S-Dihydrodaidzein and 3-(1,3-benzoxazol-2-yl)-benzamide, Two New Potential ß-estrogen Receptor Ligands with Anti-adipogenic Activity.

BACKGROUND: The elucidation of molecular pathways associated with adipogenesis has evidenced the relevance of estrogen and estrogen receptor beta (ERß). The positive effects of ERß ligands on adipogenesis, energy expenditure, lipolysis, food intake, and weight loss, make ERß an attractive target for obesity control. From ligand-based virtual screening, molecular docking, and molecular dynamic simulations, six new likely ERß ligands (C1 to C6) have been reported with potential for pharmacological obesity treatment. OBJECTIVE: In this study, the effect of molecules C1-C6 on adipogenesis using the murine 3T3-L1 cell line was evaluated. METHODS: Cell viability was assessed by MTT assays. Lipid accumulation and gene expression were investigated by ORO staining and real-time quantitative RT-PCR experiments, respectively. RESULTS: Cell viability was not significantly affected by C1-C6 at concentrations up to 10 µM. Interestingly, treatment with 10 µM of C1 (S-Dihydrodaidzein) and C2 (3-(1,3-benzoxazol-2-yl)- benzamide) for 72 h inhibited adipocyte differentiation; moreover, ORO staining evidenced a reduced intracellular lipid accumulation (40% at day 7). Consistently, mRNA expression of the adipogenic markers, PPARγ and C/EBPα, was reduced by 50% and 82%, respectively, in the case of C1, and by 83% and 59%, in the case of C2. CONCLUSION: Altogether, these results show the two new potential ß-estrogen receptor ligands, C1 and C2, to exhibit anti-adipogenic activity. They could further be used as lead structures for the development of more efficient drugs for obesity control.

Ligand-Based Virtual Screening, Molecular Docking, and Molecular Dynamic Simulations of New ß-Estrogen Receptor Activators with Potential for Pharmacological Obesity Treatment.

Obesity is a pandemic and a serious health problem in developed and undeveloped countries. Activation of estrogen receptor beta (ERß) has been shown to promote weight loss without modifying caloric intake, making it an attractive target for developing new drugs against obesity. This work aimed to predict new small molecules as potential ERß activators. A ligand-based virtual screening of the ZINC15, PubChem, and Molport databases by substructure and similarity was carried out using the three-dimensional organization of known ligands as a reference. A molecular docking screening of FDA-approved drugs was also conducted as a repositioning strategy. Finally, selected compounds were evaluated by molecular dynamic simulations. Compounds 1 (-24.27 ± 0.34 kcal/mol), 2 (-23.33 ± 0.3 kcal/mol), and 6 (-29.55 ± 0.51 kcal/mol) showed the best stability on the active site in complex with ERß with an RMSD < 3.3 Å. RMSF analysis showed that these compounds do not affect the fluctuation of the Cα of ERß nor the compactness according to the radius of gyration. Finally, an in silico evaluation of ADMET showed they are safe molecules. These results suggest that new ERß ligands could be promising molecules for obesity control.

Metastatic breast tumors downregulate miR-145 regulating the hypoxia-induced vasculogenic mimicry.

Tumor cells grow in three-dimensional (3D) channels-like structures denoted as vasculogenic mimicry (VM), which provides a route for nutrients and oxygen acquisition. VM is activated by hypoxia and associated with metastasis and poor prognosis. MetastamiRs are microRNAs regulating metastasis, however, if they control VM in breast cancer remains poorly understood. The aim of this study was to evaluate the expression of VM-associated microRNAs in tumors of metastatic breast cancer patients. Firstly, we constructed microRNAs/mRNAs coregulation networks using expression data from TCGA databases. Dozens of microRNAs regulating genes involved in VM and metastasis were found. Of these, we selected 10 microRNAs for further characterization. The presence of VM in histological samples from patients with or without metastasis was evaluated using CD31-/PAS+ immunophenotyping. Remarkably, data showed that VM was significantly increased in tumors from patients with metastasis in comparison with no-metastatic group. Gene expression analysis indicated that miR-145, miR-142-3p, miR-31, miR-148a, miR-200b-3p and miR-526b were downregulated in primary tumors from patients with metastatic disease and positive for VM. Moreover, modulated microRNAs showed a predictive clinical value in overall survival in a cohort (n=1262) of breast cancer patients. Of these, we evaluated the role of miR-145 in formation of hypoxia-induced 3D channels-like using an in vitro model that recapitulates the early stages of VM. Data showed that miR-145 mimics was able to abolish the VM development in both metastatic Hs578t and MDA-MB-231 breast cancer cells. In conclusion, manipulation of miR-145 levels may represent a therapeutic approach in metastatic breast cancer patients that developed VM.

Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model.

Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.

Breast Cancer Cells Reprogram the Oncogenic lncRNAs/mRNAs Coexpression Networks in Three-Dimensional Microenvironment.

Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments.

Proteomic and Functional Analysis of the Effects of Quinoxaline Derivatives on Entamoeba histolytica.

Quinoxalines are heterocyclic compounds that contain a benzene ring and a pyrazine ring. The oxidation of both nitrogen of the pyrazine ring results in quinoxaline derivatives (QdNO), which exhibit a variety of biological properties, including antiparasitic activity. However, its activity against Entamoeba histolytica, the protozoan that causes human amebiasis, is poorly understood. Recently, our group reported that various QdNOs produce morphological changes in E. histolytica trophozoites, increase reactive oxygen species, and inhibit thioredoxin reductase activity. Notably, T-001 and T-017 derivatives were among the QdNOs with the best activity. In order to contribute to the characterization of the antiamebic effect of QdNOs, in this work we analyzed the proteomic profile of E. histolytica trophozoites treated with the QdNOs T-001 and T-017, and the results were correlated with functional assays. A total number of 163 deregulated proteins were found in trophozoites treated with T-001, and 131 in those treated with T-017. A set of 21 overexpressed and 24 under-expressed proteins was identified, which were mainly related to cytoskeleton and intracellular traffic, nucleic acid transcription, translation and binding, and redox homeostasis. Furthermore, T-001 and T-017 modified the virulence of trophozoites, since they altered their erythrophagocytosis, migration, adhesion and cytolytic capacity. Our results show that in addition to alter reactive oxygen species, and thioredoxin reductase activity, T-001 and T-017 affect essential functions related to the actin cytoskeleton, which eventually affects E. histolytica virulence and survival.

Three-Dimensional 3D Culture Models in Gynecological and Breast Cancer Research.

Traditional two-dimensional (2D) monolayer cell cultures have long been the gold standard for cancer biology research. However, their ability to accurately reflect the molecular mechanisms of tumors occurring in vivo is limited. Recent development of three-dimensional (3D) cell culture models facilitate the possibility to better recapitulate several of the biological and molecular characteristics of tumors in vivo, such as cancer cells heterogeneity, cell-extracellular matrix interactions, development of a hypoxic microenvironment, signaling pathway activities depending on contacts with extracellular matrix, differential growth kinetics, more accurate drugs response, and specific gene expression and epigenetic patterns. In this review, we discuss the utilization of different types of 3D culture models including spheroids, organotypic models and patient-derived organoids in gynecologic cancers research, as well as its potential applications in oncological research mainly for screening drugs with major physiological and clinical relevance. Moreover, microRNAs regulation of cancer hallmarks in 3D cell cultures from different types of cancers is discussed.

A Three-Dimensional Culture-Based Assay to Detect Early Stages of Vasculogenic Mimicry in Ovarian Cancer Cells.

Vasculogenic mimicry is a cellular mechanism in which tumor cells grow and align forming complex three-dimensional (3D) channel-like structures in a hypoxic microenvironment. This phenomenon represents a novel oxygen, nutrient, and blood supply, in a similar way as occurs in classic angiogenesis. Vasculogenic mimicry has been described in numerous clinical tumors including breast, prostate, lung, and ovarian cancers where it is associated with poor prognosis; thus, it is considered as a hallmark of highly aggressive and metastatic tumors. Here, we describe a simple method to model the in vitro formation of three-dimensional cellular networks over Matrigel in SKOV3 ovarian cancer cells representing the early stages of vasculogenic mimicry.

