Distinctive Expression of Metastamirs in Breast Cancer Mesenchymal Stem Cells Isolated from Solid Tumor.

BACKGROUND: MSCs are a part of the tumor microenvironment, which secrete cytokines and chemokines. They can affect metastasis and the growth of tumors. metastamiRs are newly recognized regulatory elements of the metastasis pathway which are involved in epithelial-to-mesenchymal transition (EMT). OBJECTIVE: In the present study, we aimed to assess the expression profile of metastamiRs in the context of MSCs in correlation with their invasion and migration power. METHODS: tumor-isolated BC-MSCs and normal human mammary epithelial cells (HMECs) along with MCF-7, MDA-MB231, and MCF-10A cells were prepared and confirmed for their identity. The cells were assessed for CD44+CD24¯ percentage, Oct-4, and Survivin expression. GEO, KEGG, and TCGA databases were investigated to detect differential miR-expressions. Real-time PCR for 13 miRs was performed using LNA primers. Ultimately, Transwell-Matrigel assays as used to assess the level of migration and invasion. RESULTS: Our results indicated that some oncomiRs like miR-10b were upregulated in BC-MSCs, while the levels of miR-373 and miR-520c were similar to the MCF-10A. Generally, miR-200 family members were on lower levels compared to the other miR-suppressor (miR-146a, 146b, and 335). miR-31 and 193b were up-regulated in MCF-10A. The most invasiveness was observed in the MDA-MB231 cell line. CONCLUSION: We have demonstrated that the miR-expression levels of BC-MSCs are somewhat in between MCF-7 and MDA-MB231 miR-expression levels. This could be the logic behind the moderate level of invasion in BC-MSCs. Therefore, miR-therapy approaches such as miR-mimic or antagomiRs could be used for BC-MSCs in clinical cancer therapy.

Effect of Paclitaxel/etoposide co-loaded polymeric nanoparticles on tumor size and survival rate in a rat model of glioblastoma.

The aim of this work is to co-load paclitaxel (PTX) and etoposide (ETP) in methoxy poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (mPEG-PLGA NPs) to overcome pharmacokinetics and physiological limitations and enhance therapeutic efficacy for treating intracranial glioblastoma. Both drugs were loaded into mPEG-PLGA NPs by a nano-precipitation method. The resultant NPs demonstrated an enhanced cytotoxic effect indicated by lower IC50 values and augmented cell apoptosis to U87 and C6 glioma cell lines compared to both free drugs. Additionally, blood compatibility assays showed that the PTX/ETP co-loaded mPEG-PLGA NPs did not induce blood hemolysis, blood clotting, or platelet aggregation. In vivo anti-glioma efficacy evaluation in rats bearingintracranialC6glioma revealed a superior anti-glioma activity for the treatment with PTX/ETP co-loaded mPEG-PLGA NPs compared to other formulations, particularly a significantly longer median survival, 76 days compared to 36 days for free PTX and 37 days for free ETP treatment, respectively, and higher tumor regression, proved by magnetic resonance imaging (MRI).

Derivation of preoligodendrocytes from human-induced pluripotent stem cells through overexpression of microRNA 338.

MicroRNAs (miRNAs) control gene expression at the posttranscriptional level and have a critical role in many biological processes such as oligodendrocyte differentiation. Recent studies have shown that microRNA 338 (miR-338) is overexpressed during the oligodendrocyte development process in the central nervous system; this finding indicates a potentially important role for miR-338 in oligodendrocyte development. To evaluate this assumption, we studied the effect of miR-338 overexpression on promoting the differentiation of oligodendrocyte progenitor cells (OPCs), derived from human-induced pluripotent stem cells (hiPSC), into preoligodendrocyte. hiPSCs were differentiated into OPCs after treating for 16 days with basic fibroblast growth factor (BFGF), epidermal growth factor (FGF), and platelet-derived growth factor (PDGF)-AA. Bipolar OPCs appeared and the expression of OPC-related markers, including Nestin, Olig2, Sox10, PDGFRα, and A2B5 was confirmed by real-time polymerase chain reaction (PCR) and immunofluorescence. Then, OPCs were transduced by miR-338 expressing lentivirus or were treated with triiodothyronine (T3) for 6 days. Data obtained from real-time PCR and immunofluorescence experiment indicated that preoligodendrocyte markers such as Sox10, O4, and MBP were expressed at higher levels in transduced cells with miR-338 in comparison with the T3 group. So, the overexpression of miR-338 in iPSC-derived OPCs can promote their differentiation into preoligodendrocyte which can be used in cell therapy of myelin-related diseases.

