Nanotechnology and bioengineering approaches to improve the potency of mesenchymal stem cell as an off-the-shelf versatile tumor delivery vehicle.

Targeting actionable mutations in oncogene-driven cancers and the evolution of immuno-oncology are the two prominent revolutions that have influenced cancer treatment paradigms and caused the emergence of precision oncology. However, intertumoral and intratumoral heterogeneity are the main challenges in both fields of precision cancer treatment. In other words, finding a universal marker or pathway in patients suffering from a particular type of cancer is challenging. Therefore, targeting a single hallmark or pathway with a single targeted therapeutic will not be efficient for fighting against tumor heterogeneity. Mesenchymal stem cells (MSCs) possess favorable characteristics for cellular therapy, including their hypoimmune nature, inherent tumor-tropism property, straightforward isolation, and multilineage differentiation potential. MSCs can be loaded with various chemotherapeutics and oncolytic viruses. The combination of these intrinsic features with the possibility of genetic manipulation makes them a versatile tumor delivery vehicle that can be used for in vivo selective tumor delivery of various chemotherapeutic and biological therapeutics. MSCs can be used as biofactory for the local production of chemical or biological anticancer agents at the tumor site. MSC-mediated immunotherapy could facilitate the sustained release of immunotherapeutic agents specifically at the tumor site, and allow for the achievement of therapeutic concentrations without the need for repetitive systemic administration of high therapeutic doses. Despite the enthusiasm evoked by preclinical studies that used MSC in various cancer therapy approaches, the translation of MSCs into clinical applications has faced serious challenges. This manuscript, with a critical viewpoint, reviewed the preclinical and clinical studies that have evaluated MSCs as a selective tumor delivery tool in various cancer therapy approaches, including gene therapy, immunotherapy, and chemotherapy. Then, the novel nanotechnology and bioengineering approaches that can improve the potency of MSC for tumor targeting and overcoming challenges related to their low localization at the tumor sites are discussed.

Exploring the potential of structural modeling and molecular docking for efficient siRNA screening: A promising approach to Combat viral mutants, with a focus on HIV-1.

RNA interference (RNAi) holds immense potential for sequence-specific downregulation of disease-related genes. Small interfering RNA (siRNA) therapy has made remarkable strides, with FDA approval for treating specific human diseases, showcasing its promising future in disease treatment. Designing highly efficient siRNAs is a critical step in this process. Previous studies have introduced various algorithms and parameters for siRNA design and scoring. However, these attempts have often fallen short of meeting all essential criteria or required modifications, resulting in variable and unclear effectiveness of screened siRNAs, particularly against viral mutants with non-conserved short sequences. In this study, we present a fully optimized siRNA screening system considering all necessary parameters. Notably, we highlight the critical role of molecular docking simulations between siRNA and two functional domains of the Argonaute protein (PAZ and PIWI) in identifying the most efficient siRNAs, since the appropriate interaction between the guide siRNA strand and the RISC complex is crucial. Through our stringent method, we designed approximately 50 potential siRNAs targeting the HIV-1 vpr gene. Evaluation through XTT, qRT-PCR, and flow cytometry analysis on RAW 264.7 macrophage stable cells revealed negligible cytotoxicity and exceptional gene-silencing efficiency at both the transcriptional and translational levels for the top-ranked screened siRNAs. Given the growing interest in siRNA-based therapeutics, we anticipate that the insights from this study will contribute to improving treatment strategies against mutant viruses, particularly HIV-1.

The Role of miR-29a and miR-143 on the Anti-apoptotic MCL-1/cIAP-2 Genes Expression in EGFR Mutated Non-small Cell Lung Carcinoma Patients.

