Decellularized Placental Sponge: A Platform for Coculture of Mesenchymal Stem Cells/Macrophages to Assess an M2 Phenotype and Osteogenic Differentiation In Vitro and In Vivo.

Effective communication between immune and bone-forming cells is crucial for the successful healing of bone defects. This study aimed to assess the potential of a decellularized placental sponge (DPS) as a coculture system for inducing M1/M2 polarization in macrophages and promoting osteogenic differentiation in adipose-derived mesenchymal stem cells (AD-MSCs), both in vitro and in vivo. We prepared the DPS and conducted a comprehensive characterization of its biomechanical properties, antibacterial activity, and biocompatibility. In vitro, we examined the influence of the DPS on the polarization of macrophages cocultured with AD-MSCs through nitric oxide assays, cytokine assays, phagocytosis tests, and real-time polymerase chain reaction (PCR). For in vivo assessment, we utilized micro-CT imaging, histological evaluations, and real-time PCR to determine the impact of the DPS seeded with Wharton's jelly mesenchymal stem cells (WJ-MSCs) on bone regeneration in a calvarial bone defect model. The coculture of AD-MSCs and macrophages on the DPS led to increased production of IL-10, upregulation of CD206, Arg1, and YM1 gene expression, and enhanced phagocytic capacity for apoptotic thymocytes. Concurrently, it reduced the secretion of TNF-α and nitric oxide (NO), downregulated the expression of CD86, NOS2, and IRF5 genes, and decreased macrophage phagocytosis of yeast. These results indicated polarization of macrophages toward an M2-like phenotype. In vivo, the presence of the DPS resulted in enhanced bone formation at the defect site. Immunostaining demonstrated that both the DPS and DPS + WJ-MSC constructs induced macrophage polarization toward an M2 phenotype, as compared to the control defect. In conclusion, this immunomodulatory effect, coupled with its biocompatibility and biomechanical properties resembling natural bone, positions the DPS as an attractive candidate for further exploration in the field of bone tissue engineering and regenerative medicine.

Inhibition of MiR-155 Using Exosomal Delivery of Antagomir Can Up-Regulate PTEN in Triple Negative Breast Cancer.

BACKGROUND: The most aggressive form of breast cancer (BC) is Triple-Negative BC (TNBC), with the poorest prognosis, accounting for nearly 15% of all cases. Since there is no effective treatment, novel strategies, especially targeted therapies, are essential to treat TNBC. Exosomes are nano-sized microvesicles derived from cells and transport various intracellular cargoes, including microRNAs (miRNAs). MiRNAs, small non-coding RNA, are an influential factor in the development of cancerous transformations in cells. METHOD: Bioinformatics analysis of genes related to TNBC revealed that PTEN plays a crucial role in the disease. Relative expression of this gene was analyzed with RT-qPCR in 14 TNBC clinical samples. Electroporation was used to load miRNA antagomir into exosomes extracted from the conditioned medium. Then, the expression of miR-155 and PTEN was evaluated in MDA-MB-231 cells treated with antagomir-loaded exosomes. RESULTS: Based on the bioinformatics analysis, miR-155 is a potent inhibitor of PTEN. Following treatment with antagomir-loaded exosomes, RT-qPCR showed significantly reduced miR- 155 and increased PTEN levels in MDA-MB-231 cells. CONCLUSION: Based on the results of this study, exosomes can be effectively used as a cargo of oligonucleotides like miRNA mimics and antagomirs in targeted therapies.

Anti-cancer Effects of a Chitosan Based Nanoformulation Expressing miR-340 on 4T1 Breast Cancer Cells.

