who were pregnant had a lower antibody response in comparison to healthy individuals. Finally, antibodies developed against the S protein produced by the BNT162b2 vaccine, possessed lower neutralizing activity against the alpha, beta, gamma, and delta variants of SARS-CoV-2. In conclusion, patients with immunodeficiencies and comorbidities have a lesser antibody response, about which further studies need to be performed in order to analyze the effectiveness and duration of the humoral immunity associated with vaccination in these specific populations.

The E6 Oncoprotein of HPV16 AA-c Variant Regulates Cell Migration through the MINCR/miR-28-5p/RAP1B Axis.

The E6 oncoprotein of HPV16 variants differentially alters the transcription of the genes involved in migration and non-coding RNAs such as lncRNAs. The role of the lncRNA MINCR in cervical cancer and its relationship with variants of oncogenic HPV remain unknown. Therefore, the objective of this study was to analyze the effect of the E6 oncoprotein of the AA-c variant of HPV16 in cell migration through the MINCR/miR-28-5p/RAP1B axis. To explore the functional role of MINCR in CC, we used an in vitro model of C33-A cells with exogenous expression of the E6 oncoprotein of the AA-c variant of HPV16. Interfering RNAs performed MINCR silencing, and the expression of miR-28-5p and RAP1B mRNA was analyzed by RT-qPCR. We found that C33-A/AA-c cells expressed MINCR 8-fold higher compared to the control cells. There is an inverse correlation between the expression of miR-28-5p and RAP1B in C33-A/AA-c cells. Our results suggest that MINCR might regulate the expression of RAP1B through the inhibition of miR-28-5p in CC cells expressing the E6 oncoprotein of HPV16 AA-c. We report, for the first time, that the MINCR/miR-28-5p/RAP1B axis positively regulates cell migration in CC-derived cells that express the E6 oncoprotein of the AA-c variant of HPV16.

Prevalence and Distribution of Human Papillomavirus Genotypes (1997-2019) and Their Association With Cervical Cancer and Precursor Lesions in Women From Southern Mexico.

BACKGROUND: Cervical cancer (CC) is the fourth most common malignancy of the female genital tract. Human Papillomavirus (HPV) is the main cause of precancerous lesions and CC cases worldwide. OBJECTIVE: We assessed the prevalence and distribution of HPV types and their association with precancerous lesions and CC. METHODS: HPV genotypes were detected by 3 methods depending on the year of in which the sample was analyzed: MY09/11 RFLPs (1997 to 2010), GP5+/6+ primer systems (2005 to 2010) and INNO-LiPA HPV Genotyping Extra (2010 to 2019) in cervical samples (No-IL: 4445; LSIL: 2464; HSILs: 151 and CC: 253) from women from southern Mexico. RESULTS: The overall HPV prevalence was 54.17%, and hpv-16 was the most common genotype. In single infection, the high-risk HPV genotypes (group 1) were associated with squamous intraepitelial lesions (LSIL: HPV-39 (OR = 10.58, 95% CI 4.09-27.36, P < .001); HSIL: HPV-31 (OR = 14.76, 95% CI 6.56-33.20, P < .001); and CC: HPV-16 (OR = 25.01, 95% CI 18.83-33.21, P < .001). In multiple infections, the HPV genotypes (HPV-16 and HPV-18) were also associated with a high risk of lesions [LSIL: HPV-18 (OR = 3.45; 95% CI 1.36-8.91; P = .009); HSIL: HPV-18 (OR = 5.12; 95% CI 1.21-21.68; P = .026); and CC: HPV-16 (OR = 3.03; 95% CI 1.72-5.32; P < .001)] compared to single infection. In the analysis adjusted for age, giving birth, and cigarette smoking, a significant increase in the risk of LSIL, HSIL, and CC was maintained. CONCLUSIONS: This study provides current data on the prevalence and distribution of HPV genotypes in women from southern Mexico, which could serve as a valuable reference to guide nationwide CC screening programs and provide scientific evidence that could be useful for vaccine development efforts. Likewise, it was identified that infection with carcinogenic HPV genotypes is an independent risk factor for LSIL, HSIL, and CC.

The CpG island methylator phenotype increases the risk of high-grade squamous intraepithelial lesions and cervical cancer.

