The anti-angiogenic effect of atorvastatin loaded exosomes on glioblastoma tumor cells: An in vitro 3D culture model.

Exosomes are endogenous nanoparticles with a lipid bilayer membrane whose natural function as carriers of biological materials has attracted much attention. The ability of exosomes to cross biological barriers, especially the blood-brain barrier, has highlighted them as tools of drug delivery to brain tumors. In a previous study, we isolated and characterized exosomes derived from human endometrial mesenchymal stem cells (hEnMSCs exosomes). In the present study, we used hEnMSCs exosomes as carriers for atorvastatin and investigated its pro-apoptotic and anti-angiogenic effects on U87 glioblastoma spheroids 3D co-cultured with Human Umbilical Vein Endothelial cells (HUVECs). In the study of HUVEC proliferation by using MTT assay, cell treatments with concentrations of 5 and 10 µM of free atorvastatin and atorvastatin-loaded hEnMSCs exosomes (AtoEXOs) showed significant differences in inhibition of proliferation compared to other concentrations. Also, 5 and 10 µM of AtoEXOs inhibited HUVEC migration in both scratch closure and transwell migration assays significantly more than that of free atorvastatin. In addition, in vitro HUVEC capillary tube network formation was inhibited by 5 and 10 µM treatment of AtoEXOs significantly more that of free atorvastatin. Moreover, a significant decrease in VEGF secretion and a significant increase in Bax/Bcl2 expression ratio were observed in U87 spheroids 3D co-cultured with HUVECs, especially for 10 µM AtoEXOs compared to other treated cell groups. Our results showed that hEnMSCs exosomes loaded with atorvastatin not only mimicked the anti-tumor effects of free atorvastatin but also potentiated its anti-tumor effects on glioblastoma cells. The enhanced pro-apoptotic and anti-angiogenic capabilities of atorvastatin loaded in hEnMSCs exosomes offer promising new perspectives for the treatment of glioblastoma.

A novel natural antisense transcript at human SOX9 locus is down-regulated in cancer and stem cells.

OBJECTIVE: SOX9 is a key transcription factor with important roles in regulating proliferation and differentiation of various cell types. Dysregulation of SOX9 expression has been involved with pathogenesis of different developmental, degenerative, and neoplastic disorders. Natural antisense transcripts (NATs) are long non-coding RNAs with increasing significance in regulation of gene expression. However, the presence of a NAT at SOX9 locus has been so far unclear. RESULT: We detected a natural antisense transcript at SOX9 locus (SOX9-NAT) through strand-specific RT-PCR. In contrast to SOX9 sense RNA (mRNA), SOX9-NAT was down-regulated in cancer tissues and cell lines compared with their normal counterparts. In addition, reciprocal to SOX9 mRNA, SOX9-NAT was also down-regulated in human embryonic stem cells in comparison with human fibroblasts in vitro. CONCLUSION: The negative correlation between SOX9 mRNA and SOX9-NAT was confirmed by analyzing qPCR data, as well as RNA-Seq datasets of several human cancers. Our data suggest a functional role for SOX9-NAT in the regulation of SOX9 mRNA as a potential target in cancer treatment and regenerative medicine.

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.

A novel frameshift mutation in the EDA gene in an Iranian patient affected by X-linked hypohidrotic ectodermal dysplasia.

PURPOSE: Ectodermal dysplasias are characterized by developmental abnormalities in ectodermal structures. Hypohidrotic ectodermal dysplasias (HED) are the most common subtype. They are most commonly inherited via X-linked recessive routes. We report on a novel ectodysplasin-A (EDA) mutation that is expected to be involved in pathogenesis of HED. METHODS: Hypohidrotic ectodermal dysplasia genes, including EDA, EDAR and EDARADD, were analyzed using next-generation sequencing (NGS). The detected mutation on the EDA gene was confirmed in the patient and his mother using Sanger sequencing. RESULTS: The patient presented with adontia, absence of gum development, hyperthermia and hypohidrosis. Our genetic analysis of the patient revealed a novel frameshift hemizygous mutation (c.898_924 + 8del35ins4CTTA) on the EDA gene. The patient's mother showed a mild HED phenotype. Direct sequencing of the EDA gene in the region where her son had the mutation showed the same mutation in a heterozygous state. CONCLUSION: We identified a novel frameshift mutation in the EDA gene in an Iranian patient affected by X-linked HED. The difference between our patient's symptoms and those recorded for some previous subjects may be due to the differences in the mutations involved.