MicroRNAs (miRNAs) have a crucial role in the regulation of gene expression in tumor development, invasion, and metastasis. Herein, miRNA-340 (miR-340) has been shown to play tumor suppressor activity in breast cancer (BC). However, the clinical applications of miRNAs request the development of safe and effective delivery systems capable of protecting nucleic acids from degradation. In this study, biodegradable chitosan nanoparticles incorporating miR-340 plasmid DNA (pDNA) (miR-340 CNPs) were synthesized and characterized. Then, the anti-tumor effects of miR-340 CNPs were investigated using 4T1 BCE cells. The spherical nanoparticles (NPs) with an appropriate mean diameter of around 266 ± 9.3 nm and zeta potential of +17 ± 1.8 mV were successfully prepared. The NPs showed good stability, high entrapment efficiency and a reasonable release behavior, meanwhile their high resistance against enzymatic degradation was verified. Furthermore, NPs demonstrated appropriate transfection efficiency and could induce apoptosis, so had toxicity in 4T1 BCE cells. Also, CD47 expression on the surface of cancer cells was significantly reduced after treatment with miR-340 CNPs. The results showed that miR-340 CNPs augmented the expression of P-27 in BC cells. Furthermore, miR-340 CNPs caused down-regulation of BRP-39 (breast regression protein-39) increasingly suggested as a prognostic biomarker for neoplastic diseases like BC. In conclusion, our data show that miR-340 CNPs can be considered as a promising new platform for BC gene therapy.

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.

BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production. OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania. RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.

Impact of curcumin on ferroptosis-related genes in colorectal cancer: Insights from in-silico and in-vitro studies.

Colorectal cancer (CRC) is responsible for a significant number of cancer-related fatalities worldwide. Researchers are investigating the therapeutic potential of ferroptosis, a type of iron-dependent controlled cell death, in the context of CRC. Curcumin, a natural compound found in turmeric, exhibits anticancer properties. This study explores the effects of curcumin on genes related to ferroptosis (FRGs) in CRC. To gather CRC data, we used the Gene Expression Profiling Interactive Analysis (GEPIA) and Gene Expression Omnibus (GEO) databases, while FRGs were obtained from the FerrDb database and PubMed. We identified 739 CRC differentially expressed genes (DEGs) in CRC and discovered 39 genes that were common genes between FRGs and CRC DEGs. The DEGs related to ferroptosis were enriched with various biological processes and molecular functions, including the regulation of signal transduction and glucose metabolism. Using the Drug Gene Interaction Database (DGIdb), we predicted drugs targeting CRC-DEGs and identified 17 potential drug targets. Additionally, we identified eight essential proteins related to ferroptosis in CRC, including MYC, IL1B, and SLC1A5. Survival analysis revealed that alterations in gene expression of CDC25A, DDR2, FABP4, IL1B, SNCA, and TFAM were associated with prognosis in CRC patients. In SW480 human CRC cells, treatment with curcumin decreased the expression of MYC, IL1B, and EZH2 mRNA, while simultaneously increasing the expression of SLCA5 and CAV1. The findings of this study suggest that curcumin could regulate FRGs in CRC and have the potential to be utilized as a therapeutic agent for treating CRC.

Mesenchymal stem cell-derived exosomes enriched with miR-218 reduce the epithelial-mesenchymal transition and angiogenesis in triple-negative breast cancer cells.

BACKGROUND: The epithelial-mesenchymal transition (EMT) and angiogenesis are morphogenetic processes implicated in tumor invasion and metastasis. It is found that the aberrant expression of microRNAs (miRNAs) contributes to these processes. Exosomes are considered potential natural vehicles for miRNA delivery in cancer therapy. miR-218 is one of the tumor suppressor miRNAs and its downregulation is associated with EMT and angiogenesis. We aimed to use adipose mesenchymal stem cells-derived exosomes (ADMSC-exosomes) for miR-218 delivery to breast cancer cells and evaluate miR-218 tumor-suppressing properties in vitro. METHODS: Exosomes were isolated from conditioned media of ADMSCs. miR-218 was loaded to exosomes using electroporation. mRNA expression of target genes (Runx2 and Rictor) in MDA-MB-231 breast cancer cells was evaluated by qPCR. To explore the effects of miR-218 containing exosomes on breast cancer cells, viability, apoptosis, and Boyden chamber assays were performed. The angiogenic capacity of MDA-MB-231 cells after treatment with miR-218 containing exosomes was assessed by in vitro tube formation assay. RESULTS: miR-218 mimic was efficiently loaded to ADMSC-exosomes and delivered to MDA-MB-231 cells. Exposure to miR-218 containing exosomes significantly decreased miR-218 target genes (Runx2 and Rictor) in MDA-MB-231 cells. They increased the expression of epithelial marker (CDH1) and reduced mesenchymal marker (CDH2). miR-218 restoration using miR-218 containing exosomes reduced viability, motility, invasion, and angiogenic capacity of breast cancer cells. CONCLUSIONS: These findings suggest that ADMSC-exosomes can efficiently restore miR-218 levels in breast cancer cells and miR-218 can prevent breast cancer progression with simultaneous targeting of angiogenesis and EMT.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

TGF-ß Targeted by miR-27a Modulates Anti-Parasite Responses of Immune System.