Additive effect of metastamiR-193b and breast cancer metastasis suppressor 1 as an anti-metastatic strategy.

BACKGROUND: It has been reported that enhancing the cellular levels of miR-193b as well as breast cancer-metastasis-suppressor-1 (BRMS1) protein is associated with diminished metastatic characteristics in breast cancer. In view of these facts, as a new therapeutic intervention, we employed a restoration-based strategy using both miR-193b-3p mimic and optimized BRMS1 in the context of a chimeric construct. METHODS: miR-193b-3p and BRMS1 genes were cloned and the resulting plasmids were transfected into the MDA-MB231, MCF-7 and MCF-10A cell lines. microRNA expression levels were assessed by rea time PCR using LNA-primer and protein expression was confirmed by western blot method. Then, apoptosis, MTT, colony formation and invasion assays were carried out. RESULTS: The expression levels of miR-146a, miR-146b and miR-373 were up-regulated, while the miR-520c, miR-335 and miR-10b were down-regulated following the exogenous BRMS1 expression. The exogenous over-expression of BRMS1 was associated with higher amounts of endogenous miR-193b-3p expression and enabled more efficient targeting of the 3'UTR of uPA. Although, miR-193b-3p and BRMS1 are individually capable of suppressing breast cancer cell growth, migration and invasion abilities, their cistronic expression was capable of enhancing the ability to repress the breast cancer cells invasion. CONCLUSIONS: Our results collectively indicated the existence of an additive anti-metastatic effect between miR-193b-3p and BRMS1. Moreover, it has been hypothesized that the exogenous expression of a protein can effect endogenous expression of non-relevant microRNA. Our findings provide new grounds for miR-restoration therapy applications as an amenable anti-metastatic strategy.

Expression profile of miRNAs in urine samples of bladder cancer patients.

AIM: miRNAs have been suggested as biomarkers for bladder cancer. We aimed to find a diagnostic panel of miRNAs based on differential expression of miRNAs in urine specimens of patient with bladder cancer compared with control group. METHODS: miR-141, miR-10b, miR-34b and miR-103 were selected to assess their expression in urine samples of 66 bladder cancer patients and 53 matched controls using quantitative real time PCR. RESULTS: miR-10b and miR-34b were upregulated in cases compared with controls. The combination of four miRNAs showed a sensitivity of 75% and specificity of 63.5% with a diagnostic power of 72%. CONCLUSION: Certain miRNAs can be used as biomarkers for early diagnosis of bladder cancer.

Construction of a Recombinant Phage-vaccine Capable of Reducing the Growth Rate of an Established LL2 Tumor Model.

Over expression of the epidermal growth factor receptor (EGFR) in many human epithelial tumors has been correlated with disease progression and poor prognosis. EGFR-inhibiting immunotherapy has already been introduced in cancer therapy. Peptide displaying phage particles in eukaryotic hosts can behave as antigen carriers, able to activate the innate immune system and to elicit adaptive immunity. Herein, the M13-pAK8-VIII phagemid plasmid was engineered to contain the sequences for an EGFR mimotope along with the L2 extracellular domain of EGFR (EM-L2) which would produce the final peptide-phage vaccine. The prophylactic and therapeutic effects of this novel vaccine were evaluated on the Lewis lung carcinoma induced mouse (C57/BL6) model. The recombinant peptide was confirmed to be displayed on the surface of M13 phage as an extension for phage's PVIII protein. Immunization of mice with peptide-phage vaccine resulted in antibody production against EM-L2 and significant reduction of tumor growth rate by nearly 25 percent. In conclusion, EM-L2 displaying phage particles could be deemed as an encouraging strategy in contemporary cancer immunotherapy.

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics.

OBJECTIVES: Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays. MATERIALS AND METHODS: Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines. RESULTS: The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential. CONCLUSION: Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes.

Cross-linking gold nanoparticles aggregation method based on localised surface plasmon resonance for quantitative detection of miR-155.