BACKGROUND: High-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer, but additional alterations are necessary for its development. Abnormal DNA methylation has an important role in the origin and dissemination of cervical cancer and other human tumors. In this work, we analyzed the methylation of eight genes (AJAP1, CDH1, CDH13, MAGI2, MGMT, MYOD1, RASSF1A and SOX17) that participate in several biological processes for the maintenance of cell normality. We analyzed DNA methylation by methylation-specific PCR (MSP) and HPV infection using the INNO­LiPA genotyping kit in 59 samples diagnostic of normal cervical tissue (non-SIL), 107 low-grade squamous intraepithelial lesions (LSILs), 29 high-grade squamous intraepithelial lesions (HSILs) and 51 cervical cancers (CCs). RESULTS: We found that all samples of LSIL, HSIL, and CC were HPV-positive, and the genotypes with higher frequencies were 16, 18, 51 and 56. In general, the genes analyzed displayed a significant tendency toward an increase in methylation levels according to increasing cervical lesion severity, except for the CDH13 gene. High CpG island methylator phenotype (CIMP) was associated with a 50.6-fold (95% CI 4.72-2267.3)-increased risk of HSIL and a 122-fold risk of CC (95% CI 10.04-5349.7). CONCLUSIONS: We found that CIMP high was significantly associated with HSIL and CC risk. These results could indicate that CIMP together with HR-HPV infection and other factors participates in the development of HSIL and CC.

Inhibition of Wnt-ß-Catenin Signaling by ICRT14 Drug Depends of Post-Transcriptional Regulation by HOTAIR in Human Cervical Cancer HeLa Cells.

BACKGROUND: In Cervical cancer (CC), in addition to HPV infection, the most relevant alteration during CC initiation and progression is the aberrant activation of Wnt/ß-catenin pathway. Several inhibitory drugs of this pathway are undergoing preclinical and clinical studies. Long non-coding RNAs (lncRNAs) are associated with resistance to treatments. In this regard, understanding the efficiency of drugs that block the Wnt/ß-catenin pathway in CC is of relevance to eventually propose successful target therapies in patients with this disease. METHODS: We analyzed the levels of expression of 249 components of the Wnt/ß-catenin pathway in a group of 109 CC patients. Three drugs that blocking specific elements of Wnt/ß-catenin pathway (C59, NSC668036 and ICRT14) by TOP FLASH assays and qRT-PCR were tested in vitro in CC cells. RESULTS: 137 genes of the Wnt/ß-catenin pathway were up-regulated and 112 down-regulated in CC patient's samples, demonstrating that this pathway is dysregulated. C59 was an efficient drug to inhibit Wnt/ß-catenin pathway in CC cells. NSC668036, was not able to inhibit the transcriptional activity of the Wnt/ß-catenin pathway. Strikingly, ICRT14 was neither able to inhibit this pathway in HeLa cells, due to HOTAIR interaction with ß-catenin, maintaining the Wnt/ß-catenin pathway activated. CONCLUSIONS: These results demonstrate a mechanism by which HOTAIR evades the effect of ICRT14, a Wnt/ß-catenin pathway inhibitory drug, in HeLa cell line. The emergence of these mechanisms reveals new scenarios in the design of target therapies used in cancer.

TOP2A/MCM2, p16INK4a, and cyclin E1 expression in liquid-based cytology: a biomarkers panel for progression risk of cervical premalignant lesions.

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.

DNMTs and Impact of CpG Content, Transcription Factors, Consensus Motifs, lncRNAs, and Histone Marks on DNA Methylation.

DNA methyltransferases (DNMTs) play an essential role in DNA methylation and transcriptional regulation in the genome. DNMTs, along with other poorly studied elements, modulate the dynamic DNA methylation patterns of embryonic and adult cells. We summarize the current knowledge on the molecular mechanism of DNMTs' functional targeting to maintain genome-wide DNA methylation patterns. We focus on DNMTs' intrinsic characteristics, transcriptional regulation, and post-transcriptional modifications. Furthermore, we focus special attention on the DNMTs' specificity for target sites, including key cis-regulatory factors such as CpG content, common motifs, transcription factors (TF) binding sites, lncRNAs, and histone marks to regulate DNA methylation. We also review how complexes of DNMTs/TFs or DNMTs/lncRNAs are involved in DNA methylation in specific genome regions. Understanding these processes is essential because the spatiotemporal regulation of DNA methylation modulates gene expression in health and disease.

HOX Transcript Antisense RNA HOTAIR Abrogates Vasculogenic Mimicry by Targeting the AngiomiR-204/FAK Axis in Triple Negative Breast Cancer Cells.