Background: Immune cells and their secreted cytokines are known as the first barrier against pathogens. Leishmania major as an intracellular protozoan produces anti-inflammatory cytokines that lead to proliferation and survival of the parasite in the macrophages. miRNAs are small non-coding RNA molecules that regulate mRNAs expression. We aimed to investigate the relationship between the expression of TGF-ß and a bioinformatically candidate miRNA, in leishmaniasis as a model of TGF-ß overexpression. Methods: The miRNAs that target TGF-ß -3'UTR were predicted and scored by bioinformatic tools. After cloning of TGF-ß-3'UTR in psi-CHECK ™- 2 vector, targeting validation was confirmed using Luciferase assay. After miRNA mimic transfection, the expression of miR-27a, TGF-ß, as well as Nitric Oxide concentration was evaluated. Results: miR-27a received the highest score for targeting TGF-ß in bioinformatic predictions. Luciferase assay confirmed that miR-27a is targeting TGF-ß-3'UTR, since miR-27a transfection decreased the luciferase activity. After miRNA transfection, TGF-ß expression and Nitric Oxide concentration were declined in L. major infected macrophages. Conclusion: Bioinformatic prediction, luciferase assay, and miRNA transfection results showed that miR-27a targets TGF-ß. Since miRNA and cytokine-base therapies are developing in infectious diseases, finding and validating miRNAs targeting regulatory cytokines can be a novel strategy for controlling and treating leishmaniasis.

miRNAs Delivery for Cancer-associated Fibroblasts' Activation and Drug Resistance in Cancer Microenvironment.

Cancer-associated fibroblasts (CAFs) as a major component of cancer stroma contribute to diverse procedures of most solid tumors and might be a targeted cancer therapy approach. Their specified features, related signaling pathways, distinct biomarkers, and sub-populations need to be deciphered. There is a need for CAF extraction or induction for in vitro investigations. Some miRNAs could activate CAF-like phenotype and they also interfere in CAF-mediated drug resistance, aggressiveness, and metastatic behaviors of several cancer cell types. Due to the complex relevance of miRNA and CAFs, these non-coding oligonucleotides may serve as attractive scope for anti-cancer targeted therapies, but the lack of an efficient delivery system is still a major hurdle. Here, we have summarized the investigated information on CAF features, isolation, and induction procedures, and highlighted the miRNA-CAF communications, providing special insight into nano-delivery systems.

Exosomes derived from rapamycin-treated 4T1 breast cancer cells induced polarization of macrophages to M1 phenotype.

M2 macrophages are the most prevalent type in the tumor microenvironment and their polarization to M1 type can be used as a potential cancer immunotherapy. Here, we investigated the role of tumor microenvironment and particularly purified exosomes in M2 to M1 macrophage polarization. Rapamycin treatment on triple-negative breast cancer cells (TNBC) was performed. Tumor cells-derived exosomes (called texosomes) were isolated and characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, high-performance liquid chromatography, Fourier transform infrared, and Western blot assays. M2 mouse peritoneal macrophages were treated with rapamycin or rapamycin-texosome. Then, M1/M2 phenotype-specific marker genes and proteins were measured to assess the degree of M2 to M1 polarization. Finally, nitric oxide (NO) production, phagocytosis, and efferocytosis assays were assessed to verify the functionality of the polarized macrophages. Purified rapamycin-texosomes significantly increased the expression of the M1 markers (Irf5, Nos2, and CD86) and decreased M2 markers (Arg, Ym1, and CD206). In addition, the levels of M1-specific cytokines tumor necrosis factor alpha and interleukin 1ß (IL-1ß) were increased, whereas the levels of M2 specific cytokines IL-10 and transforming growth factor beta were declined. Furthermore, texosome treatment increased NO concentration and phagocytosis and decreased efferocytosis indicating M1 polarization. These findings suggest rapamycin-texosomes can induce M2 to M1 macrophages polarization as a potential immunotherapy for TNBC.