The survival rate of lung cancer is low due to the high frequency of drug resistance in patients with mutations in the driver genes. Overexpression of anti-apoptotic genes is one of the most prominent features of tumor drug resistance. EGFR signaling induces the expression of anti-apoptotic genes. Also, microRNAs (miRNAs) have a critical role in regulating biological functions such as apoptosis; a process mostly eluded in cancer progression. The mutation screening was performed on one thousand non-small cell lung carcinoma patients to enroll clinical samples in this study. Bioinformatics analysis predicted that miRNAs (miR-29a, miR-143) might regulate MCL-1 and cIAP-2 expression. We investigated the expression of MCL-1, cIAP-2, miR-29a, and miR-143 encoding genes in adenocarcinoma patients with or without EGFR mutations before treatment. The potential role of miR-29a and miR-143 on gene expression was evaluated by overexpression and luciferase assays in HEK-293T cells. EGFR mutations were found in 262 patients (26.2%) with a greater incidence in females (36.23% vs. 20.37%, P = 0.001). The expression levels of MCL-1 and cIAP-2 genes in patients with mutated EGFR were higher than those of wild-type EGFR. In contrast, compared to those of patients with wild-type EGFR, the expression levels of miR-29a and miR-143 were lower in the patients carrying EGFR mutations. In cell culture, overexpression of miR-29a and miR-143 significantly downregulated the expression of MCL-1 and cIAP-2. Dual-luciferase reporter experiments confirmed that miR-29a and miR-143 target MCL-1 and cIAP-2 mRNAs, respectively. Our results suggest that upregulation of EGFR signaling in lung cancer cells may increase anti-apoptotic MCL-1 and cIAP-2 gene expression, possibly through downregulation of miR-29a-3p and miR-143-3p.

HSV-1 latency-associated transcript miR-H3 and miR-H4 target STXBP1 and GABBR2 genes.

During latent infection, the HSV-1 virus generates only a single transcript, LAT, which encodes six miRNAs. The GABAergic pathway signaling system is an essential cell signaling pathway influenced by various therapeutic targets and some brain disorders, such as epilepsy. This study found that miRNAs encoding LAT might target the STXBP1 and GABBR2 genes, which are among the significant genes in the GABAergic pathway. Bioinformatic analysis utilizing TargetScan version 5.2 and the RNA22 tools uncovered miRNAs encoding LAT that can influence STXBP1 and GABBR2 transcripts. To evaluate the targeting effect of candidate microRNAs encoding LAT, namely, miR-H3 and miR-H4, LAT constructs were transfected into HEK 293T cells. The expression levels of microRNAs encoding LAT, as well as STXBP1 and GABBR2, were assayed by real-time PCR. Finally, the targeting potential of STXBP1 and GABBR2 3'UTR by LAT-encoded microRNAs was evaluated by the luciferase assay. In the current study, the bioinformatic tool TargetScan demonstrated that miR-H3 has the potential to target the transcripts of the STXBP1 and GABBR2 genes, whereas miR-H4 solely targeted GABBR2. On the other hand, the bioinformatic tool RNA22 validated the potential targeting of STXBP1 and GABBR2 by miR-H3 and miR-H4. Our findings showed that overexpression of miR-H4, miR-H3, or LAT significantly decreased STXBP1 gene expression by an average of 0.0593-fold, 0.237-fold, and 0.84-fold, respectively. Similarly, overexpression of miR-H3 or miR-H4 decreased GABBR2 expression by an average of 0.055- or 0.687-fold, respectively. Notably, targeting the GABBR2 3'UTR with the LAT transcript had no detectable effect. The evaluation of the targeting potential of STXBP1 and GABBR2 3'UTR by microRNAs encoded by LAT was conducted with a luciferase assay. Our results showed that miR-H3 overexpression reduces Renilla expression in psiCHECK2 plasmids with STXBP1 or GABBR2 3'UTR genes by 0.62- and 0.55-fold, respectively. miR-H4 reduced Renilla gene expression regulated by GABBR2's 3'UTR plasmid but had no effect on the Renilla gene expression regulated by STXBP1's 3'UTR. When the LAT transcript was overexpressed, there was a decrease in Renilla expression by 0.44-fold because of the regulation of STXBP1's 3'UTR. However, there was no significant effect observed through the control of GABBR2's 3'UTR.

Augmented antiviral activity of chlorhexidine gluconate on herpes simplex virus type 1, H1N1 influenza A virus, and adenovirus in combination with salicylic acid.

