Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

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.

Immunodiagnosis and molecular validation of Toxoplasma gondii-recombinant dense granular (GRA) 5 protein for the detection of toxoplasmosis in hemodialysis patients.

Toxoplasmosis causes serious complications in immunocompromised and pregnant women. Serological tests for the detection of toxoplasmosis are often designed from parasitic tachyzoites antigens. The process of producing these antigens is very difficult. The purpose of this study was evaluation of T. gondii-rGRA5 for the immunodiagnosis and molecular detection of Toxoplasma infection using enzyme-linked immunosorbent assay (ELISA) and LAMP methods in hemodialysis patients. The GRA5 gene was successfully expressed and purified by affinity chromatography assay and evaluated by western blot. Then it was used to design an ELISA assay. A total of 260 samples were tested for anti-Toxoplasma IgG and IgM antibodies using a commercial ELISA kit and designed ELISA kit. Finally, the LAMP method was used to evaluate the precision and reliability of the results obtained by commercial and designed ELISA kits. The consistency of the results of two methods was analyzed using the Kappa coefficient of agreement. The rGRA5 revealed higher immunoreactivity with 1:100 dilution of sera from toxoplasmosis patients. The specificity and sensitivity of the assay were 93% and 96%, respectively. According to the Kappa coefficient, there was a substantial correlation between the results of ELISA and LAMP based on rGRA5 (≈98%, p < 0.001). Also it showed that rGRA5 protein can be used as an antigenic protein for designing sero-diagnostic tests to identify Toxoplasma infection especially in hemodialysis patients.

Potential role of miR-214 in ß-catenin gene expression within hepatocellular carcinoma.

MicroRNAs (miRNAs) are important gene regulators whose dysregulations can be involved in tumorigenesis. ß-catenin, the main agent in the Wnt/ß-catenin pathway, controls various genes and its over-expression has been discovered in different kinds of cancers including Hepatocellular Carcinoma (HCC). Extensive research demonstrated that the Wnt signaling is one of the major affected pathways in HCC. This study aimed to find miRNA targeting ß-catenin gene by bioinformatic approaches and confirm this correlation to propose new therapeutic targets for HCC. Prediction of miRNAs targeting 3'-Untranslated Regions (UTR) of ß-catenin mRNA, were done using different types of credible bioinformatic databases. The luciferase assay was also recruited for further confirmation of the bioinformatic predictions. In the first step, the expression of ß-catenin was assessed in the HepG2 cell line by real-time PCR technique. Next, transduction of HepG2 cells were done by lentiviral vectors containing the desired miRNA. Then, the expression level of miRNA and the ß-catenin gene were evaluated. Based on the results obtained from different bioinformatic databases, miR-214 was selected as the potential miRNA with the highest probability in targeting ß-catenin. Furthermore, Luciferase assay results confirmed the accuracy of our bioinformatic prediction. In line with our hypothesis, after the overexpression of miR-214 in HepG2 cells, ß-catenin gene expression was reduced significantly. Gathered results indicate the miRNAs role in the down-regulation of their target genes. Hence, the results propose that miR-214 can prevent HCC development by suppressing ß-catenin and may supply a newfound approach towards HCC therapy in humans.

Sirt1 antisense transcript is down-regulated in human tumors.

Natural antisense transcripts (NATs) have recently been associated with the development of human cancers. Recent studies have shown that a natural antisense transcript (NAT) is present in Sirt1 gene which encodes a NAD-dependent deacetylase. Interestingly, expression of Sirt1 mRNA changes during development and progression of human cancers. However, it remains unclear to what extent Sirt1 antisense transcript (AS) may contribute to changes in the expression of Sirt1 mRNA. To determine this, we used quantitative measurement of RNA to reveal relationship between Sirt1 mRNA and Sirt1-AS across human cancer tissues, cell lines and stem cells. While Sirt1 mRNA level was increased in cancer cell lines and cancer tissues, the expression level of Sirt1-AS was lower in cancers compared to controls. This inverse correlation was observed in the expression of Sirt1 sense and antisense transcripts in normal and cancer tissues suggesting a functional role for Sirt1-AS in regulation of Sirt1 mRNA.

miR-141 as potential suppressor of ß-catenin in breast cancer.