Photobiomodulation, alone or combined with adipose-derived stem cells, reduces inflammation by modulation of microRNA-146a and interleukin-1ß in a delayed-healing infected wound in diabetic rats.

Diabetic wounds are categorized by chronic inflammation, leading to the development of diabetic foot ulcers, which cause amputation and death. Herewith, we examined the effect of photobiomodulation (PBM) plus allogeneic diabetic adipose tissue-derived stem cells (ad-ADS) on stereological parameters and expression levels of interleukin (IL)-1ß and microRNA (miRNA)-146a in the inflammatory (day 4) and proliferation (day 8) stages of wound healing in an ischemic infected (with 2×107 colony-forming units of methicillin-resistant Staphylococcus aureus) delayed healing wound model (IIDHWM) in type I diabetic (TIDM) rats. There were five groups of rats: group 1 control (C); group 2 (CELL) in which rat wounds received 1×106 ad-ADS; group 3 (CL) in which rat wounds received the ad-ADS and were subsequently exposed to PBM(890 nm, 80 Hz, 3.5 J/cm2, in vivo); group 4 (CP) in which the ad-ADS preconditioned by the PBM(630 nm + 810 nm, 0.05 W, 1.2 J/cm2, 3 times) were implanted into rat wounds; group 5 (CLP) in which the PBM preconditioned ad-ADS were implanted into rat wounds, which were then exposed to PBM. On both days, significantly better histological results were seen in all experimental groups except control. Significantly better histological results were observed in the ad-ADS plus PBM treatment correlated to the ad-ADS alone group (p<0.05). Overall, PBM preconditioned ad-ADS followed by PBM of the wound showed the most significant improvement in histological measures correlated to the other experimental groups (p<0.05). On days 4 and 8, IL-1 ß levels of all experimental groups were lower than the control group; however, on day 8, only the CLP group was different (p<0.01). On day 4, miR-146a expression levels were substantially greater in the CLP and CELL groups correlated to the other groups, on day 8 miR-146a in all treatment groups was upper than C (p<0.01). ad-ADS plus PBM, ad-ADS, and PBM all improved the inflammatory phase of wound healing in an IIDHWM in TIDM1 rats by reducing inflammatory cells (neutrophils, macrophages) and IL-1ß, and increasing miRNA-146a. The ad-ADS+PBM combination was better than either ad-ADS or PBM alone, because of the higher proliferative and anti-inflammatory effects of the PBM+ad-ADS regimen.

Mir-4699 promotes the osteogenic differentiation of mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) are drawing considerable attention in the field of regenerative medicine due to their differentiation capabilities. The miRNAs are among the most important epigenetic regulators of MSC differentiation. Our previous study identified miR-4699 as a direct suppressor of the DKK1 and TNSF11 gene expression. However, the precise osteogenic-related phenotype or mechanism caused by miR-4699 change has yet to be dealt with in depth. MATERIAL AND METHODS: In the present study, miR-4699 mimics were transfected into human Adipose tissue-derived mesenchymal stem cells (hAd-MSCs) and osteoblast marker gene expression (RUNX2, ALP, and OCN), was analyzed to investigate whether miR-4699 promotes osteoblast differentiation of hAd-MSCs through targeting the DKK-1 and TNFSF11. We further examined and compared the effects of recombinant human BMP2 with miR-4699 on cell differentiation. In addition to quantitative PCR, analysis of alkaline phosphatase activity, calcium content assay, and Alizarin red staining were used to explore osteogenic differentiation. To evaluate the effect of miR-4699 on its target gene (on protein level) we utilized the western blotting technique. RESULTS: The overexpression of miR-4699 in hAd-MSCs resulted in the stimulation of alkaline phosphatase activity, osteoblast mineralization, and the expression of RUNX2, ALP, and OCN osteoblast marker genes. CONCLUSION: Our findings indicated that miR-4699 supported and synergized the BMP2-induced osteoblast differentiation of mesenchymal stem cells. We suggest, thereof, the utilization of hsa-miR-4699 for further in vivo experimental investigation to reveal the potential therapeutic impact of regenerative medicine for different types of bone defects.