Three-Dimensional Organotypic Cultures Reshape the microRNAs Transcriptional Program in Breast Cancer Cells.

The 3D organotypic cultures, which depend on the growth of cells over the extracellular matrix (ECM) used as a scaffold, can better mimic several characteristics of solid cancers that influence tumor biology and the response to drug therapies. Most of our current knowledge on cancer is derived from studies in 2D cultures, which lack the ECM-mediated microenvironment. Moreover, the role of miRNAs that is critical for fine-tuning of gene expression is poorly understood in 3D cultures. The aim of this study was to compare the miRNA expression profiles of breast cancer cells grown in 2D and 3D conditions. On an on-top 3D cell culture model using a basement membrane matrix enriched with laminin, collagen IV, entactin, and heparin-sulfate proteoglycans, the basal B (Hs578T) and luminal (T47D) breast cancer cells formed 3D spheroid-like stellate and rounded mass structures, respectively. Morphological changes in 3D cultures were observed as cell stretching, cell-cell, and cell-ECM interactions associated with a loss of polarity and reorganization on bulk structures. Interestingly, we found prolongations of the cytoplasmic membrane of Hs578T cells similar to tunneled nanotubes contacting between neighboring cells, suggesting the existence of cellular intercommunication processes and the possibility of fusion between spheroids. Expression profiling data revealed that 354 miRNAs were differentially expressed in 3D relative to 2D cultures in Hs578T cells. Downregulated miRNAs may contribute to a positive regulation of genes involved in hypoxia, catabolic processes, and focal adhesion, whereas overexpressed miRNAs modulate genes involved in negative regulation of the cell cycle. Target genes of the top ten modulated miRNAs were selected to construct miRNA/mRNA coregulation networks. Around 502 interactions were identified for downregulated miRNAs, including miR-935/HIF1A and miR-5189-3p/AKT that could contribute to cell migration and the response to hypoxia. Furthermore, the expression levels of miR-935 and its target HIF1A correlated with the expression found in clinical tumors and predicted poor outcomes. On the other hand, 416 interactions were identified for overexpressed miRNAs, including miR-6780b-5p/ANKRD45 and miR-7641/CDK4 that may result in cell proliferation inhibition and cell cycle arrest in quiescent layers of 3D cultures. In conclusion, 3D cultures could represent a suitable model that better resembles the miRNA transcriptional programs operating in tumors, with implications not only in the understanding of basic cancer biology in 3D microenvironments, but also in the identification of novel biomarkers of disease and potential targets for personalized therapies in cancer.

Evaluation of the PLAC8 Gene in Mexican Women With and Without Preeclampsia and Obesity.

Preeclampsia (PE) is a leading cause of maternal-fetal mortality worldwide, and obesity is an important risk factor. Genes associated with pathophysiological events common to preeclampsia and obesity, such as PLAC8, remain to be studied; therefore, the aim of the present study was to evaluate this gene in the placentas of women affected with preeclampsia and healthy pregnant women. This case-controlled study included 71 healthy and 64 preeclampsia pregnancies. Gene expression was evaluated in primary human cytotrophoblasts (PHCT) from six normal and six preeclampsia pregnancies, and protein expression was verified in placentas from five healthy and six preeclampsia pregnancies. The whole coding and 5' regions of the PLAC8 gene were sequenced from healthy (n = 10) and preeclamptic (n = 10) pregnancies. The presence of the observed nucleotide variations was analyzed by RT-PCR in the total population. Statistical analyses were performed accordingly. Obesity was associated with severe preeclampsia (SPE) (OR = 3.34; CI 95% 1.3-8.2, p < 0.01). Significantly higher mRNA and protein expression was observed in preeclamptic vs. healthy placentas (p < 0.05). After sequencing, a single nucleotide variation was identified in 10 cases and one control (p < 0.01), which was then evaluated in the total population showing no association with preeclampsia. This preliminary study confirms the association of SPE with obesity and suggests higher expression of PLAC8 mRNA and protein in placentas from preeclampsia. No differences in nucleotide variations between cases and controls of the whole population were observed. Further research is required to evaluate the implications of higher gene/protein expression in preeclampsia and the causes of such variation.