HOX transcript antisense RNA (HOTAIR) is an oncogenic long non-coding RNA frequently overexpressed in cancer. HOTAIR can enhance the malignant behavior of tumors by sponging microRNAs with tumor suppressor functions. Vasculogenic mimicry is a hypoxia-activated process in which tumor cells form three-dimensional (3D) channel-like networks, resembling endothelial blood vessels, to obtain nutrients. However, the role of HOTAIR in vasculogenic mimicry and the underlying mechanisms are unknown in human cancers. In the current study, we investigated the relevance of HOTAIR in hypoxia-induced vasculogenic mimicry in metastatic MDA-MB-231 and invasive Hs-578t triple negative breast cancer cells. Analysis of The Cancer Genome Atlas (TCGA) database using cBioPortal confirmed that HOTAIR was upregulated in clinical breast tumors relative to normal mammary tissues. Our quantitative RT-PCR assays showed a significant increase in HOTAIR levels after 48 h hypoxia relative to normoxia in breast cancer cell lines. Remarkably, knockdown of HOTAIR significantly abolished the hypoxia-induced vasculogenic mimicry which was accompanied by a reduction in the number of 3D channel-like networks and branch points. Likewise, HOTAIR silencing leads to reduced cell migration abilities of cancer cells. Bioinformatic analysis predicted that HOTAIR has a potential binding site for tumor suppressor miR-204. Luciferase reporter assays confirmed that HOTAIR is a competitive endogenous sponge of miR-204. Congruently, forced inhibition of HOTAIR in cells resulted in augmented miR-204 levels in breast cancer cells. Further bioinformatic analysis suggested that miR-204 can bind to the 3' untranslated region of focal adhesion kinase 1 (FAK) transcript involved in cell migration. Western blot and luciferase reporter assays confirmed that FAK is a novel target of miR-204. Finally, silencing of HOTAIR resulted in low levels of cytoplasmic FAK protein and alterations in the organization of cellular cytoskeleton and focal adhesions. In summary, our results showed, for the first time, that HOTAIR mitigates cell migration and vasculogenic mimicry by targeting the miR-204/FAK axis in triple negative breast cancer cells.

Altered levels of MALAT1 and H19 derived from serum or serum exosomes associated with type-2 diabetes.

Environmental, genetic and epigenetic risk factors have been closely related to the development of type-2 diabetes (T2D). It has been reported that the expression in H19 and MALAT1 are related to metabolic diseases. To analyze the relationship between the expression of H19 and MALAT1 lncRNAs with diabetic patients. A study was conducted in subjects with T2D and nondiabetic controls, residents of Mexico City. Anthropometric measurements were made, and serum concentrations of glucose, glycosylated hemoglobin, total cholesterol, triglycerides, high- and low-density lipoprotein cholesterol were analyzed. Total RNA was extracted from serum and serum exosomes. The H19 and MALAT1 expression levels were quantified by RT-qPCR. A significant reduction in the expression of MALAT1 from serum or serum exosomes were found in patients with T2D, metabolic syndrome and low levels of HDL-c. Significant increase in H19 levels was found in diabetic subjects with poor glycemic control. Additionally, the principal component analyzes showed that serum MALAT1 expression was associated with total cholesterol and HDL-c levels, and the exosomes H19 expression was associated with waist circumference. The results obtained suggest that MALAT1 expression levels could be an epigenetic biomarker of diabetes risk or of its comorbidities.

HOTAIR Knockdown Decreased the Activity Wnt/ß-Catenin Signaling Pathway and Increased the mRNA Levels of Its Negative Regulators in Hela Cells.