Exosomal delivery of 7SK long non-coding RNA suppresses viability, proliferation, aggressiveness and tumorigenicity in triple negative breast cancer cells.

AIMS: RN7SK (7SK), a highly conserved non-coding RNA, serves as a transcription regulator via interaction with a few proteins. Despite increasing evidences which support the cancer-promoting roles of 7SK-interacting proteins, limited reports address the direct link between 7SK and cancer. To test the hypothetic suppression of cancer by overexpression of 7SK, the effects of exosomal 7SK delivery on cancer phenotypes were studied. MATERIALS AND METHODS: Exosomes derived from human mesenchymal stem cells were loaded with 7SK (Exo-7SK). MDA-MB-231, triple negative breast cancer (TNBC), cell line was treated with Exo-7sk. Expression levels of 7SK were evaluated by qPCR. Cell viability was assessed via MTT and Annexin V/PI assays as well as qPCR assessment of apoptosis-regulating genes. Cell proliferation was evaluated by growth curve analysis, colony formation and cell cycle assays. Aggressiveness of TNBCs was evaluated via transwell migration and invasion assays and qPCR assessment of genes regulating epithelial to mesenchymal transition (EMT). Moreover, tumor formation ability was assessed using a nude mice xenograft model. KEY FINDINGS: Treatment of MDA-MB-231 cells with Exo-7SK resulted in efficient overexpression of 7SK; reduced viability; altered transcription levels of apoptosis-regulating genes; reduced proliferation; reduced migration and invasion; altered transcription of EMT-regulating genes; and reduced in vivo tumor formation ability. Finally, Exo-7SK reduced mRNA levels of HMGA1, a 7SK interacting protein with master gene regulatory and cancer promoting roles, and its bioinformatically-selected cancer promoting target genes. SIGNIFICANCE: Altogether, as a proof of the concept, our findings suggest that exosomal delivery of 7SK may suppress cancer phenotypes via downregulation of HMGA1.

MicroRNA-122 Is More Effective than Rapamycin in Inhibition of Epithelial-mesenchymal Transition and mTOR Signaling Pathway in Triple Negative Breast Cancer.

The fundamental mechanism responsible for the aggressiveness of metastatic cancers such as triple-negative breast cancer (TNBC) is the epithelial-mesenchymal transition (EMT). In cancer microenvironments, the Phosphoinositide 3-kinases (PI3K)-Akt- mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating the EMT mechanism. The current study focuses on the impacts of rapamycin, a newly retargeted chemotherapeutic agent against mTOR, and MicroRNA (miR)-122 on the aggressive behavior of TNBC.  The half-maximal inhibitory concentration (IC50) of rapamycin on 4T1 cells was determined using an MTT assay. Also, miR-122 was transiently transfected into 4T1 cells to study its effect on the pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression level of central mTOR and EMT-related cascade genes. Moreover, cell mobility and migration were evaluated using scratch and migration assays, respectively. Both rapamycin and miR-122 significantly decreased the expression levels of PI3K, AKT, and mTOR, as well as ZeB1 and Snail genes. However, no significant change was observed in Twist gene expression. Furthermore, scratch and migration assays revealed that the migration of 4T1 cells was markedly reduced, especially following miR-122 induction. Our experimental findings and gene enrichment studies indicated that miR-122 mainly operates on multiple metabolic pathways, as well as EMT and mTOR, while rapamycin has restricted targets in cancer cells.  Consequently, miR-122 can be considered a potential cancer microRNA therapy option, which can be validated in the future in animal studies to demonstrate its efficacy in cancer control.

Screening and introduction of key cell cycle microRNAs deregulated in colorectal cancer by integrated bioinformatics analysis.

Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men worldwide. Impaired cell cycle regulation leads to many cancers and is also approved in CRC. Therefore, cell cycle regulation is a critical therapeutic target for CRC. Furthermore, miRNAs have been discovered as regulators in a variety of cancer-related pathways. This study is designed to investigate how miRNAs and mRNAs interact to regulate the cell cycle in CRC patients. Utilizing the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Expression Omnibus (GEO), and Therapeutic Target Database (TTD), cell cycle-associated genes were identified and evaluated. Seven of the 22 differentially expressed genes (DEGs) implicated in the cell cycle in three GSEs (GSE24514, GSE10950, and GSE74604) were identified as potential therapeutic targets. Then, using PyRx software, we performed docking proteins with selected drugs. The results demonstrated that these drugs are appropriate molecules for targeting cell cycle DEGs. Tarbase, miRTarbase, miRDIP, and miRCancer databases were used to find miRNAs that target the indicated genes. The ability of these six miRNAs to impact the cell cycle in colorectal cancer may be concluded. These miRNAs were found to be downregulated in SW480 cells when compared to the normal tissue. Our data imply that a precise selection of bioinformatics tools can facilitate the identification of miRNAs that impact mRNA translation at different stages of the cell cycle. The candidates can be investigated more as targets for cell cycle arrest in cancers.

Effect of Calcitriol Treated Mesenchymal Stem Cells as an Immunomodulation Micro-environment on Allergic Asthma in a Mouse Model.

BACKGROUND: Allergic asthma is a chronic inflammatory illness of the respiratory system characterized by an increase in the number of inflammatory cells in the airways and trouble breathing. Mesenchymal stem cells (MSCs) have the potential to be used in inflammatory diseases as a cellular immunosuppressive treatment. They express calcitriol receptors and communicate with other immunocytes, which increases their anti-inflammatory activity. This study aimed to determine the effects of calcitriol-treated MSC treatment on allergic asthma pathways in a mouse model. METHODS: To generate a mouse model of asthma, the mice were sensitized intraperitoneally with ovalbumin (OVA) and aluminum hydroxide emulsion and then challenged intra-nasally with OVA. On day 14, experimental mice received tail vein injections of calcitriol-treated MSCs in PBS prior to allergen exposure. The cytokines assays including IL-4, 10, 12, 17, TGF-ß and IFN-γ, splenocytes proliferation, and histological examination of lungs samples were performed. The mice were sensitized with OVA and the response to dexamethasone treatment was compared. RESULTS: Calcitriol-treated MSCs significantly increased the levels of IL-12, TGF-ß, and IFN-γ compared to non-treated MSCs groups. Moreover, calcitriol-treated and non-treated MSCs significantly decreased IL-4 and IL-17 compared to asthmatic groups. The results of the histopathological examination showed that calcitriol-treated MSCs reduced the accumulation of inflammatory cells and bronchial wall thickening in comparison with the asthma group. CONCLUSION: Using the allergic asthma model, we were able to show that calcitriol-treated MSCs had an inhibitory impact on airway inflammation. Our findings suggest that the injection of calcitrioltreated MSCs may be a viable treatment option for allergic asthma.

Identification of miR-3182 and miR-3143 target genes involved in the cell cycle as a novel approach in TNBC treatment: A systems biology approach.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis, lacking therapeutic targets. miRNAs play crucial roles in TNBC through regulating various mechanisms, including cellular growth and proliferation. This study aims to identify critical target genes of two novel miRNAs (miR-3143 and miR-3182) involved in the cell cycle of TNBC as possible therapeutic targets and investigates their regulatory and therapeutic roles through a systems biology approach and in vitro experiment. Datasets related to the TNBC cell line (MDA-MB-231) were screened and retrieved, and Gene regulatory networks were constructed. Significant regulatory motifs were detected and analyzed using the FANMOD and Cytoscape analyzer, and the clusters and seeds were identified using the MCODE. Functional enrichment analysis was also performed using DAVID and STRING. The most critical genes were determined using the analysis of GRN motifs and PPI clusters. The essential genes involved in the cell cycle were selected and verified using the bc-GenExMiner v4.7. We overexpressed miR-3143 and miR-3182 in the MDA-MB-231 cell line using human umbilical cord mesenchymal stem cell (HUCMSC)-miRNA loaded exosomes, and the expression of the critical target genes was investigated using RT-qPCR. We identified eight critical genes as potential therapeutic targets. Their expression decreased by overexpression of miR-3143 and miR-3182 in RT-qPCR. The identified critical genes have probably significant roles in the pathogenesis of TNBC through the cell cycle. We suggest that the overexpression of miR-3143 and miR-3182 could be a new therapeutic candidate in TNBC and is worth more investigation.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

PURPOSE: In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2. METHODS: Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed. RESULTS: The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively. CONCLUSIONS: The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.

