The Involvement of microRNAs in Bone Remodeling Signaling Pathways and Their Role in the Development of Osteoporosis.

Bone remodeling, crucial for maintaining the balance between bone resorption and formation, relies on the coordinated activity of osteoclasts and osteoblasts. During osteoclastogenesis, hematopoietic stem cells (HSCs) differentiate into the osteoclast lineage through the signaling pathways OPG/RANK/RANKL. On the other hand, during osteoblastogenesis, mesenchymal stem cells (MSCs) differentiate into the osteoblast lineage through activation of the signaling pathways TGF-ß/BMP/Wnt. Recent studies have shown that bone remodeling is regulated by post-transcriptional mechanisms including microRNAs (miRNAs). miRNAs are small, single-stranded, noncoding RNAs approximately 22 nucleotides in length. miRNAs can regulate virtually all cellular processes through binding to miRNA-response elements (MRE) at the 3' untranslated region (3'UTR) of the target mRNA. miRNAs are involved in controlling gene expression during osteogenic differentiation through the regulation of key signaling cascades during bone formation and resorption. Alterations of miRNA expression could favor the development of bone disorders, including osteoporosis. This review provides a general description of the miRNAs involved in bone remodeling and their significance in osteoporosis development.

Skin pigmentation related variants in Mexican population and interaction effects on serum 25(OH)D concentration and vitamin D deficiency.

Skin pigmentation is negatively associated with circulating vitamin D (VD) concentration. Therefore, genetic factors involved in skin pigmentation could influence the risk of vitamin D deficiency (VDD). We evaluated the impact genetic variants related to skin pigmentation on VD in Mexican population. This cross-sectional analysis included 848 individuals from the Health Worker Cohort Study (ratio males to females ~ 1:3). Eight genetic variants: rs16891982 (SLC45A2), rs12203592 (IRF4), rs1042602 and rs1126809 (TYR), rs1800404 (OCA2), rs12913832 (HERC2), rs1426654 (SLC24A5), and rs2240751 (MFSD12); involved in skin pigmentation were genotyped. Skin pigmentation was assessed by self-report. Linear and logistic regression were used to assess the association between the variants of interest and VD and VDD, as appropriate. In our study, eight genetic variants were associated with skin pigmentation. A genetic risk score built with the variants rs1426654 and rs224075 was associated with lower VD levels (ß = - 1.38, 95% CI - 2.59, - 0.17, p = 0.025). Nevertheless, when examining gene-gene interactions, we observed that rs2240751 × rs12203592 were associated with VD levels (P interaction = 0.021). Whereas rs2240751 × rs12913832 (P interaction = 0.0001) were associated with VDD. Our results suggest that skin pigmentation-related gene variants are associated with lower VD levels in Mexican population. These results underscore the importance of considering genetic interactions when assessing the impact of genetic polymorphisms on VD levels.

Association of MARC1, ADCY5, and BCO1 Variants with the Lipid Profile, Suggests an Additive Effect for Hypertriglyceridemia in Mexican Adult Men.

Epidemiological studies have reported that the Mexican population is highly susceptible to dyslipidemia. The MARC1, ADCY5, and BCO1 genes have recently been involved in lipidic abnormalities. This study aimed to analyze the association of single nucleotide polymorphisms (SNPs) rs2642438, rs56371916, and rs6564851 on MARC1, ADCY5, and BCO1 genes, respectively, with the lipid profile in a cohort of Mexican adults. We included 1900 Mexican adults from the Health Workers Cohort Study. Demographic and clinical data were collected through a structured questionnaire and standardized procedures. Genotyping was performed using a predesigned TaqMan assay. A genetic risk score (GRS) was created on the basis of the three genetic variants. Associations analysis was estimated using linear and logistic regression. Our results showed that rs2642438-A and rs6564851-A alleles had a risk association for hypertriglyceridemia (OR = 1.57, p = 0.013; and OR = 1.33, p = 0.031, respectively), and rs56371916-C allele a trend for low HDL-c (OR = 1.27, p = 0.060) only in men. The GRS revealed a significant association for hypertriglyceridemia (OR = 2.23, p = 0.022). These findings provide evidence of an aggregate effect of the MARC1, ADCY5, and BCO1 variants on the risk of hypertriglyceridemia in Mexican men. This knowledge could represent a tool for identifying at-risk males who might benefit from early interventions and avoid secondary metabolic traits.

Effect of Hypoxia in the Transcriptomic Profile of Lung Fibroblasts from Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.

Unravelling the Contribution of the rs7041 and rs4588 Polymorphisms of the GC Gene and Serum VDBP Levels for Developing Metabolic Syndrome in the Mexican Population.

