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Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression
Behnaz Maleki, Mahdi Noureddini, Somayeh Saadat, Javad Verdi, Alireza Farrokhian, Hossein Ghanbarian, Ebrahim Cheraghi, Behrang Alani,
Volume 12, Issue 1 (Vol.12 No.1 Apr 2023)
Abstract
Background: Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs).

Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days.

Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed.

Conclusions: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.

In Vitro Differentiation of Endometrium Stem Cells into Cardiomyocytes: The Putative Effect of miR-17-5p, miR-26b-5p, miR-32-5p, and SMAD6
Somayeh Saadat, Mahdi Noureddini, Behnaz Maleki, Naeim Ehtesham, Alireza Farrokhian, Javad Verdi, Ebrahim Cheraghi, Hossein Ghanbaraian, Behrang Alani,
Volume 13, Issue 2 (Vol.13 No.2 Jul 2024)
Abstract
Background: The important role of SMAD6 and several microRNAs (miRNAs), such as miR-17-5p, miR-26b-5p, and miR-32-5p, has been demonstrated in controlling the proliferation and differentiation of cardiomyocytes (CMs). Hence, this study was designed to assess the role of these regulatory factors in cardiac cell generation from human endometrium-derived mesenchymal stem cells (hEMSCs).

Methods: To induce transdifferentiation into CMs, hEMSCs were treated with a cardiac-inducing medium containing 5-azacytidine and bFGF for 30 days. Immunofluorescence staining and qRT-PCR, respectively, were used to measure the protein levels of SMAD6 and the mRNA expression of SMAD6 and the three miRNAs every six days.

Results: Our findings demonstrated the mesenchymal stem cell properties of hEMSCs and their ability to differentiate into various types of mesenchymal stem cells. The differentiated hEMSCs exhibited morphological features resembling CMs. During the induction period, the number of positive cells for SMAD6 protein and the expression level of miR-26b-5p increased and peaking on days 24 and 30, while the expression levels of miR-17-5p and miR-32-5p decreased. The Pearson correlation coefficients revealed that SMAD6 level is inversely correlated with miR-17-5p and miR-32-5p and directly correlated with miR-26b-5p.

Conclusion: Our results indicate that miR-17-5p, miR-26b-5p, miR-32-5p, and SMAD6 are potentially involved in the molecular signaling pathways of transdifferentiation of hEMSCs to CMs.

Algae-Mediated Green Synthesis of Dextran-Coated Titanium Nanoparticles and Their Cytotoxic Potential Against MCF7 Breast Cancer Cells
Raghad Jasim Fayyad, Entkhab Muhsen Abed Ali Alanisi, Alaa Naseer Mohammed Ali Mohammed Ali,
Volume 13, Issue 3 (Vol.13 No.3 Oct 2024)
Abstract
Background: The green synthesis of nanoparticles through algae-mediated processes offers an eco-friendly, cost-effective, and scalable approach for producing nanomaterials with potential applications in cancer therapy. The present study investigated the algae-mediated green synthesis of dextran-coated titanium oxide nanoparticles (TiO2NPs) and evaluated their cytotoxic effects against MCF-7 breast cancer cells.

Methods: Chlorella vulgaris was isolated and identified. The polymerase chain reaction (PCR)-amplification of the 18S ribosomal RNA gene was used to confirm the isolate. Dextran from C. vulgaris was used to prepare coated TiO2NPs, characterized using three techniques. The cytotoxicity of the dextran-coated TiO2NPs was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay on MCF7- breast cancer cells at various concentrations (25, 50, and 75%) and exposure times (24, 48, and 72 hours). The bioactive compounds in the algal extract were also identified by gas chromatography-mass spectrometry (GC-MS).

Results: Chlorella vulgaris was successfully isolated as confirmed by the 345-bp PCR-amplified fragment. The characterization of the TiO2NPs confirmed the successful nanoparticle formation. A cluster of nanocrystalline particles had an average diameter of 71.44 nm. Compositional analysis revealed 15.85% atomic percentage for titanium. The dextran-coated TiO2NPs exhibited an impressive cytotoxicity rate of up to 99% at optimal concentration (25%) and exposure time (48 hours). Additionally, GC-MS analysis identified bioactive compounds in the algal extract, such as fatty acids, which may contribute to the observed anticancer effects.

Conclusion: The study demonstrated the potential of algae-mediated TiO2NPs in cancer co-therapy, enhancing treatment effectiveness and reducing the side effects of traditional therapies.

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