Coculture of adipose-derived mesenchymal stem cells/macrophages on decellularized placental sponge promotes differentiation into the osteogenic lineage.

BACKGROUND: Several factors like three-dimensional microstructure, growth factors, cytokines, cell-cell communication, and coculture with functional cells can affect the stem cells behavior and differentiation. The purpose of this study was to investigate the potential of decellularized placental sponge as adipose-derived mesenchymal stem cells (AD-MSCs) and macrophage coculture systems, and guiding the osteogenic differentiation of stem cells. METHODS: The decellularized placental sponge (DPS) was fabricated, and its mechanical characteristics were evaluated using degradation assay, swelling rate, and pore size determination. Its structure was also investigated using hematoxylin and eosin staining and scanning electron microscopy. Mouse peritoneal macrophages and AD-MSCs were isolated and characterized. The differentiation potential of AD-MSCs co-cultured with macrophages was evaluated by RT-qPCR of osteogenic genes on the surface of DPS. The in vivo biocompatibility of DPS was determined by subcutaneous implantation of scaffold and histological evaluations of the implanted site. RESULTS: The DPS had 67% porosity with an average pore size of 238 µm. The in vitro degradation assay showed around 25% weight loss during 30 days in PBS. The swelling rate was around 50% during 72 h. The coculture of AD-MSCs/macrophages on the DPS showed a significant upregulation of four differentiation osteogenic lineage genes in AD-MSCs on days 14 and 21 and a significantly higher mineralization rate than the groups without DPS. Subcutaneous implantation of DPS showed in vivo biocompatibility of scaffold during 28 days follow-up. CONCLUSIONS: Our findings suggest the decellularized placental sponge as an excellent bone substitute providing a naturally derived matrix substrate with biostructure close to the natural bone that guided differentiation of stem cells toward bone cells and a promising coculture substrate for crosstalk of macrophage and mesenchymal stem cells in vitro.

Downregulation of NRARP exerts anti-tumor activities in the breast tumor cells depending on Wnt/ß-catenin-mediated signals: The role of miR-130a-3p.

The Notch-regulated ankyrin repeat protein (NRARP) functions as a molecular link between Notch and Wnt signaling pathways. Although it has recently been identified to be overexpressed in breast cancer (BC), the molecular mechanisms that regulate NRARP remain unknown. Since microRNAs (miRNAs) regulate gene expression post-transcriptionally, miRNA dysregulation could explain the abnormal gene expression. Here, we identified miR-130a-3p as an NRARP regulator and evaluated its effects on the behavior of BC cells. Quantitative real-time PCR was performed to assess the transcriptional levels of miR-130a-3p and NRARP in BC cells. Next, miR-130a-3p was transiently transfected into BC cells to assess its influence on NRARP expression. Owing to the positive regulatory effects of NRARP on the Wnt/ß-catenin signaling pathway, we also analyzed the expression levels of five Wnt/ß-catenin pathway genes and one downstream target gene in BC cells. We then assessed anti-tumor activities of miR-130a-3p in BC cells using the MTT proliferation assay, the soft agar colony formation assay for anchorage-independent growth (AIG), as well as scratch and transwell assays for cell migration. The results showed that miR-130a-3p was downregulated in BC cells, whereas NRARP was upregulated. Overexpression of miR-130a-3p inhibited the expression of NRARP and some Wnt/ß-catenin signaling pathway genes, as well as exerted anti-tumor effects as evidenced by decreased cell proliferation, AIG, and migration of BC cells. In conclusion, the tumor-suppressive function of miR-130a-3p in BC may be mediated by inhibiting NRARP and Wnt/ß-catenin signaling pathway. As a result, miR-130a-3p could be introduced as a therapeutic target for miRNA therapy in BC.