Metabolic syndrome (MetS) is a multifactorial disorder integrated by a constellation of cardiovascular risk factors. The genetic and environmental determinants of MetS are not fully elucidated. This study investigated the association of two common single nucleotide polymorphisms (SNPs) on GC, rs7041 and rs4588, derived haplotypes, and serum vitamin D binding protein (VDBP) levels with the susceptibility to suffer MetS in Mexican adults. We included 1924 individuals; clinical and biochemical data were obtained through standard methods. Genotyping was performed through predesigned TaqMan assays. Logistic regression models were used to assess the associations of interest. Prevalence of MetS was 52.9% in the whole population, being more frequent in women. We observed that some association results differed between sexes. The GG genotype of the rs7041 was associated with increased odds of MetS in women. For the rs4588, the CA genotype had a protective effect against MetS in women. The haplotype GC2 was associated with reduced odds for MetS and some of its components in women. Our data suggest that VDBP serum levels were influenced by genotypes/haplotypes and this interplay seems to influence the risk of MetS. Our data provide reliable evidence regarding the association of GC polymorphisms with MetS risk in Mexican women.

MicroRNA-1270 Inhibits Cell Proliferation, Migration, and Invasion via Targeting IRF8 in Osteoblast-like Cell Lines.

Osteoporosis (OP) is the most common bone disease affecting elderly individuals. The diagnosis of this pathology is most commonly made on the basis of bone fractures. Several microRNAs (miRNAs/miRs) have been identified as possible biomarkers for the diagnosis and treatment of OP. miRNAs can regulate gene expression, and determining their functions can provide potential pharmacological targets for treating OP. A previous study showed that miR-1270 was upregulated in monocytes derived from postmenopausal women with OP. Therefore, the present study aimed to uncover the role of miR-1270 in regulating bone metabolism. To reveal the mechanism underlying the regulatory effect of miR-1270 on interferon regulatory factor 8 (IRF8) expression, luciferase assay, reverse transcription-quantitative PCR, and Western blot analysis were performed. The results suggest that miR-1270 could regulate the mRNA and protein expression levels of IRF8 by directly binding to its 3'-untranslated region. The effects of miR-1270 overexpression and IRF8 silencing on cell proliferation, migration, and invasion were also evaluated. To the best of our knowledge, the current study was the first to support the crucial role of miR-1270 in bone metabolism via modulation of IRF8 expression. In addition, miR-1270 overexpression could attenuate human osteoblast-like cells' proliferation and migration ability.

The Polyphenols α-Mangostin and Nordihydroguaiaretic Acid Induce Oxidative Stress, Cell Cycle Arrest, and Apoptosis in a Cellular Model of Medulloblastoma.

Medulloblastoma is a common malignant brain tumor in the pediatric age. The current therapeutics present serious collateral effects. Polyphenols α-mangostin and nordihydroguaiaretic acid (NDGA) exert potent antitumoral activity in different cancer models, although their antitumoral effects have not been described in medulloblastoma cells yet. This study aimed to examine the proapoptotic effects of these polyphenols on human medulloblastoma cells. Medulloblastoma cell line Daoy was incubated with increasing concentrations of α-mangostin or NDGA for 24 h. The cell viability was analyzed using crystal violet and trypan blue dyes. Determination of the glutathione (GSH)/glutathione disulfide (GSSG) ratio and levels of carbonylated proteins was performed to evaluate the oxidative stress. Cell cycle progression and induction of cell death by fluorochrome-couple and TUNEL assays were evaluated using flow cytometry assays. Individual treatments with α-mangostin or NDGA decreased the viability of Daoy cells in a dose-dependent manner, inducing G2/M and S-G2/M cell cycle arrest, respectively. Both polyphenols induced cell death and increased oxidative stress. Very interestingly, α-mangostin showed more potent effects than NDGA. Our results indicate that α-mangostin and NDGA exert important cytostatic and cytotoxic effects in the Daoy cell line. These data highlight the potential usefulness of these compounds as an alternative strategy in medulloblastoma treatment.

Expression profiles of the Wnt/ß-catenin signaling-related extracellular antagonists during proliferation and differentiation in human osteoblast-like cells.

