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Abstract MicroRNA expression in breast cancer (BC) is explored both as a potential biomarker and for therapeutic purposes. Recent studies have revealed that miR-203a-3p is involved in BC, and importantly contributes to BC chemotherapy responses; however, the regulatory pathways of miR-203a in BC remain elusive. Hence, we aimed to investigate the miR-203a regulatory mechanisms and their potential functions in the progress of BC. To this end, the miR-203a potential involving pathways was predicted by databases analyzing its target genes. The relations between miR-203a, the phosphatidylinositol 3′-kinase (PI3K)–Akt, and Wnt signaling pathways were mechanistically investigated. Our results revealed that miR-203a inhibited the activation of the PI3K/Akt and Wnt pathways and reduced its downstream cell cycle signals, including Cyclin D1 and c-Myc. Moreover, the overexpression of miR-203a drastically arrested the cell cycle at subG1 and G1 phases, decreased the viability, proliferation, and migration, and increased apoptosis of BC cells. Therefore, miR-203a-3p may be considered a tumor suppressor factor and a potential biomarker or therapeutic target for BC.

Abstract Background GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. Methods To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. Results Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. Conclusions To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

Abstract HER-2/neu (HER2) is a member of the epidermal growth factor receptors family, encoding a protein with tyrosine kinase activity. Following the gene amplification or increased HER2 transcription, carcinogenesis has been observed in some cancers. Genetic and epigenetic changes occurring in enhancer sequences can deeply affect the expression and transcriptional regulation of downstream genes, which can cause some physiological and pathological changes, including tumor progression. A therapeutic approach that directly targets the genomic sequence alterations is of high importance, with low side effects on healthy cells. Here, we employed the CRISPR/Cas9 method to genetically knockout an expressed putative enhancer (GH17J039694; we coined it as Her2-Enhancer1) located within the HER2 gene, 17q12: 39,694,339–39,697,219 (UCSC-hg38). We then investigated the potential regulatory effect of Her2-Enhancer1 on HER2 and HER2-interacting genes. To evaluate the cis and trans effects of Her2-Enhancer1, genetic manipulation of this region was performed in HER2-positive and -negative breast cancer cells. Our bioinformatics and real-time PCR data revealed that this putative enhancer region is indeed expressed, and acts as an expressed enhancer. Further functional analysis on edited and unedited cells revealed a significant alteration in the expression of HER2 variants, as well as some other target genes of HER2. Moreover, the apoptosis rate was considerably elevated within the edited cells. As we expected, Western blot analysis confirmed a reduction in protein levels of HER2, GRB7, the gene interacting with HER2, and P-AKT in the PI3K/AKT pathway. Altogether, our findings revealed an enhancer regulatory role for Her2-Enhancer1 on HER2 and HER2-interacting genes; and that this region has a potential for targeted therapy of HER2-positive cancers.

Abstract Background Carcinoma of the breast, a prevailing factor in female mortality worldwide, involves dysregulation of lncRNAs and microRNAs. Aim The main goal of this research was to predict and experimentally examine the LINC01405 expression status in breast cancer subtypes, along with investigation of its interaction with miR‐29b and miR‐497‐5p that results in regulating PI3‐Kinase, WNT, and TGF‐beta signaling pathways. Methods and Results We performed a meta‐analysis of five GEO datasets, encompassing microarray and RNA‐seq data, to identify differentially expressed genes. The Cancer Genome Atlas transcriptome dataset was also analyzed to determine essential gene modules, associated with different stages of breast cancer by weighted gene co‐expression networks. In addition, networks of drug‐gene interactions were constructed to explore potential treatment options. LINC01405 as a microRNA sponge was chosen and examined. furthermore, downstream target genes were discovered. Experimental validation consisted of plasmid constructs used in cell culture experiments, RT‐qPCR for expression analysis, and cell cycle assays. Our bioinformatics findings showed higher LINC01405 expression in Basal‐like triple‐negative breast carcinoma. In contrast, lower expression in Luminal samples was observed compared with normal samples, which was consistently observed in both breast cancer tissues and cell lines. LINC01405 expression level was correlated with miR‐29b and miR‐497 levels. The MDA‐MB‐231 cell line demonstrated higher LINC01405 expression and lower miR‐29b and miR‐497 expression levels. However, SKBR3 and MCF7 cells had lower LINC01405 expression and higher miR‐29b and miR‐497 levels, suggesting a regulatory role for LINC01405 as a competing endogenous RNA. This was experimentally confirmed when LINC01405 was overexpressed in SKBR3 cells, and the common target genes of miR‐29b and miR‐497 were upregulated. Additionally, LINC01405 upregulation led to the increased cell populations, proliferation, and upregulation of critical cancer‐related genes, including AKT1, AKT3, mTOR, WNT3A, SMAD3, CYCLIN D1, CYCLIN D2, BCL2, and GSK3B. Conclusion We revealed the differential expression of LINC01405 in several types of breast cancer and its role in regulating signaling pathways, potentially via scavenging miRNAs. These findings clarified the role of LINC01405 in breast cancer development and identified potential therapeutic targets.