MiR-155 plays a critical role in the formation of cancers and other diseases. In this study, the authors aimed to design and fabricate a biosensor based on cross-linking gold nanoparticles (AuNPs) aggregation for the detection and quantification of miR-155. Also, they intended to compare this method with SYBR Green real-time polymerase chain reaction (PCR). Primers for real-time PCR, and two thiolated capture probes for biosensor, complementary with miR-155, were designed. Citrate capped AuNPs (18.7 ± 3.6 nm) were synthesised and thiolated capture probes immobilised to AuNPs. The various concentrations of synthetic miR-155 were measured by this biosensor and real-time PCR method. Colorimetric changes were studied, and the calibration curves were plotted. Results showed the detection limit of 10 nM for the fabricated biosensor and real-time PCR. Also, eye detection using colour showed the weaker detection limit (1 µM), for this biosensor. MiR-133b as the non-complementary target could not cause a change in both colour and UV-visible spectrum. The increase in hydrodynamic diameter and negative zeta potential of AuNPs after the addition of probes verified the biosensor accurately fabricated. This fabricated biosensor could detect miR-155 simpler and faster than previous methods.

Serum Levels of miR-155, miR-326, and miR-133b as Early Diagnostic Biomarkers for the Detection of Human Acute Heart Allograft Rejection in Comparison with Serum Cardiac Troponin T.

BACKGROUND: Acute heart allograft rejection occurs as a result of antibody-mediated rejection that presents during the first month after transplantation. Finding a non-invasive biomarker is essential for diagnosis of heart allograft rejection. In this research, we intended to compare expression levels of several microRNAs across cardiac troponin T levels between rejected patients (who died before one month following transplantation), non-rejected patients (who survived for at least one month after transplantation), and non-transplanted patients (CABG surgery patients). METHODS: Serum levels of miR-155, miR-326, and miR-133b were evaluated by the q-RT-PCR method. Furthermore, cardiac troponin T levels were measured by a highly sensitive electrochemiluminescence assay. Finally, the data were analyzed by independent sample t-test using SPSS 21® computer software. Results: It was observed that miR-326 and miR-155 expression levels increased after 24h and 72h of surgery in rejected patients compared with the two other groups, but these increases were not statistically significant. Moreover, the decrease in miR-133b expression level was non-significant after transplantation in the rejected group compared with the non-rejected group. However, cTnT levels in rejected patients increased significantly compared with the other groups (P < .05). After ROC curve analysis, the cTnT marker with the most area under the curve (AUC = 1.00, 95% confidence interval, 1.00 to 1.00; P = .006), had the best discriminatory power, and among microRNAs, miR-326 had the largest area under curve (AUC = 0.81), and consequently the highest discriminatory power. CONCLUSIONS: We demonstrated that troponin T can be a more efficient biomarker than miRNAs for early prediction of human death caused by acute heart rejection, and the ROC curves analysis verified this finding.

Generation of the Fluorescent HPV16 E7 Protein for Detection of Delivery In vitro.

BACKGROUND: Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies. OBJECTIVE: The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro. METHODS: Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line. RESULTS: Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection. CONCLUSION: Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.

Critical-sized full-thickness skin defect regeneration using ovine small intestinal submucosa with or without mesenchymal stem cells in rat model.

Decellularized extracellular matrices (ECM) based materials are routinely used for a variety of clinical applications. Hereof, in vivo application of decellularized ovine small intestinal submucosal (DOSIS) layer as, a scaffold is yet to be investigated. In this study, the effectiveness of the DOSIS scaffold, with or without rat bone marrow mesenchymal stem cells (BM-MSCs), in full-thickness wound healing of critical-sized defect was experimentally studied in a rat model. The experimental groups included; group I (control), group II (DOSIS), and group III (BM-MSCs-seeded DOSIS). Wound healing of all groups was examined and compared clinically and histopathologically on days 7, 14, and 21 postoperation. Our results represented BM-MSCs-seeded DOSIS accelerated wound contraction and healing compared to both the DOSIS alone and control groups. Epithelization was close to completion 21 days postoperation in DOSIS alone. In OSIS with BM-MSCs group, epithelization was faster and had fully taken place at the subsequent time points. DOSIS layer, as cell-free form with low substantially DNA content, accelerated healing of rat skin wound defects that was created at critical-size and full-thickness. In conclusion, decellularized OSIS alone and in combination with BM-MSCs has the potential to be used as a wound graft material in skin regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2177-2190, 2018.