BACKGROUND: The excessive usage of chlorhexidine gluconate (CHG) as a broad-spectrum antimicrobial reagent can have a negative impact on the environment and on human health. The aim of this study was to investigate the effectiveness of some plant-derived compounds in reducing the CHG concentration required to exert its antiviral activity. METHODS: Antiviral assays were conducted according to EN 14476 (2019) against herpes simplex virus type 1 (HSV-1), H1N1 influenza A virus, and adenovirus type 5 (Ad-5) as enveloped and non-enveloped viral models to assess the synergistic interaction of CHG and natural additive compounds. RESULTS: The effective concentration of 0.247 mM CHG against HSV-1 was decreased tenfold in combination with 0.0125 mM salicylic acid, with a titer reduction of 1.47 ⋅ 104 CCID50/ml. The time required for complete inactivation of HSV-1 particles was reduced to 15 min when the virus was exposed to 0.061 mM CHG and 0.0125 mM salicylic acid. Additionally, the presence of salicylic acid protected the CHG activity against interfering substances. CONCLUSION: Our supplemented CHG formulation showed immediate antiviral effectiveness, which is important for management of the infections. CHG can be combined with salicylic acid to exhibit synergistic antiviral activity at lower concentrations and reduce the time required for inactivation. Furthermore, in the presence of interfering substances, the combination has higher antiviral activity than CHG alone.

Mir-122 upregulation and let-7f downregulation combination: The effects on hepatic differentiation of hiPSCs on the PCL-Gel-HA nanofibrous scaffold.

Cell therapy and tissue engineering as promising candidates for the liver transplantation dilemma are of special interest. Induced pluripotent stem cells (iPSCs) are one of the best sources in this field, but their differentiation methods to hepatocytes have remained challenging. We transduced human iPSCs (hiPSCs) with miR-122 and off-let-7f (hiPSCsmiR-122 + off-let-7f ) to evaluate how they can differentiate hiPSCs to hepatocyte-like cells (HLCs) without any extrinsic growth factor. Additionally, we studied the effect of Poly ɛ-caprolactone-gelatin-hyaluronic acid (PCL-Gel-HA) nanofibrous scaffold as an extracellular matrix (ECM) simulator on differentiation improvement. Definitive endoderm markers (FOXA2 and SOX17), as well as hepatic markers (AFP, Albumin, CK18, HNF4α) expression, were significantly higher in hiPSCsmiR-122 + off-let-7f derived HLCs (hiPSCs-HLCs) compared to the control group (miR-scramble transduced hiPSCs: hiPSCsscramble ). hiPSCs-HLCs indicated hepatocyte morphological characteristics and positive immunostaining for AFP, Albumin and HNF4α. Albumin and urea secretion were significantly higher in hiPSCs-HLCs than hiPSCsscramble . Comparing these markers in the PCL-Gel-HA group with the tissue culture plate (TCP) group revealed that PCL-Gel-HA could improve differentiation towards HLCs significantly. Regarding our results, these microRNAs can be used to differentiate hiPSCs to the functional hepatocytes for disease modelling, drug screening and cell-based therapy in future studies.

Wnt/ß-catenin signaling pathway is involved in early dopaminergic differentiation of trabecular meshwork-derived mesenchymal stem cells.

Permanent degeneration and loss of dopaminergic (DA) neurons in substantia nigra is the main cause of Parkinson's disease. Considering the therapeutic application of stem cells in neurodegeneration, we sought to examine the neurogenic differentiation potential of the newly introduced neural crest originated mesenchymal stem cells (MSCs), namely, trabecular meshwork-derived mesenchymal stem cells (TM-MSCs) compared to two other sources of MSCs, adipose tissue-derived stem cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The three types of cells were therefore cultured in the presence and absence of a neural induction medium followed by the analysis of their differentiation potentials. Our results showed that TM-MSCs exhibited enhanced neural morphologies as well as higher expressions of MAP2 as the general neuron marker and Nurr-1 as an early DA marker compared to the adipose tissue-derived mesenchymal stem cells (AD-MSCs) and bone marrow-derived stem cells (BMSCs). Also, analysis of Nurr-1 immunostaining showed more intense Nurr-1 stained nuclei in the neurally induced TM-MSCs compared to those in the AD-MSCs, BMSCs, and noninduced control TM-MSCs. To examine if Wnt/beta-catenin pathway drives TM-MSCs towards a DA fate, we treated them with the Wnt agonist (CHIR, 3 µM) and the Wnt antagonist (IWP-2, 3 µM). Our results showed that the expressions of Nurr-1 and MAP2, as well as the Wnt/beta-catenin target genes, c-Myc and Cyclin D1, were significantly increased in the CHIR-treated TM-MSCs, but significantly reduced in those treated with IWP-2. Altogether, we declare first a higher neural potency of TM-MSCs compared to the more commonly used MSCs, BMSCs and ADSCs, and second that Wnt/beta-catenin activation directs the neurally induced TM-MSCs towards a DA fate.