Abstract Melatonin is a pleiotropic molecule that can influence various aspects of plant performance. Recent studies have exhibited that it mediates plant defensive responses, probably through managing redox homeostasis. We tried to track the regulatory effects of melatonin on the antioxidant machinery of Linum album cell culture. To this, different concentrations of melatonin were applied, and the oxidative status of cells was investigated by measuring the levels of oxidative molecules and antioxidant agents. The results showed that H2O2 content did not change at the low melatonin levels, while it increased at the high concentrations. It can be correlated with the low melatonin dosages capacity to remove excessive amounts of H2O2, while the high melatonin dosages exhibit toxicity effects. In contrast, the NO enhancement occurred at 50 μM melatonin, proposing its role in triggering melatonin-induced defensive responses. The MDA results stated that NO led to oxidative stress in melatonin-treated cells at 50 μM melatonin. Antioxidant enzyme POD was activated by melatonin treatment, while SOD enzyme behaved reversely which can explain the changes in the H2O2 level. In addition, the analysis of the phenolics profile showed that the contents of phenolic acids, flavonoids, and lignans enhanced following an increase in PAL enzyme activity. The increased level of phenolic hormone SA can indicate that melatonin affects the defensive responses in L. album cells through a SA-dependent pathway. In general, it seems that melatonin, by modulating NO and SA levels, can induce the activity of antioxidant enzymes and the production of phenolics, especially lignans, in L. album cells.

Cardiac fibrosis (c-fibrosis) is a critical factor in cardiovascular diseases, leading to impaired cardiac function and heart failure. This study aims to optimize the isoproterenol (ISO)-induced c-fibrosis model and evaluate the therapeutic efficacy of dendrosomal nano-curcumin (DNC) in both in-vitro and in-vivo conditions. Also, we were looking for the differentially expressed genes following the c-fibrosis induction. At the in-vitro condition, primary cardiac fibroblasts were exclusively cultured on collagen-coated or polystyrene plates and, were treated with ISO for fibrosis induction and post-treated or co-treated with DNC. RT-qPCR and flow cytometry analysis indicated that DNC treatment attenuated the fibrotic effect of ISO treatment in these cells. At the in-vivo condition, our findings demonstrated that ISO treatment effectively induces cardiac (and pulmonary) fibrosis, characterized by pro-fibrotic and pro-inflammatory gene expression and IHC (α-SMA, COL1A1, and TGFβ). Interestingly, fibrosis symptoms were reduced following the pretreatment, co-treatment, or post-treatment of DNC with ISO. Additionally, the intensive RNAseq analysis suggested the COMP gene is differentially expressed following the c-fibrosis and our RT-qPCR analysis suggested it as a novel potential marker. Overall, our results promise the application of DNC as a potential preventive or therapy agent before and after heart challenges that lead to c-fibrosis. [ABSTRACT FROM AUTHOR]

Strand debonding patterns and release (prestress transfer) practices can result in significant local stresses at prestressed girder ends, which are not accounted for in design. This article presents a numerical and analytic study of various single-webbed prestressed concrete girder shapes for which large amounts of prestressing are necessitated by the use of 0.7 in. (17.8 mm) diameter strands. Shapes with wide bottom flanges are shown to potentially exhibit large stresses that effectively pry, or peel, the portions of the flanges extending from the web away from the web. Such peeling stresses can be mostly mitigated by partially debonding strands in the recommended pattern from the outside in. Similarly, releasing or cutting strands in a uniform manner mitigates peeling stresses. While releasing all strands simultaneously is optimal, it is unlikely to be practical for large prestressed components. For conventional release operations, the results presented in this article indicate that a symmetrical top-down method would not result in significant peeling stress. Moreover, prestressed girder end region detailing requirements aimed at providing adequate flange confinement and strand anchorage at the ultimate limit would be adequate to control peeling stresses, including those resulting from the inadvertent use of a poor release sequence. [ABSTRACT FROM AUTHOR]