Triple negative breast cancer (TNBC) is well known for its heterogeneous features and lack of targeted therapy. A variety of cell signaling pathways have been linked to the initiation and progression of these tumors where canonical Wnt signaling is one of the main candidate pathways. Considering past literatures on this matter and negative reports regarding mutations in ß-catenin gene (CTNNB1), we focus our attention to another level of gene expression control level, microRNAs (miRNAs). For proper miRNA target detection, we utilized bioinformatics as a relatively new and reliable tool for miRNA: mRNA prediction. MDA-MB-231 (invasive breast cancer) and MCF-10A (normal breast) cell lines were chosen as models. We used different bioinformatic tools such as TargetScan, miRanda, etc. For miRNA targeting CTNNB1-3´UTR confirmation, luciferase assay was carried out. miRNA expression was induced in cell lines through viral constructs expressing desired miRNA. Quantitative real-time PCR was performed for the measurement of expression levels of selected miRNA and target gene. miR-141 was selected via expanded search among various bioinformatic tools. miR-141 expression level was downregulated in MDA-MB-231 cell line, and CTNNB1 gene expression was upregulated. After transduction with viral construct, miR-141 expression was elevated in both cell lines, and gene expression was notably decreased. ß-Catenin can be considered as one of the main players in these tumors' pathogenesis. Also, it is the potential target of miR-141, in which its downregulation was detected in cell lines, and can be considered as a promising new targeted approach toward TNBC.

The anti-cancer properties of miR-340 plasmid-chitosan complexes (miR-340 CC) on murine model of breast cancer.

MiRNA-340 (miR-340) has been found to have tumour-suppressing effects in breast cancer (BC). However, for clinical use, miRNAs need to be delivered safely and effectively to protect them from degradation. In our previous study, we used chitosan complexes as a safe carrier with anti-cancer properties to deliver miR-340 plasmid into 4T1 cells. This study explored further information concerning the anti-cancer impacts of both chitosan and miR-340 plasmid in a murine model of BC. Mice bearing 4T1 cells were intra-tumorally administered miR-340 plasmid-chitosan complexes (miR-340 CC). Afterwards, the potential of miR-340 CC in promoting anti-tumour immune responses was evaluated. MiR-340 CC significantly reduced tumour size, inhibited metastasis, and prolonged the survival of mice. MiR-340 CC up-regulates P-27 gene expression related to cancer cell apoptosis, and down-regulates gene expressions involved in angiogenesis and metastasis (breast regression protein-39 (BRP-39)) and CD163 as an anti-inflammatory macrophages (MQs) marker. Furthermore, CD47 expression as a MQs immune check-point was remarkably decreased after miR-340 CC treatment. The level of IL-12 in splenocytes of miR-340 CC treated mice increased, while the level of IL-10 decreased, indicating anti-cancer immune responses. Our findings display that miR-340 CC can be considered as a promising therapy in BC.

Cytotoxic Effects of a Novel tagged Apoptin on Breast Cancer Cell Lines.

Backgrounds: Apoptin can induce tumor cell-specific apoptosis in a broad range of human tumor cells and is a potential anticancer therapeutic candidate to kill tumor cells. Materials and Methods: We designed two structures of apoptin fusion protein, SUMO-PTD4-Apoptin, and PTD4-Apoptin. To express these fusion proteins, E. coli BL21(DE3) was employed. MTT assay, Flow cytometry, and cell cycle analysis were used to investigate the function of proteins on two breast cancer cell lines (MDA-MB-231 and MCF-7) and MCF 10A cell line (as normal cells). Results: Expression of the recombinant SUMO-PTD4-Apoptin and PTD4-Apoptin in E. coli BL21(DE3) was successful. MTT assay results showed that the IC50 was 6.4 µg/ml for SUMO-PTD4-Apoptin in MDA-MB-231 and was 9.3 after 24 h of treatment in MCF-7. The specific cytotoxicity in both cell lines is significant in comparison with MCF-10A, which is used as a normal cell line (IC50 = 29.4). The IC50 for PTD4-Apoptin was 11.07 µg/ml after 24 h of treatment in MDA-MB-231, while the IC50 of PTD4-Apoptin for MCF7 cells was not significantly different from normal cells. The flow cytometry analysis displayed a significant increment in the apoptosis and late apoptosis number in the MDA-MB-231 cells after treatment with SUMO-PTD4-Apoptin and PTD4-Apoptin protein. PTD4-Apoptin and SUMO-PTD4-Apoptin treatment of MDA-MB-231 cells caused a noteworthy increase in the G0-G1 phase and a reduction in the cell population of S and M/G2. Conclusion: This study demonstrates that the fusion of PTD4-Apoptin to SUMO-PTD4-Apoptin could provide an effective method to help enhance the expression and solubility of heterologous Apoptin in E. coli. BL21 (DE3).