The potential role of miR-1290 in cancer progression, diagnosis, prognosis, and treatment: An oncomiR or onco-suppressor microRNA?

Cancer is one of the leading causes of death in humans because of the lack of early diagnosis, distant metastases, and the resistance to adjuvant therapies, including chemotherapy and radiotherapy. In addition to playing an essential role in tumor progression and development, microRNAs (miRNAs) can be used as a robust biomarker in the early detection of cancer. MiR-1290 was discovered for the first time in human embryonic stem cells, and under typical physiological situations, plays an essential role in neuronal differentiation and neural stem cell proliferation. Its coding sequence is located at the 1p36.13 regions in the first intron of the aldehyde dehydrogenase 4 gene member A1. miR-1290 is out of control in many cancers such as breast cancer, colorectal cancer, esophageal squamous cell carcinoma, gastric cancer, lung cancer, pancreatic cancer, and plays a vital role in their development. Therefore, it is suggested that miR-1290 can be considered as a potential diagnostic and therapeutic target in many cancers. In addition to the importance of miR-1290 in the noninvasive diagnosis of various cancers, this systematic review study discussed the role of miR-1290 in altering the expression of different genes involved in cancer development and chemo-radiation resistance. Moreover, it considered the regulatory effect of natural products on miR-1290 expression and the interaction of lncRNAs by miR-1290.

Postbiotics of Lactobacillus casei target virulence and biofilm formation of Pseudomonas aeruginosa by modulating quorum sensing.

Among various anti-virulence aspects, the efficacy of the bioactive constituents of probiotics, referred to as postbiotics, to affect quorum sensing (QS)-modulated signaling of pathogens, is considered as a safe natural approach. The present study investigated the potential QS-inhibitory activity of lyophilized postbiotics from Lactobacillus casei sub sp. casei PTCC 1608 on virulence phenotypes and biofilm of two strains and three clinical isolates of Pseudomonas aeruginosa. The effect of L. casei postbiotics (LCP) at sub-minimum inhibitory concentration on the expression of QS genes including lasR/I, rhlR/I, pqsA, pqsR and virulence genes including pelF (pellicle/biofilm glycosyltransferase PelF), lasB (elastase LasB) and toxA (exotoxin A) was evaluated. The viability of mouse fibroblastic NIH/3T3 cell line treated with sub-MICS of LCP was also investigated. Postbiotics were characterized using mass spectrometry-based analyses. The QS-attenuation effect of pure lactic acid as the major constituent of LCP was determined on P. aeruginosa strains. Neutralized postbiotics and crude bacteriocin did not exhibit any antibacterial activity. It was found that sub-MICS of LCP could more drastically attenuate the tested virulence phenotypes and biofilm formation than lactic acid. Biofilm inhibition was confirmed using scanning electron microscopy. The rhlI, rhlR, and pelF genes were down-regulated after treatment with LCP. No cytotoxicity effect was observed on NIH/3T3 cell line. The findings demonstrated that postbiotics of L. casei could reduce the virulence and biofilm development of P. aeruginosa and suggested a novel safe natural source for the expansion of anti-virulence treatments.

Induction of the antioxidant defense system using long-chain carotenoids extracted from extreme halophilic archaeon, Halovenus aranensis.

The field of microbial pigments is an emerging area in natural products science. Carotenoids form a major class of such pigments and are found to be diversely synthesized by microorganisms that reside in hypersaline ecosystems to provide resistance against oxidative stress. Human cells can benefit from compounds such as carotenoids as antioxidant agents through either their capability to quench free radicals or their effect on promoting the antioxidant defense pathway. In this study, the antioxidant effectiveness of carotenoid extract from an extremely halophilic archaeon Halovenus aranensis strain EB27T has been evaluated using different approaches. Finally, the ability of the extracted pigment to induce the antioxidant defense pathway of human primary skin fibroblast cells was studied. Hvn. aranensis carotenoid extract exhibited strong effectiveness such that at 2 µg/ml, the carotenoid extract fully neutralized the oxidative stress of hydrogen peroxide at its EC50 based on MTT assay. Results from real-time PCR of relevant genes, luciferase bioreporter of oxidative stress, and the western blot analysis further confirmed the antioxidant capability of the carotenoids. It was also shown the carotenoid extract had more antioxidant activity compared to ß-carotene the same concentration. Results suggest the carotenoid extract from this archaeon to have high potential for clinical and industrial applications.