Bone formation is a dynamic process directed by osteoblast activity. The transition from the proliferation to differentiation stage during osteoblast maturation involves the downregulation of the Wnt/ß-catenin signaling pathway, and extracellular antagonists are important for the regulation of Wnt signaling. However, the expression levels of Wnt antagonists in these stages of human osteoblast maturation have not been fully elucidated. Therefore, the aim of the present study was to investigate the expression levels of extracellular Wnt antagonists during proliferation and differentiation in osteoblast-like cell lines. The results demonstrated an overlap between the differential expression of secreted Frizzled-related protein (SFPR)2, SFRP3, SFRP4 and Dickkopf (DKK) 2 genes during the differentiation stage in the MG-63 and Saos-2 cells. Furthermore, high expression levels of DKK3 in MG-63 cells, Wnt inhibitory factor 1 (WIF1) in Saos-2 cells and DKK4 in hFOB 1.19 cells during the same stage (differentiation), were observed. The upregulated expression levels of Wnt antagonists were also correlated with the high expression of anxin 2 during the differentiation stage. These findings suggested that Wnt-related antagonists could modulate the Wnt/ß-catenin signaling pathway. By contrast, DKK1 was the only gene that was found to be upregulated during the proliferation stage in hFOB 1.19 and Saos-2 cells. To the best of our knowledge, the present study provides, for the first time, the expression profile of Wnt antagonists during the proliferation stage and the initial phases of differentiation in osteoblast-like cell lines. The current results offer a basis to investigate potential targets for bone-related Wnt-signaling modulation in bone metabolism research.

Single-nucleotide polymorphism rs10036727 in the SLIT3 gene is associated with osteoporosis at the femoral neck in older Mexican postmenopausal women.

AIMS: Osteoporosis (OP) remains a major public health problem worldwide. The most serious complications of this disease are fragility fractures, which increase morbidity and mortality. Management of OP represents an economic burden for health systems. Therefore, it is necessary to develop new screening strategies to identify the population at risk and implement preventive measures. We previously identified the SNPs rs3801387 in WNT16, rs7108738 in SOX6, rs10036727 in SLIT3 and rs7584262 in PKDCC as associated with bone mineral density in postmenopausal women through a genome-wide association study. The aim of this study was to validate those SNPs in two independent cohorts of non-related postmenopausal women. MATERIALS AND METHODS: We included 1160 women classifying them as normal, osteopenic or osteoporotic and a group with hip fragility fracture. Genotyping was performed using predesigned TaqMan assays. RESULTS: The variants rs10036727 and rs7108738 showed a significant association with BMD at the femoral neck. SLIT3 has been previously proposed as a potential biomarker and therapeutic resource. CONCLUSIONS: Our results provide new evidence regarding a possible involvement of SLIT3 in bone metabolisms and encourage the development of more studies in different populations to support these observations.

Exposure to biomass smoke, cigarettes, and alcohol modifies the association between tumour necrosis factor (-308G/A, -238G/A) polymorphisms and tuberculosis in Mexican carriers.

INTRODUCTION: Exposure to biomass smoke, cigarettes, alcohol, and the impairment of immunoregulation are considered to be risk factors for tuberculosis. Tumour necrosis factor (TNF) -308G/A and -238G/A gene polymorphisms have been associated with tuberculosis. However, the results remain inconsistent. The aim of this study was to determine the association between TNF polymorphisms and tuberculosis in the presence of biomass smoke, cigarettes, and alcohol in a Mexican population. MATERIAL AND METHODS: TNF polymorphisms were determined in 118 tuberculosis patients and 223 controls. We performed a univariate, bivariate, stratified analysis. Odds ratios, confidence intervals, and p-values were calculated. RESULTS: Occupational biomass smoke exposure was associated with tuberculosis between the patients and controls (OR = 1.70, 95% CI: 1.08-2.70, p = 0.02). We also found an association of the -308A allele carriers between the patients and controls without exposure to occupational (p = 0.04, OR = 0.16, 95% CI: 0.01-0.92) and in-home (p = 0.02, OR = 0.14, 95% CI: 0.01-0.81) biomass smoke, as well as an association with alcohol (p = 0.01, OR = 0.24, 95% CI: 0.05-0.75). The haplotype analysis revealed an association of the -308A/-238G haplotype between patients and nonconsanguineous controls without exposure to occupational (p = 0.02, OR = 0.12, 95% CI: 0.01-0.99) and in-home (p = 0.01, OR = 0.1, 95% CI: 0.01-0.9) biomass smoke, cigarette use (p = 0.04, OR = 0.28, 95% CI: 0.08-0.98), and alcohol (p = 0.02, OR = 0.22, 95% CI: 0.05-0.88) intake. CONCLUSIONS: The TNF -308A allele and the -308A/-238G haplotype are associated with tuberculosis, as are exposure to biomass smoke, cigarettes, and alcohol. No association for the -238G/A polymorphism was found. Our results provide insight into a possible protective role of TNF polymorphisms in tuberculosis in our population.