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.

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.

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.

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.

Overexpression of pigment epithelium-derived factor in breast cancer cell-derived exosomes induces M1 polarization in macrophages.

M2 macrophages, the major component of tumor microenvironment, are recognized as important player in tumor progression. M2 macrophages mediate this effect by promoting tumor angiogenesis, tumor metastasis, and suppression of tumor immunity. Reprogramming of M2 macrophages can serve as a promising strategy in cancer immunotherapy. In this study, we constructed pigment epithelium-derived factor (PEDF) expressing vector and transfected MDA-MB-231 cells with this construct. Then, exosomes were isolated from transfected cells and M2 macrophages were incubated with isolated exosomes from transfected cell. The effect of isolated exosomes on macrophage polarization was examined by real-time PCR and ELISA. The results demonstrated reprogramming of M2 macrophages after incubation with isolated exosomes from PEDF transfected cells. M2-to-M1 repolarization of macrophages was confirmed by upregulation of CD80, IRF5, MCP1, and IL-1ß and repression of CD206, Arg, TGM2, and TGF-ß. Therefore, these findings revealed that introducing PEDF into exosomes by genetic manipulation of parent cells may be a potential approach for reprogramming of M2 macrophages in cancer.

Effects of Sambucus ebulus Extract on Cell Proliferation and Viability of Triple- Negative Breast Cancer: An In Vitro and In Vivo Study.

BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancer (BC) cases and is a severe type of BC. Since medicinal herbs containing biocompatible substances that are accepted by patient more than chemical therapeutics, they can be considered a safe option for treating BC. OBJECTIVE: This study evaluated the effect of Sambucus Ebulus (S. ebulus) extract on a model of TNBC. METHODS: S. ebulus extract was prepared using petroleum ether, ethyl acetate, and methanol. The petroleum ether extract was fractionated and analyzed using vacuum liquid chromatography and GC-MS, respectively. MDAMB- 231 and MCF-10A were used as TNBC and normal breast cells, respectively. Flowcytometry and MTT assays were performed to evaluate cell cycle, apoptosis, and viability of the cells. Gene expression analysis was performed using RT-qPCR. Nude mouse allograft tumor models were used, and pathological sections were evaluated. RESULTS: The findings indicated that S. ebulus extract remarkably decreased cell proliferation and viability. The extract had no toxicity to the normal breast cells but efficiently killed the cancer cells. Cell cycle- and apoptosisrelated gene expression showed that fraction 4 of S. ebulus extract significantly increased the expression of Bax, Bak, P53, and c-MYC. CONCLUSION: This study showed satisfactory results of the effect of S. ebulus extract on clearing BC cells both in vitro and in vivo. Thus, S. ebulus extract may be a safe herbal compound for eliminating BC cells without toxicity to host cells.

Assessment of AXL and mTOR genes expression in medullary thyroid carcinoma (MTC) cell line in relation with over expression of miR-144 and miR-34a.

OBJECTIVES: The aim of the present study was to investigate the expression of AXL and mTOR genes and their targeting microRNAs (miRNAs) including miR-34a and miR-144 in Medullary Thyroid Carcinoma (MTC) cell line, TT, and determine the effect of these two miRNAs on their target genes to introduce new molecular markers or therapeutics. METHODS: The expression of miR-34a, miR-144, and their targets genes including AXL and mTOR was evaluated by quantitative Real-time PCR. Luciferase assay was performed to confirm the interaction between miRNAs and their target mRNAs. The expression level of AXL and mTOR was evaluated before and after miRNAs induction in TT cell line compared with Cos7 as control cells. RESULTS: The expression of AXL and mTOR were up-regulated significantly, while miR-34a and miR-144 were down-regulated in TT cell line compared to Cos7. After transduction, the overexpression of miR-34a and 144 caused down-regulation of both genes. Luciferase assay results showed that the mTOR is targeted by miR-34a and miR-144 and the intensity of luciferase decreased in the presence of miRNAs. CONCLUSIONS: Based on the results of the present study and since AXL and mTOR genes play a critical role in variety of human cancers, suppression of these genes by their targeting miRNAs, especially miR-34a and miR-144, can be propose as a new strategy for MTC management. However, more studies are needed to approve the hypothesis.