The miR-429 suppresses proliferation and migration in glioblastoma cells and induces cell-cycle arrest and apoptosis via modulating several target genes of ERBB signaling pathway.

BACKGROUND: Glioblastoma multiforme (GBM) is an aggressive and lethal brain cancer, which is incurable with standard cancer treatments. miRNAs have great potential to be used for gene therapy due to their ability to modulate several target genes simultaneously. We found miR-429 is downregulated in GBM and has several predicted target genes from the ERBB signaling pathway using bioinformatics tools. ERBB is the most over-activated genetic pathway in GBM patients, which is responsible for augmented cell proliferation and migration in GBM. METHODS AND RESULTS: Here, miR-429 was overexpressed using lentiviral vectors in U-251 and U-87 GBM cells and it was observed that the expression level of several oncogenes of the ERBB pathway, EGFR, PIK3CA, PIK3CB, KRAS, and MYC significantly decreased, as shown by real-time PCR and western blotting. Using the luciferase assay, we showed that miR-429 directly targets MYC, BCL2, and EGFR. In comparison to scrambled control, miR-429 had a significant inhibitory effect on cell proliferation and migration as deduced from MTT and scratch wound assays and induced cell-cycle arrest and apoptosis in flow cytometry. CONCLUSIONS: Altogether, miR-429 seems to be an efficient suppressor of the ERBB genetic signaling pathway and a potential therapeutic for GBM.

Application of iPSCs derived pancreatic ß-like cells using pancreatic bio-scaffold.

This study aimed toengineer a pancreatic tissue. Intact rat pancreases were successfully decellularized, and were reseeded with human-induced pluripotent stem cells using different 2D and 3D culture growth factors. The differentiation process was assessed for the presence of a pancreas-like tissue. The histology and SEM analysis revealed cell attachment in all samples, except for the Exp4, and the Flow-cytometry provided 87% viability for the differentiated cells. In Exp1, PDX1 with the positive expression of 2.87±0.06 was dramatically higher than Exp2 with a 2.44±0.06 reaction. NGN3-reactions were 8±0.1 and 6.6±0.2 in Exp1 and Exp2 at P < 0.05, respectively. C-peptide with the expression of 7.5±0.7 in Exp3 was almost equal to that in Exp1 and Exp2. Glucagon (5.1±1) and PDX1 (3.2±0.82) in Exp3 indicated no significant difference. The significant upregulations of pancreatic endocrine markers (PDX1 and NGN3), and the cell-specific glucose transporter (GLUT2) were observed in the differentiated IPCs in the 3D culture of Exp2 after 21 days. The highest insulin and C-peptide concentrations were observed in Exp2. In Exp3, insulin secretion in response to high glucose and 10 mM arginine was 42.43 ±6.34 µU/ml. A decellularized pancreas in the presence of hiPSCs and growth factors could be efficiently used as a natural scaffold.

MSC-derived exosomes carrying a cocktail of exogenous interfering RNAs an unprecedented therapy in era of COVID-19 outbreak.

BACKGROUND: The onset of the SARS-CoV-2 pandemic has resulted in ever-increasing casualties worldwide, and after 15 months, standard therapeutic regimens are yet to be discovered. MAIN BODY: Due to the regenerative and immunomodulatory function of MSCs, they can serve as a suitable therapeutic option in alleviating major COVID-19 complications like acute respiratory distress syndrome. However, the superior properties of their cognate exosomes as a cell-free product make them preferable in the clinic. Herein, we discuss the current clinical status of these novel therapeutic strategies in COVID-19 treatment. We then delve into the potential of interfering RNAs incorporation as COVID-19 gene therapy and introduce targets involved in SARS-CoV-2 pathogenesis. Further, we present miRNAs and siRNAs candidates with promising results in targeting the mentioned targets. CONCLUSION: Finally, we present a therapeutic platform of mesenchymal stem cell-derived exosomes equipped with exogenous iRNAs, that can be employed as a novel therapeutic modality in COVID-19 management aiming to prevent further viral spread within the lung, hinder the virus life cycle and pathogenesis such as immune suppression, and ultimately, enhance the antiviral immune response.

Downregulation of hepatitis C virus replication by miR-196a using lentiviral vectors.