RNAa-mediated epigenetic attenuation of the cell senescence via locus specific induction of endogenous SIRT1.

SIRT1, a known regulator of cellular senescence, is a therapeutic target for age related disorders and its upregulation is a strategy to improve the cell therapeutic potentials of human mesenchymal stem cell (MSCs). Knockdown of natural antisense transcripts via small activating RNAs (RNAa) is an emerging approach for safe and locus specific gene regulation. We have recently identified a natural antisense transcript at human SIRT1 locus (SIRT1-NAT), the expression of which shows a negative correlation with that of SIRT1. To test the hypothetic upregulation of SIRT1 via knockdown of SIRT1-NAT, in this study we designed a single stranded oligonucleotide (SIRT1-antagoNAT) against the antisense transcript, transfection of which efficiently knocked down the SIRT1-NAT and induced SIRT1 transcription in human MSCs. In addition, activation of SIRT1 transfection via knockdown of SIRT1-NAT in human MSCs enhanced their proliferation and differentiation potentials, reduced senescence associated ß-galactosidase activity and reversed the senescence associated molecular alterations. Our findings introduce an RNAa mediated approach for epigenetic induction of endogenous SIRT1 and the consequent attenuation of senescence. Further studies should evaluate the therapeutic potentials of this approach against various age related disorders.

SARS-CoV-2 mRNA-vaccine candidate; COReNAPCIN®, induces robust humoral and cellular immunity in mice and non-human primates.

At the forefront of biopharmaceutical industry, the messenger RNA (mRNA) technology offers a flexible and scalable platform to address the urgent need for world-wide immunization in pandemic situations. This strategic powerful platform has recently been used to immunize millions of people proving both of safety and highest level of clinical efficacy against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we provide preclinical report of COReNAPCIN®; a vaccine candidate against SARS-CoV-2 infection. COReNAPCIN® is a nucleoside modified mRNA-based vaccine formulated in lipid nanoparticles (LNPs) for encoding the full-length prefusion stabilized SARS-CoV-2 spike glycoprotein on the cell surface. Vaccination of C57BL/6 and BALB/c mice and rhesus macaque with COReNAPCIN® induced strong humoral responses with high titers of virus-binding and neutralizing antibodies. Upon vaccination, a robust SARS-CoV-2 specific cellular immunity was also observed in both mice and non-human primate models. Additionally, vaccination protected rhesus macaques from symptomatic SARS-CoV-2 infection and pathological damage to the lung upon challenging the animals with high viral loads of up to 2 × 108 live viral particles. Overall, our data provide supporting evidence for COReNAPCIN® as a potent vaccine candidate against SARS-CoV-2 infection for clinical studies.

Adipose derived mesenchymal stem cell exosomes loaded with miR-10a promote the differentiation of Th17 and Treg from naive CD4+ T cell.