MicroRNA-548-3p overexpression inhibits proliferation, migration and invasion in osteoblast-like cells by targeting STAT1 and MAFB.

Osteoporosis is the most common bone disease and a public health issue with increasing prevalence in Mexico. This disease is caused by an imbalance in the bone remodelling process mediated by osteoclast and osteoblast. MicroRNAs have emerged as key players during the differentiation of both types of cells specialized involved in bone metabolism. We found high expression levels of miR-548x-3p in circulating monocytes derived from postmenopausal osteoporotic women. This study aimed to analyse the functional characterization of miR-548x-3p roles in the bone remodelling process. We validated by RT-qPCR, the elevated levels of miR-548x-3p in circulating monocytes derived from osteoporosis women. Through bioinformatics analysis, we identify MAFB and STAT1 as potential target genes for miR-548x-3p. Both genes showed low levels of expression in circulating monocytes derived from osteoporotic women. In addition, we demonstrated the binding of miR-548x-3p to the 3'-UTR of both mRNAs. MiR-548x-3p was overexpressed in osteoblasts-like cell lines decreasing the levels of MAFB and STAT1 mRNA and protein. We found that miR-548x-3p overexpression inhibits the proliferation, migration and invasion of the cell lines evaluated. Our results identified, by the first time, the potential role of miR-548x-3p as a modulator of the bone remodelling process by regulating the expression of MAFB and STAT1.

Serum miRNAs miR-140-3p and miR-23b-3p as potential biomarkers for osteoporosis and osteoporotic fracture in postmenopausal Mexican-Mestizo women.

Osteoporosis is a metabolic bone disorder characterized by low bone mineral density and decreased bone strength, leading to an increased risk of fractures with a consequent increase in morbidity and mortality. The current methods to estimate the fracture risk are very limited. microRNAs (miRNAs) have been considered as good biomarkers for many pathological processes, including osteoporosis. Some circulating miRNAs are associated with regulation of bone formation and differentiation of bone cells. The aim of this study, was to analyze the expression of miRNAs in serum of patients with osteoporosis (n = 20) and healthy controls (n = 20). Expression of 754 miRNAs was analyzed through quantitative real time RT-PCR arrays. Seven miRNAs showed significant differences between groups. The microRNAs miR-23b-3p, miR-140-3p and miR-885-5p were selected based on fold change and p-values (40.5, p = 0.038, 20.7, p = 0.045, and 2.2, p = 0.002; respectively) for validation in independent serum samples from patients with osteopenia (n = 28), osteoporosis (n = 26) and osteoporotic hip fracture (n = 21). After validation, we confirm differences across the groups for miR-23b-3p and miR-140-3p. Our data pointed miR-140-3p and miR-23b-3p as potential biomarkers candidates for osteoporosis in postmenopausal women.

A Pilot Genome-Wide Association Study in Postmenopausal Mexican-Mestizo Women Implicates the RMND1/CCDC170 Locus Is Associated with Bone Mineral Density.

To identify genetic variants influencing bone mineral density (BMD) in the Mexican-Mestizo population, we performed a GWAS for femoral neck (FN) and lumbar spine (LS) in Mexican-Mestizo postmenopausal women. In the discovery sample, 300,000 SNPs were genotyped in a cohort of 411 postmenopausal women and seven SNPs were analyzed in the replication cohort (n = 420). The combined results of a meta-analysis from the discovery and replication samples identified two loci, RMND1 (rs6904364, P = 2.77 × 10-4) and CCDC170 (rs17081341, P = 1.62 × 10-5), associated with FN BMD. We also compared our results with those of the Genetic Factors for Osteoporosis (GEFOS) Consortium meta-analysis. The comparison revealed two loci previously reported in the GEFOS meta-analysis: SOX6 (rs7128738) and PKDCC (rs11887431) associated with FN and LS BMD, respectively, in our study population. Interestingly, rs17081341 rare in Caucasians (minor allele frequency < 0.03) was found in high frequency in our population, which suggests that this association could be specific to non-Caucasian populations. In conclusion, the first pilot Mexican GWA study of BMD confirmed previously identified loci and also demonstrated the importance of studying variability in diverse populations and/or specific populations.

Association between PNPLA3 (rs738409), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), GCKR (rs780094), and elevated transaminase levels in overweight/obese Mexican adults.