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis and hepatocellular carcinoma. Cellular microRNAs (miRNAs) directly modulate the viral infectivity and indirectly through targeting virus-related host factors. They play an essential role in the progression of different stages of HCV infection. The roles of miR-196 family in HCV infection and hepatocellular carcinoma progression remain poorly understood. Using ViTa databases, miR-196a as a high-score miRNA targeting the NS5 A region of HCV genome was selected. Using dual luciferase assay and an established cell-cultured HCV (HCVcc) system, the effect of miR-196a on HCV genome was assessed. In silico analysis demonstrated the significant role of miR-196a in the downregulation of HCV replication. Using dual luciferase assay, the liver-specific miR-196a and NS5 A gene binding was confirmed. To assess the experimental role of miR-196a, an HCVcc system was established in the Huh 7.5 cell lines. The HCV-RNA 1b derived from an infected patient was transfected into Huh 7.5 cells containing miR-196a lentiviral vectors (Huh 7.5/miR-196a), mocks (Huh 7.5/mock vector), and naïve Huh 7.5 cells. The rate of reduction of the HCV genome replication was assessed using relative real-time PCR assay. These results represent miR-196a overexpression and its roles in regulating HCV genome replication. However, miR-196a may inhibit HCV replication and accelerate the early stages of apoptosis. Overexpression of miR-196a in Huh 7.5 replicon cell is a potential new strategy to prevent hepatitis C infection. The results of this study suggest that miR-196a directly downregulates HCV replication and may serve as a new antiviral therapy.

Inhibitory effect of flavonoid xanthomicrol on triple-negative breast tumor via regulation of cancer-associated microRNAs.

Breast cancer is the leading cause of cancer death in women worldwide. Due to the side effects of current chemo-reagents on healthy tissues, it is essential to search for alternative compounds with less toxicity and better efficacy. In the present study, we have investigated the anticancer effects of flavonoid xanthomicrol on the mice breast cancer model using MTT assay, cell cycle and Annexin/PI analysis, colony formation assay, H&E staining, immunohistochemistry, and miRNA analysis. Our results demonstrated that xanthomicrol decreased the cell viability and clonogenic capability, induced G1-arrest and apoptosis in the breast cancer cells in vitro, and caused a significant reduction in the volume and weight of mice tumors in vivo. In addition, xanthomicrol reduced the expression of TNFα, VEGF, MMP9, and Ki67, while upregulating the expression of apoptotic markers such as Bax, caspase3, and caspase9. Finally, the expression of miR21, miR27, and miR125, known as oncomirs, decreased significantly after xanthomicrol administration, while the expression of miR29 and miR34, functioning as tumor suppressors, increased significantly (p < .001). Our data demonstrated that xanthomicrol can induce apoptosis and suppress angiogenesis in breast cancer cells due to its inhibitory effect on oncomirs and its stimulatory effect on tumor suppressor miRNAs.

MicroRNA-4731-5p delivered by AD-mesenchymal stem cells induces cell cycle arrest and apoptosis in glioblastoma.

Glioblastoma multiforme (GBM) exhibits the most malignant brain tumor with very poor prognosis. MicroRNAs (miRNAs) are regulatory factors that can downregulate the expression of multiple genes. Several miRNAs acting as tumor-suppressor genes have been identified so far. The delivery of miRNA by mesenchymal stem cell (MSC) due to their ability to specifically target tumors is a new, hopeful therapeutic approach for glioblastoma. The objective of our study is the investigation of the effect of lentivirus-mediated microRNA-4731 (miR-4731) genetic manipulated adipose-derived (AD)-MSC on GBM. The downregulation of miR-4731 in human GBM tumor was detected using the GEO dataset. To evaluate the function of miR-4731, we overexpressed miR-4731 using lentiviral vectors in U-87 and U-251 GBM cell lines. The effects of miR-4731 on cell proliferation and cell cycle of glioma cells were analyzed by wound test and flow-cytometry assay. miR-4731 inhibited the proliferation of GBM cancer cells. Coculturing was used to study the antiproliferative effect of miR-4731-AD-MSCs on GBM cell lines. Direct and indirect coculture of GBM cell lines with miR-4731-AD-MSCs induced cell cycle arrest and apoptosis. Our findings suggest that AD-MSCs expressing miR-4731 have favorable antitumor characteristics and should be further explored in future glioma therapy.

The potency of hsa-miR-9-1 overexpression in photoreceptor differentiation of conjunctiva mesenchymal stem cells on a 3D nanofibrous scaffold.