Promoter methylation status of RORC, IL17A, and TNFA in peripheral blood leukocytes in adolescents with obesity-related asthma.

A higher Th17-immune response characterises obesity and obesity-related asthma phenotype. Nevertheless, obesity-related asthma has a more significant Th17-immune response than obesity alone. Retinoid-related orphan receptor C (RORC) is the essential transcription factor for Th17 polarisation. Previous studies have found that adolescents with obesity-related asthma presented upregulation of RORC, IL17A, and TNFA. However, the mechanisms that cause these higher mRNA expression levels in this asthmatic phenotype are poorly understood. Methylation directly regulates gene expression by adding a methyl group to carbon 5 of dinucleotide CpG cytosine. Thus, we evaluated the relationship between RORC, IL17A, and TNFA methylation status and mRNA expression levels to investigate a possible epigenetic regulation. A total of 102 adolescents (11-18 years) were studied in the following four groups: 1) healthy participants (HP), 2) allergic asthmatic participants (AAP), 3) obese participants without asthma (OP), and 4) non-allergic obesity-related asthma participants (OAP). Real-time qPCR assessed the methylation status and gene expression levels in peripheral blood leukocytes. Remarkably, the OAP and AAP groups have lower promoter methylation patterns of RORC, IL17A, and TNFA than the HP group. Notably, the OAP group presents lower RORC promoter methylation status than the OP group. Interestingly, RORC promoter methylation status was moderately negatively associated with gene expression of RORC (r s = -0.39, p < 0.001) and IL17A (r s = -0.37, p < 0.01), respectively. Similarly, the promoter methylation pattern of IL17A was moderately negatively correlated with IL17A gene expression (r s = -0.3, p < 0.01). There is also a moderate inverse relationship between TNFA promoter methylation status and TNFA gene expression (r s = -0.3, p < 0.01). The present study suggests an association between lower RORC, IL17A, and TNFA gene promoter methylation status with obesity-related asthma and allergic asthma. RORC, IL17A, and TNFA gene promoter methylation patterns are moderately inversely correlated with their respective mRNA expression levels. Therefore, DNA methylation may regulate RORC, IL17A, and TNF gene expression in both asthmatic phenotypes.

Unraveling the relevance of the polyadenylation factor EhCFIm25 in Entamoeba histolytica through proteomic analysis.

We recently reported that silencing of the polyadenylation factor EhCFIm25 in Entamoeba histolytica, the protozoan which causes human amoebiasis, affects trophozoite proliferation, death, and virulence, suggesting that EhCFIm25 may have potential as a new biochemical target. Here, we performed a shotgun proteomic analysis to identify modulated proteins that could explain this phenotype. Data are available via ProteomeXchange with identifier PXD027784. Our results revealed changes in the abundance of 75 proteins. Interestingly, STRING analysis, functional GO-term annotations, KEGG analyses, and literature review showed that modulated proteins are mainly related to glycolysis and carbon metabolism, cytoskeleton dynamics, and parasite virulence, as well as gene expression and protein modifications. Further studies are needed to confirm the hypotheses emerging from this proteomic analysis, to thereby acquire a comprehensive view of the molecular mechanisms involved.

Deciphering the Long Non-Coding RNAs and MicroRNAs Coregulation Networks in Ovarian Cancer Development: An Overview.

