Molecular Approach for HIV-1 Replication Inhibition: Assessment of Different siRNAs Targeting Tat and Nef Genes to Effectively Suppress their Expression.

BACKGROUND: Inhibition of viral genes through siRNA seems to be promising for treatment of complicated viral infections like human immunodeficiency virus (HIV-1). HIV-1 Tat (Trans Activator of Transcription) and Nef (Negative regulatory Factor) proteins are very interesting targets for designing siRNAs. METHODS: The effectiveness of suppressing Tat and Nef was investigated using three specific siTATs and three siNEFs. They were used to transfect the developed stable and infected Human Embryonic Kidney cells (HEK293) as an ex-vivo model. Both stable and virus infected HEK293 cells were transfected with each siTAT and siNEF. The inhibitory effect was evaluated using qRT-PCR, western blot analysis, and HIV P24 ELISA. RESULTS: siTAT-100, siTAT-162, and siNEF-136 and at a concentration of 100 nM/mL showed the most inhibitory effect on their target genes. CONCLUSIONS: Utilization of more developed molecular inhibition strategies such as RNAi or even a combination of different molecular approaches could be promising to overcome emerging HIV escape mutants.

Expression pattern of key microRNAs in patients with newly diagnosed chronic myeloid leukemia in chronic phase.

INTRODUCTION: Chronic myeloid leukemia (CML) is caused by reciprocal translocation in hematopoietic stem cells (HSCs). This translocation forms the BCR-ABL1 oncogene, which alters several signaling pathways that control malignancy. CML has three phases: chronic, accelerated, and blast crisis. The microRNAs (miRNAs or miRs) are noncoding RNAs that downregulate their target gene by targeting 3' UTR of mRNA or through translational inhibition. It has been shown that miRNAs regulate many biological processes, and dysregulation of these regulatory RNAs is involved in disease development, particularly in cancer. The important role of miRNAs as therapeutic agents and biomarkers has been demonstrated in CML patients at different phases of the disease. METHODS: Stem-loop reverse transcription polymerase chain reaction was used to characterize differentially expressed miRNAs of leukocytes in the peripheral blood of 50 newly diagnosed CML patients in chronic phase. RESULTS: Some onco-miRNAs were found to be downregulated (miR-155 and miR-106), and some tumor suppressor miRs (miR-16-1, miR-15a, miR-101, miR-568) were upregulated. CONCLUSION: These results show that very few miRNAs alone would be good candidates for CML diagnosis independently of conflicting results, but together could be an additional tool for CML diagnosis. Moreover, miRNAs might be good candidates for prognosis prediction and CML therapy.

Megakaryocytic differentiation of CD133+ hematopoietic stem cells by down-regulation of microRNA-10a.

MicroRNAs (miRNA), as a class of non-coding RNAs, play a crucial role in normal hematopoiesis. Recent studies on miRNA profiling during hematopoiesis showed miR-10a down-regulation in megakaryocytic differentiation. Here we investigated whether decrease in miR-10a can differentiate umbilical cord blood CD133+ cells to megakaryocytic series. We ectopically induced miR-10a down-regulation by locked nucleic acid anti-miR-10a transfection of CD133+ cells. The megakaryocytic differentiation was evaluated by CD42/CD61 and CD41 surface expression and colonogenic capacity in Megacult media. In addition, real-time polymerase chain reaction was done for detection of miR-10a and its target HoxA1 mRNA. HOXA1 protein expression was detected by flow cytometry as mean fluorescent intensity that shows the antibody reaction proteins. Our findings showed megakaryocytic differentiation of about 28% of umbilical cord blood CD133+ stem cells with bright expression of CD42/CD61 and CD41 in parallel with increase in HoxA1 mRNA and protein level. Colony forming of CD61+ cells in Megacult demonstrated the colonogenic capacity of differentiated progenitor cells. In conclusion, MiR-10a has a role in megakaryocyte differentiation of stem cells via HOXA1 transcription factor targeting.

Bypassing the maturation arrest in myeloid cell line U937 by over-expression of microRNA-424.

Micro RNAs are a class of small non-coding RNAs which has been recently shown to play a crucial role in major cellular processes such as development and differentiation through post-transcriptional regulation. The role of these epigenetic elements has also been demonstrated in hematopoietic lineage differentiation and there is a large body of evidence that miR-424 is responsible for monocyte differentiation. Our goal was to examine the effect of miR-424 over-expression on defeating the maturation blockage in monoblastic cell line U937. The permanent over-expression of miR-424 was established using a retroviral vector construct containing the precursor of miR-424 sequence. Induction of differentiation process was monitored by assaying changes in cell morphology, and expression of cell surface markers using light microscopy, quantitative RT-PCR, and flow cytometry for monocyte markers such as CD11b and CD14. The cells showed monocytic characteristics 14 days after transduction, and CD11b and CD14 expression were significantly increased, confirmed by flow cytometry QRT-PCR and RT-PCR results. In conclusion, miR-424 over-expression is an effective factor in maturation of the monoblastic U937 cells and it has the ability of directing them into cells, expressing monocyte/macrophage characteristics.

Differentiation MicroRNAs Affect Stemness Status of USSCs.

BACKGROUND: MicroRNAs are endogenous non-coding RNAs with important regulatory and cell fate functions. Many studies have shown that several microRNAs are obviously up-regulated during stem cell differentiation. The question rises here is weather inhibiting differentiation will affect the stemness and self renewal status of stem cells. METHODS: miRCURY ™LNA microRNA inhibitor (anti-miR-145 and anti-let7g) are a sequence-specific and chemically modified oligonucleotide that specifically target and knockdown miR-145 and let7g miRNA molecules. Unrestricted somatic stem cells (USSCs) were isolated from umbilical cord blood and treated with LNAs. The effect of anti-miRNA transfection was assessed by quantitative real-time PCR. RESULTS: Real-time PCR showed that LNA was efficiently introduced into the cells and reduced miR145 and Let7g expression levels to 40% and 10% in relation to corresponding scramble control, respectively. Gene expression analysis as to self renewal and expansion showed more than 3.5 fold up regulation in Oct4 in cells treated with mir145 inhibition. Similarly a significant up to 2.5 fold up-regulation in Oct4 and cMyc expression was observed in samples treated with anti-let7g. CONCLUSION: Suppression in differentiation inducing microRNAs (miR-145 and let7g) can enhance the self renewal and stemness status of USSCs at transcriptional level.

Nanofibrous poly(epsilon-caprolactone)/poly(vinyl alcohol)/chitosan hybrid scaffolds for bone tissue engineering using mesenchymal stem cells.

In the present study, based on a biomimetic approach, novel 3D nanofibrous hybrid scaffolds consisting of poly(epsilon-caprolactone), poly(vinyl alcohol), and chitosan were developed via a multi-jet electrospinning method. The influence of chemical, physical, and structural properties of the scaffolds on the differentiation of mesenchymal stem cells into osteoblasts, and the proliferation of the differentiated cells were investigated. Osteogenically induced cultures revealed that cells were well-attached, penetrated into the construct and were uniformly distributed. The expression of early and late phenotypic markers of osteoblastic differentiation was upregulated in the constructs cultured in osteogenic medium.