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation.

Fundamentals of protein and cell interactions in biomaterials.

The extracellular matrix (ECM) is an active and complex microenvironment with outstanding biomechanical, biophysical, and biochemical characteristics, which can indirectly or directly controls cell adhesion, migration, proliferation, and differentiation, as well as partaking in regeneration and homeostasis of organs and tissues. The ECM has captivated a great deal of attention with the rapid progress of tissue engineering (TE) in the field of regenerative medicine (RM). Approaches to TE, RM and cancer therapy center on the necessity to deliver cell signals to direct cell proliferation and differentiation. These "external signals" are induced from cell-cell, and cell-ECM, interactions, as well as from physico-chemical, mechanical stimuli and growth factors. With the advent of new biomaterials such as casein, we gave a general insight into cell-ECM protein interactions in biomaterials and their applications in TE, RM and cancer therapy. An account of the main ECM molecules and cellular receptors with emphasis on integrins and its ligands was given, their effect on the induction of particular signal transduction pathways is also elucidated.

Lung cancer and miRNAs: a possible remedy for anti-metastatic, therapeutic and diagnostic applications.

INTRODUCTION: Lung cancer still accounts for the leading cause of cancer-related deaths worldwide and despite the emerging advances in diagnostic and therapeutic techniques it remains to be a serious global public health concern. Micro-ribonucleic acids (microRNAs) are responsible for invasion and metastasis of various tumors including lung cancer which underscores the necessity of understanding their functions. Areas covered: Herein, we aim to summarize the recent advances made in our understanding of the miRNAs with special reference to lung cancer. Moreover, the role of miRNAs in crucial cellular processes will be elucidated. Various applications of the miRNAs would be explained and different kinds of them would be discussed to delineate their significance in lung cancer biology, therapy and diagnosis. Expert commentary: the miRNA study in the field of respiratory disease and specially lung cancer has emerged lately. Given the several miRNAs, which are in the clinical trials, this field is passing through its maturation phase which ultimately could rise to a robust tool for lung cancer therapy, diagnosis and prevention.

The Role of Stem Cells in the Treatment of Cerebral Palsy: a Review.

Cerebral palsy (CP) is a neuromuscular disease due to injury in the infant's brain. The CP disorder causes many neurologic dysfunctions in the patient. Various treatment methods have been used for the management of CP disorder. However, there has been no absolute cure for this condition. Furthermore, some of the procedures which are currently used for relief of symptoms in CP cause discomfort or side effects in the patient. Recently, stem cell therapy has attracted a huge interest as a new therapeutic method for treatment of CP. Several investigations in animal and human with CP have demonstrated positive potential of stem cell transplantation for the treatment of CP disorder. The ultimate goal of this therapeutic method is to harness the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. During the recent years, there have been many investigations on stem cell therapy. However, there are still many unclear issues regarding this method and high effort is needed to create a technology as a perfect treatment. This review will discuss the scientific background of stem cell therapy for cerebral palsy including evidences from current clinical trials.

Improved human endometrial stem cells differentiation into functional hepatocyte-like cells on a glycosaminoglycan/collagen-grafted polyethersulfone nanofibrous scaffold.

Liver tissue engineering (TE) is rapidly emerging as an effective technique which combines engineering and biological processes to compensate for the shortage of damaged or destroyed liver tissues. We examined the viability, differentiation, and integration of hepatocyte-like cells on an electrospun polyethersulfone (PES) scaffold, derived from human endometrial stem cells (hEnSCs). Natural polymers were separately grafted on plasma-treated PES nanofibers, that is, collagen, heparan sulfate (HS) and collagen-HS. Galactosilated PES (PES-Gal) nanofibrous were created. The engineering and cell growth parameters were considered and compared with each sample. The cellular studies revealed increased cell survival, attachment, and normal morphology on the bioactive natural polymer-grafted scaffolds after 30 days of hepatic differentiation. The chemical and molecular assays displayed hepatocyte differentiation. These cells were also functional, showing glycogen storage, α-fetoprotein, and albumin secretion. The HS nanoparticle-grafted PES nanofibers demonstrated a high rate of cell proliferation, differentiation, and integration. Based on the observations mentioned above, engineered tissue is a good option in the future, for the commercial production of three-dimensional liver tissues for clinical purposes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2516-2529, 2017.