AIMS: The balance between various CD4+ T cell subsets through highly regulated differentiation of naïve T cells is critical to ensure proper immune response, disruption of which may cause autoimmunity and cancers. miR-10a has been reported to regulate the fate of naïve T cells. Mesenchymal stem cells (MSC) derived exosomes are known effective immunomodulators and ideal vehicles for delivery of microRNAs. This study was aimed to examine the impacts of miR-10a on CD4+ cell fate upon exosomal delivery in combination with immunomodulatory effects of MSCs. MAIN METHODS: Exosomes isolated form adipose tissue derived mesenchymal stem cells (AD-MSC-Exo) were transfected with miR-10a and added to naïve T cells purified from mouse spleen. AD-MSC-Exos were characterized and the efficacy of miR-10a delivery was evaluated. The expression levels of T-bet, GATA3, RORγt, and Foxp3 and the secreted levels of IFN-γ, IL-4, IL-17, and TGF-ß respectively specific to Th1, Th2, Th17 and Treg, were assessed by qPCR and ELISA. KEY FINDINGS: Being transferred by AD-MSC-Exo, miR-10a was effectively induced in CD4+ T cells. Upon treatment with miR-10a loaded exosomes, the expression levels of RORγt and Foxp3 were enhanced and that of T-bet was reduced. Similarly, the secreted levels of IL-17, and TGF-ß were increased and that of IFN-γ was decreased. SIGNIFICANCE: Our data indicate that miR-10a loaded exosomes, promote Th17 and Tregs response while reduce that of Th1. Promotion of both Th17 and Tregs in concert, mediated by the combined effect of miR-10a and MSC-Exo, indicate new therapeutic potentials, particularly in line with novel anti-tumor immunotherapeutic strategies.

Inhibition of miR-34a reduces cellular senescence in human adipose tissue-derived mesenchymal stem cells through the activation of SIRT1.

AIMS: Human adipose derived mesenchymal stem cells (hAD-MSCs) as the most promising target for cell therapy and regenerative medicine, face senescence as a major drawback resulting in their limited proliferation and differentiation potentials. To evaluate the efficacy of miR-34a silencing as an anti-senescence strategy in hAD-MSCs, in this study common hallmarks of senescence were assessed after transient inhibition of miR-34a in hAD-MSCs. MATERIALS AND METHODS: The expression levels of miR-34a in hAD-MSCs at different passages were evaluated by real-time quantitative PCR. hAD-MSCs at passage 2 and passage 7 were transfected with miR-34a inhibitor. Doubling time assay, colony forming assay, and cell cycle analysis were performed to evaluate cell proliferation rate. The activity of senescence associated ß-galactosidase (SA-ß-gal) was assessed by histochemical staining. Moreover, the senescence associated molecular alterations including that of pro-senescence (P53, P21 and P16) and anti-senescence (SIRT1, HTERT and CD44) genes were examined by quantitative RT-PCR and western blot assays. To evaluate the differentiation potentials of MSCs, following adipogenic and osteogenic induction, the expression levels of lineage specific markers were analyzed by qPCR. KEY FINDINGS: Our results showed that inhibition of miR-34a enhances the proliferation, promotes the adipogenic and osteogenic differentiation potency, reduces the senescence associated-ß gal activity, and reverses the senescence associated molecular alterations in hAD-MSCs. SIGNIFICANCE: In this study, we showed that inhibition of miR-34a reduces the cellular senescence through the activation of SIRT1. Our findings support the silencing of miR-34a as an anti-senescence approach to improve the therapeutic potentials of hAD-MSCs.

Analysis of Chemokine Receptor Gene Expression in Esophageal Cancer Cells Compared with Breast Cancer with Insights into Metastasis.

BACKGROUND: Chemokine receptors have been shown to play an important role in the development and metastatic spread of various malignancies. In this study, the gene expression profile of some key chemokine receptors involved in metastasis has been investigated in esophageal and breast cancer cell lines. METHODS: In a descriptive study, gene expression profile of CCR1, CCR6, CCR7, CCR9, CXCR1, and CXCR4 in human esophageal cancer cell line (KYSE-30) and human breast cancer cell line (MCF7) were analyzed using real-time PCR and their results were compared accordingly. RESULTS: We demonstrated for the first time the expression of CCR1, CCR6, CCR7, CCR9, CXCR1, and CXCR4 at transcriptional level in human esophageal cancer cell line. The expression of CCR1, CCR7 and CXCR4 were lower in esophageal compared with breast cancer cells, although without significant difference. CCR9 was highly expressed in esophageal cancer cells as compared to the breast cancer cells (P < 0.05). Similarly, the expression of CCR6 and CXCR1 were higher, although without significant difference. CONCLUSION: Esophageal cancer cells like breast cancer express some key chemokine receptors involved in metastasis. Targeting of proposed receptors in esophageal cancer may be a novel strategy for prevention of cancer metastasis.