There is scarce information about the link between specific single-nucleotide polymorphisms (SNPs) and risk of liver disease among Latinos, despite the disproportionate burden of disease among this population. Our aim was to investigate nine SNPs in or near the following genes: PNPLA3, LYPLAL1, PPP1R3B, GCKR, NCAN, IRS1, PPARG, and ADIPOR2 and examine their association with persistently elevated alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels in Mexican adults. Data and samples were collected from 741 participants in the Mexican Health Worker Cohort Study, in Cuernavaca, Mexico. We identified 207 cases who had persistently elevated levels of ALT or AST (≥40 U/L) and 534 controls with at least two consecutive normal ALT or AST results in a 6 month period, during 2004-2006 and 2011-2013. TaqMan assays were used to genotype the SNPs. The risk allele of PNPLA3 rs738409 was found to be associated with persistently elevated levels of ALT or AST, adjusting for age, sex, BMI, type 2 diabetes, and ancestry: (OR 2.28, 95 % CI 1.13, 4.58). A significant association was found between the LYPLAL1, PPP1R3B, and GCKR risk alleles and elevated ALT or AST levels among overweight/obese adults. These results suggest that among Mexicans, the PNPLA3 (rs738409), LYPLAL1 (rs12137855), PPP1R3B (rs4240624), and GCKR (rs780094) polymorphisms may be associated with a greater risk of chronic liver disease among overweight adults. This study is the first to examine these nine SNPs in a sample of adults in Mexico.

Probenecid Sensitizes Neuroblastoma Cancer Stem Cells to Cisplatin.

We used both in vitro cultures of neuroblastoma cell lines and nude-mice xenotransplants to explore the effects of co-administration of cisplatin and probenecid. Probenecid sensitized neuroblastoma cells, including tumor cells with stem features, to the effects of cisplatin, both in vitro and in vivo. This effect was mediated by an increase in the apoptotic cell death and a concomitant decrease in cell proliferation. This effect is accompanied by modulation of the mRNA and protein of the drug efflux transporters MDR1, MRP2, and BCRP. The co-administration of probenecid with cisplatin should be explored as a possible therapeutic strategy.

Analysis of association of MEF2C, SOST and JAG1 genes with bone mineral density in Mexican-Mestizo postmenopausal women.

BACKGROUND: Osteoporosis, a disease characterized by low bone mineral density (BMD), is an important health problem in Mexico. BMD is a highly heritable trait, with heritability estimates of 50-85%. Several candidate genes have been evaluated to identify those involved in BMD variation and the etiology of osteoporosis. This study investigated the possible association of single-nucleotide polymorphisms (SNPs) in the MEF2C, SOST and JAG1genes with bone mineral density (BMD) variation in postmenopausal Mexican-Mestizo women. METHODS: Four hundred unrelated postmenopausal women were included in the study. Risk factors were recorded and BMD was measured in total hip, femoral neck and lumbar spine using dual-energy X-ray absorptiometry. In an initial stage, a total of twenty-five SNPs within or near SOST gene and seven SNPs in the JAG1 gene were genotyped using a GoldenGate assay. In a second stage, three MEF2C gene SNPs were also genotyped and SOST and JAG1 gene variants were validated. Real time PCR and TaqMan probes were used for genotyping. RESULTS: Linear regression analyses adjusted by age, body mass index and ancestry estimates, showed that five SNPs in the SOST gene were significantly associated with BMD in total hip and femoral neck but not lumbar spine. The lowest p value was 0.0012, well below the multiple-test significance threshold (p=0.009), with mean effect size of -0.027 SD per risk allele. We did not find significant associations between BMD and MEF2C/JAG1 gene variants [rs1366594 "A" allele: ß=0.001 (95% CI -0.016; 0.017), P=0.938; rs2273061 "G" allele: ß=0.007 (95% CI -0.007; 0.023), p=0.409]. CONCLUSIONS: SOST polymorphisms may contribute to total hip and femoral neck BMD variation in Mexican postmenopausal women. Together, these and prior findings suggest that this gene may contribute to BMD variation across populations of diverse ancestry.

Survivin isoform Delta Ex3 regulates tumor spheroid formation.

Survivin is an important member of the Inhibitor of Apoptosis Proteins (IAPs) family and has essential roles in apoptosis and cell cycle progression. This gene is commonly upregulated in human cancer and provides an exciting diagnostic and therapeutic target. Survivin is expressed as several isoforms that are generated by alternative splicing, and some of these present antagonistic activities. Currently, information regarding the regulation of these isoforms is lacking. In this study, we sought to analyze survivin Delta Ex3 expression in a three-dimensional model of avascular tumors and its overexpression effects in processes such as proliferation, clonogenicity and apoptosis. We found a positive correlation between spheroid growth and survivin Delta Ex3 expression during the exponential phase. We demonstrated that this isoform not only decreased apoptosis but also inhibited tumor spheroid formation by decreasing proliferation and clonogenic survival. These results point toward a dual and antagonistic effect of this spliced survivin isoform in cancer development.