Role of protease and protease inhibitors in cancer pathogenesis and treatment.

Cancer is the second cause of death in 2015, and it has been estimated to surpass heart diseases as the leading cause of death in the next few years. Several mechanisms are involved in cancer pathogenesis. Studies have indicated that proteases are also implicated in tumor growth and progression which is highly dependent on nutrient and oxygen supply. On the other hand, protease inhibitors could be considered as a potent strategy in cancer therapy. On the basis of the type of the key amino acid in the active site of the protease and the mechanism of peptide bond cleavage, proteases can be classified into six groups: cysteine, serine, threonine, glutamic acid, aspartate proteases, as well as matrix metalloproteases. In this review, we focus on the role of different types of proteases and protease inhibitors in cancer pathogenesis.

Nano-structures mediated co-delivery of therapeutic agents for glioblastoma treatment: A review.

Glioblastoma is a malignant brain tumor and leads to death in most patients. Chemotherapy is a common method for brain cancer in clinics. However, the recent advancements in the chemotherapy of brain tumors have not been efficient enough. With the advancement of nanotechnology, the used drugs can enhance chemotherapy efficiency and increase the access to brain cancers. Combination of therapeutic agents has been recently attracted great attention for glioblastoma chemotherapy. One of the early benefits of combination therapies is the high potential to provide synergistic effects and decrease adverse side effects associated with high doses of single anticancer drugs. Therefore, brain tumor treatments with combination drugs can be considered as a crucial approach for avoiding tumor growth. This review investigates current progress in nano-mediated co-delivery of therapeutic agents with focus on glioblastoma chemotherapy prognosis.

Application of immuno-PCR for the detection of early stage cancer.

Cancer detection in premalignant stage is directly related with increase survival rate. Several biomarkers have been investigated and characterized for monitoring changes inside the cancerous cells. Although enzyme-linked immunosorbent assay (ELISA) is the method of choice in clinical practice for detecting biomarkers in serum/urine samples. However, in certain malignancies the amount of biomarkers before reaching metastasis are too low to be detected by conventional ELISA. The seminal work of Sano et al. led to the development of highly sensitive and powerful detection method, the immuno-PCR (iPCR), which can detect very small amount of antigens/biomarkers. In spite of, several publications on iPCR sensitivity, it has not been recommended for clinical use and is limited to the scientific community only. In order to evaluate the importance of iPCR, we have made an effort to collect published studies, supporting the use of iPCR in detecting premalignant cancer.

Inducible expression of indoleamine 2,3-dioxygenase attenuates acute rejection of tissue-engineered lung allografts in rats.

Lung disease remains one of the principal causes of death worldwide and the incidence of pulmonary diseases is increasing. Complexity in treatments and shortage of donors leads us to develop new ways for lung disease treatment. One promising strategy is preparing engineered lung for transplantation. In this context, employing new immunosuppression strategies which suppresses immune system locally rather than systemic improves transplant survival. This tends to reduce the difficulties in transplant rejection and the systemic impact of the use of immunosuppressive drugs which causes side effects such as serious infections and malignancies. In our study examining the immunosuppressive effects of IDO expression, we produced rat lung tissues with the help of decellularized tissue, differentiating medium and rat mesenchymal stem cells. Transduction of these cells by IDO expressing lentiviruses provided inducible and local expression of this gene. To examine immunosuppressive properties of IDO expression by these tissues, we transplanted these allografts into rats and, subsequently, evaluated cytokine expression and histopathological properties. Expression of inflammatory cytokines IFNγ and TNFα were significantly downregulated in IDO expressing allograft. Moreover, acute rejection score of this experimental group was also lower comparing other two groups and mRNA levels of FOXP3, a regulatory T cell marker, upregulated in IDO expressing group. However, infiltrating lymphocyte counting did not show significant difference between groups. This study demonstrates that IDO gene transfer into engineered lung allograft tissues significantly attenuates acute allograft damage suggesting local therapy with IDO as a strategy to reduce the need for systemic immunosuppression and, thereby, its side effects.