Evaluating the influence of Human Umbilical Cord Mesenchymal Stem Cells-derived exosomes loaded with miR-3182 on metastatic performance of Triple Negative Breast Cancer cells.

AIMS: Deregulation of microRNA (miRNA) function has been linked to numerous human cancers, such as Triple Negative Breast Cancer (TNBC). Exosomes, a subgroup of extracellular vehicles (EVs), can efficiently deliver many different cargo types to the target cell and have an extensive role in delivering therapeutic cargo for treatment. The present study intended to interrogate the effects of exosomal delivery of miR-3182 on TNBC cellular processes. MAIN METHODS: Human Umbilical Cord Mesenchymal Stem Cells (HUCMSCs) were cultured and exosomes were isolated and characterized using TEM, SEM, DLS, and Western blot. Exosomes were transfected with miR-3182 and added to the treatment groups. The expression level of miR-3182 and their target genes including mTOR and S6KB1 were evaluated using RT-qPCR. The effects of miR-3182 loaded HUCMSC-exosomes treatment on the cellular aspect of MDA-MB-231 cells including their viability, migration potency, cell cycle status and apoptosis were investigated. KEY FINDINGS: According to the results, exosomal miR-3182 significantly abolished cell proliferation and migration (P < 0.05). miR-3182 loaded exosomes also induced apoptosis in TNBC cells by down-regulating mTOR and S6KB1 genes (P < 0.05). SIGNIFICANCE: In nutshell, miR-3182-loaded HUCMSC-exosomes can suppress TNBC invasion, suggesting that exosomes containing miR-3182 could be a reliable therapeutic paradigm in TNBC therapy.

Conditioned medium derived from seminal extracellular vesicles-exposed endometrial stromal cells induces inflammatory cytokine secretion by macrophages.

OBJECTIVE: Seminal plasma (SP) contains large numbers of sub-cellular structures called extracellular vesicles (EV) which have been postulated to have immunological functions due to their bioactive contents including proteins and small non-coding RNAs. Although the response of endometrial cells to seminal EV (SEV) is recently being elucidated, the impact of these signaling vesicles on stroma-immune crosstalk is still unknown. Herein, we aimed to investigate the effect of conditioned medium (CM) derived from SEV-exposed endometrial stromal cells (eSC) on cytokine secretion by macrophages. STUDY DESIGN: SEV were isolated from SP samples of healthy donors and characterized by common methods needed for EV characterization, including size determination by dynamic light scattering (DLS), transmission electron microscopy (TEM), and western blot analysis of EV markers. Endometrial biopsies were obtained from healthy individuals and eSC were isolated and characterized. EV internalization assay was performed by labeling the SEV with PKH67 green fluorescent dye. Then, the eSC were exposed to SEV and the CM was collected. Finally, the CM from SEV-exposed eSC was added to the macrophage culture and the level of inflammatory (interleukin (IL)-1α and IL-6) and anti-inflammatory (IL-10) cytokines were measured in the culture supernatant of macrophages. RESULTS: The results demonstrated that the CM derived from SEV-exposed eSC induce IL-1α and IL-6 secretion by the macrophages, while the secretion of IL-10 was reduced. CONCLUSION: Our results support the idea that the stroma-immune interaction is affected by SEV. This effect may be a part of immunoregulatory function of SP inside upper female genital tract and have an obvious impact during peri-implantation period.

Comparative Expression Profile Analysis of Apoptosis-Related miRNA and Its Target Gene in Leishmania major Infected Macrophages.