BACKGROUND/AIMS: HOTAIR is a long non-coding RNA that promotes the development of human cancer. TET1 enzyme is involved in DNA demethylation by oxidation of 5-methylcytocine and it is considered a tumor suppressor in some types of cancer. HOTAIR and TET1 are involved in modulation of the Wnt/ß-catenin signaling pathway, but their role in cervical cancer remains to be elucidated. The aim of this work was to analyze the effect of HOTAIR in TET1 expression, Wnt/ß-catenin signaling, and expression, methylation and hidroxymethylation of some negative regulators of this pathway in HeLa cells. METHODS: HOTAIR and TET expression were analyzed by RT-qPCR and western blot. The HOTAIR knockdown was done with DsiRNA and the activity of the Wnt/ß-catenin signaling pathway through luciferase assays and ß-catenin nuclear translocation. The mRNA levels of SNAIL, EDN3, CYCD1, SPRY2 (targets of Wnt/ß-catenin pathway) PCDH10, SOX17, AJAP1, and MAGI2 (negative regulators of Wnt/ß-catenin pathway) were evaluated by RT-qPCR. The DNA methylation and hidroxymethylation of negative regulators of the Wnt/ß-catenin pathway were evaluated by methylation-specific PCR and chemical modification, followed by digestion and quantitative PCR. RESULTS: HOTAIR knockdown in HeLa cells decreased the activity of Wnt/ß-catenin signaling pathway. It increased the mRNA levels of Wnt/ ß-catenin negative regulators through a decrease in their promoter's methylation pattern. TET1 enzyme was also down-regulated in HOTAIR knockdown cells. CONCLUSION: Our study suggests a mechanism in which HOTAIR promotes the over-activation of Wnt/ß-catenin signaling pathway by downregulation of PCDH10, SOX17, AJAP1 and MAGI2 and also TET.

Multiple infections by EBV, HCMV and Helicobacter pylori are highly frequent in patients with chronic gastritis and gastric cancer from Southwest Mexico: An observational study.

The chronic inflammation and damage to the gastric epithelium induced by Helicobacter pylori (H. pylori) are the main risk factors for gastric cancer development. Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) induce chronic inflammation and have been found in gastric tumors. The objectives this observational study were to determine the frequency of multiple infections by Helicobacter pylori, Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) and to relate the infection by EBV and HCMV with H. pylori vacA/cagA genotypes in patients with chronic gastritis or gastric cancer. DNA from H. pylori, EBV and HCMV was detected by PCR in biopsies from 106 Mexican patients with chronic gastritis and 32 from gastric cancer. The cagA status and the vacA genotypes of H. pylori were determined by PCR. In chronic gastritis and gastric cancer EBV was found in 69.8% and 87.5%, HCMV in 52.8% and 53.1%, and H. pylori in 48.1% and 40.6%, respectively. In chronic gastritis, 53% of H. pylori patients were EBV and 33% were both EBV/HCMV; in gastric cancer, 92.3% of H. pylori-infected individuals were EBV and 46.1% were EVB/HCMV. All the intestinal- and mixed-type tumors and the 83.3% of diffuse-type tumors were EBV. No significant differences were found between single infections or coinfections with the diagnosis or the cancer type. The H. pylori genotypes were not related to EBV or HCMV infection. The frequency of dual infections by H. pylori, EBV and HCMV is higher in patients from southwest Mexico than other populations. It is likely that these pathogens act synergistically to induce inflammation and gastric cancer.

Methylation of the L1 gene and integration of human papillomavirus 16 and 18 in cervical carcinoma and premalignant lesions.

High-risk human papillomavirus (HPV) is the primary cause of cervical carcinoma (CC). Viral integration into the host chromosomes is associated with neoplastic progression, and epigenetic changes may occur as a result. The objective of the present study was to analyze HPV L1 gene methylation and to compare the use of quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH) and L1 methylation analysis as methods for detecting HPV integration. Cervical scrapes or biopsy samples positive for HPV 16 or 18, from 187 female patients with CC, squamous intraepithelial lesions (SILs) or no intraepithelial lesion (non-IL) were analyzed. Methylation of the L1 gene was determined using bisulfite modification followed by PCR, and HPV integration was subsequently analyzed. HPV 16 L1 gene methylation was revealed to increase with histological grade, with statistically significant differences observed as follows: Low-grade SIL vs. CC, P<0.0001 and non-IL vs. CC, P<0.0001. HPV 18 L1 gene methylation also increased according to histological grade, however, no statistically significant differences were observed. Methylation at CpG site 5608 of the HPV 16 L1 gene was associated with all grades of cervical lesions, whereas methylation at CpG site 5617 demonstrated the strongest association with CC (odds ratio, 42.5; 95% confidence interval, 4.7-1861; P<0.0001). The concordance rates between the various methods for the detection of the physical status of HPV 16 and HPV 18 were 96.1% for qPCR and ISH, 76.7% for qPCR and L1 gene methylation, and 84.8% for ISH and L1 gene methylation. In conclusion, methylation of the HPV 16 L1 gene increases significantly according to the grade of the cervical lesion, and methylation at CpG sites 5608 and 5617 of this gene may be used as prognostic biomarkers. ISH and L1 gene methylation have good concordance with qPCR with regards to the detection of HPV integration. Therefore, these are useful methods in determining the physical state of HPV.

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.