Background: Hsa-miR-495 (miR-495) has been extensively investigated in cancer initiation and progression. On the other hand, our bioinformatics analysis suggested that miR-495 exerts its effects through targeting of TGFβ signaling components. Methods & Results: In order to investigate such an effect, miR-495 precursor was overexpressed in HEK293T, SW480, and HCT116 cells, which was followed by downregulation of TGFβR1, TGFβR2, SMAD4, and BUB1 putative target genes, detected by RT-qPCR. Also, luciferase assay supported the direct interaction of miR-495 with 3′UTR sequences of TGFβR1, TGFβR2, SMAD4, and BUB1 genes. Furthermore, a negative correlation of expression between miR-495-3p and some of these target genes was deduced in a set of colorectal and breast cancer cell lines. Then, flow cytometry analysis showed that the overexpression of miR-495 in HCT116 and HEK293T resulted in an arrest at the G1 phase. Consistently, western blotting analysis showed a significant reduction of the Cyclin D1 protein in the cells overexpressing miR-495, pointing to downregulation of the TGFβ signaling pathway and cell cycle arrest. Finally, microarray data analysis showed that miR-495-3p is significantly downregulated in colorectal tumors, compared to the normal pairs. Conclusions: Overall, the results of the current study introduced miR-495-3p as a cell cycle progression suppressor, which may negatively regulate TGFβR1, TGFβR2, SMAD4, and BUB1 genes. This finding suggests miR-495-3p as a tumor suppressor candidate for further evaluation.

Abstract Cardiac fibrosis in the failing heart is modulated by activated myofibroblasts, and is a pathology marked by their deposition of extracellular matrix proteins. The TGFβ signaling pathway is important in stimulating fibrosis and therefore seems an attractive new target for anti-fibrotic therapy. The relationship between ncRNAs and TGFβ signaling pathway has been extensively studied. Here, we have provided several lines of evidence to prove that the fibrosis process could be regulated by miR-331 through targeting TGFβ signaling. First, bioinformatics analysis and dual luciferase assay validated a direct interaction between the miR-331 and TGFβ-R1 3′UTR sequence which results in the downregulation of TGFβ signaling pathway. Second, miR-331 expression was inversely related to the expression of a number of genes which are involved in extracellular matrix (ECM) production and deposition processes, both in the in vivo and in vitro fibrosis models. Third, in cultured mouse and human cardiac myofibroblasts (CMyoFbs) under ISO treatment, overexpression of miR-331 decreased the expression level of fibrosis-related genes. Consistently, western blot analysis confirmed that miR-331 overexpression ended in both Smad3 and Col1A1 protein level reduction in mouse cardiac myofibroblasts. Finally, flow cytometry analysis, cyclin D1 and D2 gene expression analysis, and wound-healing assay confirmed the inhibitory effect of miR-331 against cell proliferation and migration in ISO-treated cardiac myofibroblasts. Taken together, accumulative results showed that miR-331 reduced the level of fibrosis-related proteins in cardiac myofibroblasts culture via regulating TGFβ signaling pathway.

Gastric cancer is one of the common causes of cancer mortality worldwide, with a low survival rate for the affected people. Recent studies have revealed the key role of long non-coding RNAs (lncRNAs) in the development and progression of many cancers, including gastric cancer. Looking for the potential molecular regulators of gastric cancer incidence and progression, LINC02381 was identified as a downregulated lncRNA in gastric cancer tissues by analysis of available microarray and RNA-seq data and RT-qPCR confirmed this differential expression. MiR-21, miR-590, and miR-27a miRNAs were predicted to be sponged by LINC02381, and dual luciferase assay verified LINC02381 as a competitive endogenous RNA (CeRNA), which binds to them. Furthermore, we found that increased expression of LINC02381 attenuates Wnt pathway activity. Also, functional analysis indicates that LINC02381 arrests cell cycle, increases apoptosis and caspase activity, and reduces cell survival and proliferation rate of the human gastric cancer cell lines AGS and MKN45. Moreover, EMT analysis showed that LINC02381 is involved in gastric cancer progression and inhibits metastasis. Overall, this work for the first time introduces LINC02381 as a CeRNA involved in gastric cancer and provides novel insight into the molecular pathogenesis of gastric cancer.

Cardiomyocyte (CM) maturation, which is characterized by structural, functional, and metabolic specializations, is the last phase of CM development that prepares the cells for efficient and forceful contraction throughout life. Over the past decades, CM maturation has gained increased attention due to the fact that pluripotent stem cell-derived CMs are structurally, transcriptionally, and functionally immature and embryonic-like, which causes a defect in cell replacement therapy. The current challenge is to discover and understand the molecular mechanisms, which control the CM maturation process. Currently, emerging shreds of evidence emphasize the role of long noncoding RNAs (lncRNAs) in regulating different aspects of CM maturation, including myofibril maturation, electrophysiology, and Ca2+ handling maturation, metabolic maturation and proliferation to hypertrophy transition. Here, we describe the structural and functional characteristics of mature CMs. Furthermore, this review highlights the lncRNAs as crucial regulators of different aspects in CM maturation, which have the potential to be used for mature CM production. With the current advances in oligonucleotide delivery; lncRNAs may serve as putative therapeutic targets to produce highly mature CMs for research and regenerative medicine.