BACKGROUND: Cutaneous Leishmaniasis (CL) is an emerging uncontrollable and neglected infectious disease worldwide including Iran. The aim of this study was to investigate the expression profile of apoptosis-related miRNA and its target gene in macrophages. METHODS: This study was carried out in the Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran from January 2016 to November 2018. Applying literature reviews, bioinformatics software, and microarray expression analysis, we selected miRNA-24-3p interfering in apoptosis pathway. The expression profile of this miRNA and target gene were investigated in Leishmania major (MRHO/IR/75/ER)-infected primary and RAW 264.7 macrophages (IBRC-C10072) compared with non-infected macrophages (control group) using quantitative Real-time PCR. RESULTS: Results of bioinformatics analysis showed that miR-24-3p as anti-apoptotic miRNA inhibits pro-apoptotic genes (Caspases 3 and 7). Microarray expression data presented in Gene Expression Omnibus (GEO) revealed a significant difference in the expression level of selected miRNA and its target gene between two groups. QRT-PCR results showed that the expression of miR-24-3p was upregulated in L. major infectioned macrophages that approved the results of bioinformatics and microarray analysis. CONCLUSION: Parasite can alter miRNAs expression pattern in the host cells to establish infection and its survival. Alteration in miRNAs levels likely plays an important role in regulating macrophage functions following L. major infection. These results could highlight current understanding and new insights concerning the gene expression in macrophages during leishmaniasis and will help to development of novel strategies for control and treatment of CL.

Expression of Bioinformatically Candidate miRNAs including, miR-576-5p, miR-501-3p and miR-3143, Targeting PI3K Pathway in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an invasive and lethal form of breast cancer. PI3K pathway, which often activated in TNBC patients, can be a target of miRNAs. The purpose of this study was bioinformatic prediction of miRNAs targeting the key genes of this pathway and evaluation of the expression of them and their targets in TNBC. MATERIALS AND METHODS: We predicted miRNAs targeting PIK3CA and AKT1 genes using bioinformatics tools. Extraction of total RNA, synthesis of cDNA and quantitative real-time polymerase chain reaction were performed from 18 TNBC samples and normal adjacent tissues and cell lines. RESULTS: Our results demonstrated that miR-576-5p, miR-501-3p and miR-3143 were predicted to target PIK3CA, AKT1 and both of these mRNAs, respectively and were down-regulated while their target mRNAs were up-regulated in clinical samples and cell lines. The analysis of the receiver operating characteristic curve was done for the evaluation of the diagnostic value of predicted miRNAs in TNBC patients. CONCLUSION: The findings of our study demonstrated the reverse correlation between miRNAs and their target genes and therefore the possibility of these miRNAs to be proposed as new candidates for TNBC targeted therapies.

Is miR-144 an effective inhibitor of PTEN mRNA: a controversy in breast cancer.

Breast cancer is the first common cancer among women worldwide. One of the major signaling pathways playing a role in the onset and progression of this disease is PI3K/Akt/mTOR, which can be inhibited by PTEN. miRNAs are small non-coding molecules that regulate the expression of their targets by inhibition or suppression, and thus, their dysregulated expression results in the development of cancer. Using various software applications predicting miRNAs and evaluating GEO microarray data, miR-144 was selected as an inhibitor of PTEN. The expression of miR-144 and PTEN was evaluated in 18 triple negative breast cancer (TNBC) clinical samples and cell lines including 4T1, MDA-MB-231, MDA-MB-468, SK-BR-3, and MCF-7 in comparison with normal cells. PTEN and miR-144 expression analysis revealed their elevated expression in MCF-7 cells. MDA-MB-468, SK-BR-3, and MDA-MB-231 cells showed decreased levels of PTEN and increased levels of miR-144. In contrast, 4T1 cells had an increased expression of PTEN and decreased expression of miR-144. In clinical samples, miR-144 was up-regulated in 22% of the cases and PTEN was down-regulated in 78% of the cases. The results showed that the expression of PTEN and miR-144 was inversely correlated in metastatic breast cancer cell lines. However, in TNBC clinical samples, there was no correlation between the expression of miR-144 and PTEN. Literature shows that there are other influencing factors affecting the expression of miRNAs. Therefore, care should be taken in interpreting the results of gene expression studies and its relation with cancer diagnosis/prognosis.