Autophagy knock down: as a booster for the replication of viruses in cell culture

Background: Autophagy suppression recently has been known to have a remarkable effect for cellular adjustment and viability in the final stages of cancer. On the other hand, autophagy has the potential effect in preventing many viruses from replication. Beclin1 is the most substantial constituent in autophagy apparatus regulation. This study was intended to investigate the beclin1 siRNA knockdown effect on the extent of activity of the oncolytic vesicular stomatitis virus (VSV) as a model in cell culture. Materials and Methods: In the current study, the cancer cell line , HeLa (cervical squamous cancer cell line ) was infected by VSV, followed by beclin1 siRNA vector transfection. The potential change in the expressions of gene beclin1 in transfected cells, as well as untransfected ones were examined by real time PCR, and also the titer of viruses was compared in cells with and without transfection.Results: The results revealed that the amount of putative gene beclin1 expression in HeLa cells decreased greatly due to siRNA suppressive impact, and also the sensitivity of the cells to VSV oncolytic effect increased upon decrease in beclin1gene expression.Conclusion: It seems that autophagy suppression by using siRNA with VSV is a substantial aid for increase in virus titer in cancer cell lines

The Involvement of Mir-210 in Unrestricted Somatic Stem Cells Differentiation into Osteoblasts

Introduction: Bone surgery as a current bone treatment method is not always successful to fulfil bone repair in bone degenerative diseases orextensive injuries. Due to the limited capacity of bone remodeling, the demand for alternative approaches remains to be met. Thus, efforts in exvivo generation of bone forming cells, osteoblasts, and their further application in cell therapy as a promising approach are of vital prominencefrom a scientific perspective. Though several studies have focused on microRNA roles in osteoblast differentiation in various cell recourses, yetnone has reported miR-210 enhancing role in human mesynchymal stem cells (MSCs) so far.Materials and Methods: Hence, we wished toexamine the nature of the relationship between osteoblast differentiation and miR-210 in unique human mesynchymal stem cells, unrestrictedsomatic stem cells (USSCs). Osteoblast markers at gene level namely, Runx2, col I in addition to osteocalcin were assessed using qRT-PCR, andAlizarin Red S staining was also carried out to observe histochemical changes 7 days following miR-210 transduction.Results: The conclusion that follows from our findings represents a marked increase in osteoblast differentiation markers. Interestingly, for the first time, human USSCsdifferentiation into osteoblasts was performed in our research.Conclusion: our study may provide helpful insights into surmounting bonerelated issues by combination of both gene and cell therapy

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma: Supplementary Issue: Biomarkers for Colon Cancer

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC.Peer reviewe

Analysis of microRNA signatures using size-coded ligation-mediated PCR

The expression pattern and regulatory functions of microRNAs (miRNAs) are intensively investigated in various tissues, cell types and disorders. Differential miRNA expression signatures have been revealed in healthy and unhealthy tissues using high-throughput profiling methods. For further analyses of miRNA signatures in biological samples, we describe here a simple and efficient method to detect multiple miRNAs simultaneously in total RNA. The size-coded ligation-mediated polymerase chain reaction (SL-PCR) method is based on size-coded DNA probe hybridization in solution, followed-by ligation, PCR amplification and gel fractionation. The new method shows quantitative and specific detection of miRNAs. We profiled miRNAs of the let-7 family in a number of organisms, tissues and cell types and the results correspond with their incidence in the genome and reported expression levels. Finally, SL-PCR detected let-7 expression changes in human embryonic stem cells as they differentiate to neuron and also in young and aged mice brain and bone marrow. We conclude that the method can efficiently reveal miRNA signatures in a range of biological samples

Expression Change of miR-214 and miR-135 during Muscle Differentiation

Objective: MicroRNAs (miRNAs) are a class of small non-coding RNAs that play pivotal roles in many biological processes such as regulating skeletal muscle development where alterations in miRNA expression are reported during myogenesis. In this study, we aimed to investigate the impact of predicted miRNAs and their target genes on the myoblast to myocyte differentiation process. Materials and Methods: This experimental study was conducted on the C2C12 cell line. Using a bioinformatics approach, miR-214 and miR-135 were selected according to their targets as potential factors in myoblast to myocyte differentiation induced by 3% horse serum. Immunocytochemistry (ICC) was undertaken to confirm the differentiation process and quantitative real-time polymerase chain reaction (PCR) to determine the expression level of miRNAs and their targets. Results: During myoblast to myocyte differentiation, miR-214 was significantly downregulated while miRNA-135, Irs2, Akt2 and Insr were overexpressed during the process. Conclusion: miR-214 and miR-135 are potential regulators of myogenesis and are involved in skeletal muscle development through regulating the IRS/PI3K pathway

Development of Insulin Resistance through Induction of miRNA-135 in C2C12 Cells

Abstract Objective: Micro-RNAs (miRNAs) are a class of posttranscriptional regulators that play crucial roles in various biological processes. Emerging evidence suggests a direct link between miRNAs and development of several diseases including type 2 diabetes (T2D). In this study, we aimed to investigate the effect of predicted miRNA and target genes on insulin resistance. Materials and Methods: This experimental study was conducted on the C2C12 cell line. Using bioinformatics tools miRNA-135 and two respective target genes-insulin receptor (Insr) and vesicle associated membrane protein 2 (Vamp2)- were selected as potential factors involved in insulin resistance process. Levels of glucose uptake miRNA expression and respective gene targets were determined after cell transfaction by miR-135. Results: It was determined that Insr gene expression was significantly down-regulated in miR-135 transfected C2C12 cell line (P≤0.05). Interestingly; these transfected cells have shown a significant difference in glucose uptake incomparision the positive control cells, while it was similar to the insulin resistant cell line (P≤0.05). In contrast, no significant alteration of Vamp2 gene expression was observed. Conclusion: Our data indicated no change on the Vamp2 expression level after miRNA transfection, while expression level of Insr was reduced and miR-135 expression was contrarily increased leading to poor stimulation of glucose uptake through insulin, and development of insulin resistance phenotype in C2C12 cell line

Accelerated reconstruction of rat calvaria bone defect using 3D-printed scaffolds coated with hydroxyapatite/bioglass

Abstract Self-healing and autologous bone graft of calvaraial defects can be challenging. Therefore, the fabrication of scaffolds for its rapid and effective repair is a promising field of research. This paper provided a comparative study on the ability of Three-dimensional (3D) printed polycaprolactone (PCL) scaffolds and PCL-modified with the hydroxyapatite (HA) and bioglasses (BG) bioceramics scaffolds in newly bone formed in calvaria defect area. The studied 3D-printed PCL scaffolds were fabricated by fused deposition layer-by-layer modeling. After the evaluation of cell adhesion on the surface of the scaffolds, they were implanted into a rat calvarial defect model. The rats were divided into four groups with scaffold graft including PCL, PCL/HA, PCL/BG, and PCL/HA/BG and a non-explant control group. The capacity of the 3D-printed scaffolds in calvarial bone regeneration was investigated using micro computed tomography scan, histological and immunohistochemistry analyses. Lastly, the expression levels of several bone related genes as well as the expression of miR-20a and miR-17-5p as positive regulators and miR-125a as a negative regulator in osteogenesis pathways were also investigated. The results of this comparative study have showed that PCL scaffolds with HA and BG bioceramics have a great range of potential applications in the field of calvaria defect treatment