Non-coding RNAs are emergent elements from the genome, which do not encode for proteins but have relevant cellular functions impacting almost all the physiological processes occurring in eukaryotic cells. In particular, microRNAs and long non-coding RNAs (lncRNAs) are a new class of small RNAs transcribed from the genome, which modulate the expression of specific genes at transcriptional and posttranscriptional levels, thus adding a new regulatory layer in the flux of genetic information. In cancer cells, the miRNAs and lncRNAs interactions with its target genes and functional pathways are deregulated as a consequence of epigenetic and genetic alterations occurring during tumorigenesis. In this review, we summarize the actual knowledge on the interplay of lncRNAs with its cognate miRNAs and mRNAs pairs, which interact in coregulatory networks with a particular emphasis on the mechanisms underlying its oncogenic behavior in ovarian cancer. Specifically, we reviewed here the evidences unraveling the relevant roles of lncRNAs/miRNAs pairs in altered regulation of cell migration, angiogenesis, therapy resistance, and Warburg effect. Finally, we also discussed its potential clinical implications in ovarian cancer and related endocrine disease therapies.

(-)-Epicatechin protects from amebic liver abscess development in hamster.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x106E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Current Advances in the Development of Diagnostic Tests based on Aptamers in Parasitology: A Systematic Review.

Aptamers are single-stranded DNA or RNA sequences of 20-80 nucleotides that interact with different targets such as: proteins, ions, viruses, or toxins, through non-covalent interactions and their unique three-dimensional conformation. They are obtained in vitro by the systematic evolution of ligands by exponential enrichment (SELEX). Because of their ability of target recognition with high specificity and affinity, aptamers are usually compared to antibodies. However, they present many advantages that make them promising molecules for the development of new methods for the diagnosis and treatment of human diseases. In medical parasitology, aptamers also represent an attractive alternative for the implementation of new parasite detection methods, easy to apply in endemic regions. The aim of this study was to describe the current advances in the development of diagnostic tests based on aptamers in parasitology. For this, articles were selected following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, with specific inclusion and exclusion criteria. The 26 resulting articles deal with the use of aptamers for the detection of six important protozoa that affect human health. This systematic review clearly demonstrates the specificity, sensitivity and selectivity of aptamers and aptasensors, that certainly will soon become standard methods in medical parasitology.

Proteomics approaches to understand cell biology and virulence of Entamoeba histolytica protozoan parasite.

Entamoeba histolytica is the primitive eukaryotic parasite responsible of human amoebiasis, a disease characterized by bloody intestinal diarrhea and invasive extraintestinal illness. The knowledge of the complete genome sequence of virulent E. histolytica and related non-pathogenic species allowed the development of novel genome-wide methodological approaches including protein expression profiling and cellular proteomics in the so called post-genomic era. Proteomics studies have greatly increased our understanding of the cell biology of this ancient parasite. This review summarizes the current works concerning proteomics studies on cell biology, life cycle, virulence and pathogenesis, novel therapies, and protein expression regulation mechanisms in E. histolytica parasite. Also, we discuss the use of proteomics data for the development of novel therapies, the identification of potential disease biomarkers and differential diagnosis between species. SIGNIFICANCE: Entamoeba histolytica is the unicellular protozoan parasite responsible of human amoebiasis, a serious disease with worldwide distribution characterized by bloody intestinal diarrhea and invasive extraintestinal illness including peritonitis and liver, pulmonary and brain abscesses. The post-genomic era allowed the development of proteomic studies including protein expression profiling and cellular proteomics. These proteomics studies have greatly increased our understanding on cell biology, life cycle (cyst-trophozoite conversion), virulence, pathogenesis, novel therapies, and protein expression regulation mechanisms in E. histolytica. Importantly, proteomics has revealed the identity of proteins related to novel therapies, and the identification of potential disease biomarkers and proteins with use in diagnosis between species. Hopefully in the coming years, and through the use of more sophisticated omics tools, including deep proteomics, a more complete set of proteins involved in the aforementioned cellular processes can be obtained to understand the biology of this ancient eukaryote.

A novel protective role for microRNA-3135b in Golgi apparatus fragmentation induced by chemotherapy via GOLPH3/AKT1/mTOR axis in colorectal cancer cells.