MiRNAs are small non-coding RNAs that are ordinarily involved in modulating mRNAs and stem cell differentiation. 3D nanofibrous scaffolds have an important role in the differentiation of stem cells due to their similarity to the extracellular matrix (ECM). In the present study, we tried to introduce a new approach to guiding the differentiation of conjunctiva mesenchymal stem cells (CJMSCs) into photoreceptor-like cells by hsa-miR-9-1 delivery on both 2D and 3D substrates. First, the CJMSCs were transduced by a lentiviral vector carrying miR-9 (pCDH + hsa-miR-9-1) and then cell transduction efficacy verified by using fluorescent microscopy, flow cytometry, and qPCR analyses. Silk Fibroin-poly-L-lactic acid (SF-PLLA) scaffold was fabricated by the electrospinning technique while the scaffold characteristics including morphology, chemical properties, and biocompatibility were evaluated by SEM, FTIR, and MTT assays, respectively. Then, the miR-9-CJMSCs were seeded on both TCPS and the scaffold; photoreceptor gene and protein expressions were evaluated by RT-qPCR and immunostaining after 14 and 21 days of transduction. More than 80% of CJMSCs were transduced and miR-9 expression was significantly higher in miR-9-CJMSCs compared with empty vector (EV)-CJMSCs. SEM and FTIR confirmed the fabrication of the SF/PLLA hybrid structure. RT-qPCR and immunostaining analyses showed that the specific photoreceptor genes and proteins were expressed in miR-9 transduced CJMSCs. Mir-9 induced CJMSCs into photoreceptor-like cells in a time-dependent manneron on both TCPS and nanofibrous scaffold.We have proved that hsa-miR-9-1 has the potency to guide the photoreceptor differentiation of mesenchymal stem cells and promote retinal regeneration.

Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene.

BACKGROUND: Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA- pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group. RESULTS: Designed shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector. CONCLUSIONS: Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

miRandb: a resource of online services for miRNA research.

Recent discovery of thousands of small and large noncoding RNAs, in parallel to technical improvements enabling scientists to study the transcriptome in much higher depth, has resulted in massive data generation. This burst of information prompts the development of easily accessible resources for storage, retrieval and analysis of raw and processed data, and hundreds of Web-based tools dedicated to these tasks have been made available. However, the increasing number and diversity of bioinformatics tools, each covering a specific and specialized area, as well as their redundancies, represent potential sources of complication for end users. To overcome these issues, we are introducing an easy-to-follow classification of microRNA (miRNA)-related bioinformatics tools for biologists interested in studying this important class of small noncoding RNAs. We also developed our miRNA database miRNA algorithmic network database (miRandb) that is a meta-database, which presents a survey of > 180 Web-based miRNA databases. These include miRNA sequence, discovery, target prediction, target validation, expression and regulation, functions and their roles in diseases, interactions in cellular pathways and networks and deep sequencing. miRandb recapitulates the diverse possibilities and facilitates that access to the different categories of miRNA resources. Researchers can easily select the category of miRNA information and desired organism, in result eligible databases with their features are presented. This database introducing an easy-to-follow classification of available resources that can facilitate selection of appropriate resources for miRNA-related bioinformatics tools. Finally, we described current shortages and future necessities that assist researchers to use these tools easily. Our database is accessible at http://mirandb.ir.

Trimethyl chitosan-hyaluronic acid nano-polyplexes for intravitreal VEGFR-2 siRNA delivery: Formulation and in vivo efficacy evaluation.

As vascular endothelial growth factor in choroidal neovascularization is a major cause of visual loss of the elderlies and diabetics, gene therapy may offer an alternative treatment. However, siRNA instability and inefficient delivery are the main hindrances. To address this issue, we developed a nano-sized siRNA loaded therapeutic delivery system. The chitosan-hyaluronic acid nano-polyplexes were prepared by the modified ionic gelation method. The obtained nano-polyplex with a narrow size distribution, indicated no significant cytotoxicity in the MTT test and proper cellular uptake in confocal images. The RT-PCR analysis indicated remarkable gene silencing on HUVEC cells. The intravitreally administered nano-polyplexes in rabbits overcame both the vitreous and retina barriers and reached the posterior tissues efficiently. Intravitreal injections of the VEGFR-2 siRNA nano-polyplexes significantly reduced the size of the laser-induced choroidal neovascularization, compared to the control group. Consequently, the developed formulation can be a promising candidate for intravitreal delivery of siRNA.