Chemotherapy activates a novel cytoplasmic DNA damage response resulting in Golgi apparatus fragmentation and cancer cell survival. This mechanism is regulated by Golgi phosphoprotein-3 (GOLPH3)/Myo18A/F-actin axis. Analyzing the functions of miR-3135b, a small non-coding RNA with unknown functions, we found that its forced overexpression attenuates the Golgi apparatus fragmentation induced by chemotherapeutic drugs in colorectal cancer (CRC) cells. First, we found that miR-3135b is downregulated in CRC cell lines and clinical tumors. Bioinformatic predictions showed that miR-3135b could be regulating protein-encoding genes involved in cell survival, resistance to chemotherapy, and Golgi dynamics. In agreement, ectopic transfection of miR-3135b in HCT-15 cancer cells significantly inhibited cell proliferation, sensitized cells to 5-fluoruracil (5-FU), and promoted late apoptosis and necrosis. Also, miR-3135b overexpression impaired the cell cycle progression in HCT-15 and SW-480 cancer cells. Because GOLPH3, a gene involved in maintenance of Golgi structure, was predicted as a potential target of miR-3135b, we studied their functional relationships in response to DNA damage induced by chemotherapy. Immunofluorescence and cellular ultrastructure experiments using antibodies against TGN38 protein, a trans-Golgi network marker, showed that 5-FU and doxorubicin treatments result in an apoptosis-independent stacks dispersal of the Golgi ribbon structure in both HCT-15 and SW-480 cells. Remarkably, these cellular effects were dramatically hindered by transfection of miR-3135b mimics. In addition, our functional studies confirmed that miR-3135b binds to the 3'-UTR of GOLPH3 proto-oncogene, and also reduces the levels of p-AKT1 (Ser473) and p-mTOR (Ser2448) signaling transducers, which are key in cell survival and autophagy activation. Moreover, we found that after treatment with 5-FU, TGN38 factor coimmunolocalizes with beclin-1 autophagic protein in discrete structures associated with the fragmented Golgi, suggesting that the activation of pro-survival autophagy is linked to loss of Golgi integrity. These cellular effects in autophagy and Golgi dispersal were reversed by miR-3135b. In summary, we provided experimental evidence suggesting for the first time a novel role for miR-3135b in the protection of chemotherapy-induced Golgi fragmentation via GOLPH3/AKT1/mTOR axis and protective autophagy in colorectal cancer cells.

miR-927 has pro-viral effects during acute and persistent infection with dengue virus type 2 in C6/36 mosquito cells.

Dengue virus (DENV) is an important flavivirus that is transmitted to humans by Aedes mosquitoes, where it can establish a persistent infection underlying vertical and horizontal transmission. However, the exact mechanism of persistent DENV infection is not well understood. Recently miR-927 was found to be upregulated in C6/36-HT cells at 57 weeks of persistent infection (C6-L57), suggesting its participation during this type of infection. The aim of this study was to determine the role of miR-927 during infection with DENV type 2. The results indicate an overexpression of miR-927 in C6-L57 cells and acutely infected cells according to the time of infection and the m.o.i. used. The downregulation of miR-927 in C6-L57 cells results in a reduction of both viral titre and viral genome copy number. The overexpression of miR-927 in C6-L40 and C6/36 cells infected at an m.o.i. of 0.1 causes an increase in both viral titre and viral genome copy number, suggesting a pro-viral activity of miR-927. In silico prediction analysis reveals target mRNAs for miR-927 are implicated in post-translational modifications (SUMO), translation factors (eIF-2B), the innate immune system (NKIRAS), exocytosis (EXOC-2), endocytosis (APM1) and the cytoskeleton (FLN). The expression levels of FLN were the most affected by both miR-927 overexpression and inhibition, and FLN was determined to be a direct target of miR-927 by a dual-luciferase gene reporter assay. FLN has been associated with the regulation of the Toll pathway and either overexpression or downregulation of miR-927 resulted in expression changes of antimicrobial peptides (Cecropins A and G, and Defensin D) involved in the Toll pathway response.