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MicroRNAs (miRNA) are a large family of small single-stranded RNA molecules found in all multicellular organisms. Early studies have been shown that miRNA are involved in cancer development and progression, and this role can be done by working as an oncogenes and tumor suppressor genes, so manipulation of this molecules can be a promising approach in cancer therapy, and experimental results represented that the modification in breast cancer phenotype is possible by miRNA expression alteration. miR-16, which is located in 13q14 chromosome, plays critical roles as a tumor suppressor by targeting several oncogenes which regulate cell cycle and apoptosis. Hence, in the present study, we investigated whether miR-16 could decline growth and survival of MCF-7 cell line as model of human breast cancer. MCF-7 cell line was infected with lentiviruses containing miR-16 precursor sequence. The effects of ectopic expression of miR-16 on breast cancer phenotype were examined by cell cycle analysis and apoptosis assays. miR-16 cytotoxicity effect was measured by the MTT assay. We showed that the miR-16 overexpression reduces Cyclin D1 and BCL2 at messenger RNA (mRNA) and protein levels in MCF-7 cell line. In addition, this is found that enforced expression of miR-16 decreases cell growth and proliferation and induces apoptosis in MCF-7 cells. In conclusion, our results revealed that upregulation of miR-16 would be a potential approach for breast cancer therapy.

The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (*50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (*90%). Moreover dependency of hMSCs to size of transfected nucleic acid and timedependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, Electronic supplementary material The online version of this article

Despite interesting in silico evidence, the specific role of mir-221 in osteogenesis has not been studied. We evaluated the osteogenic induction of transient-transfected anti-mir-221 in human unrestricted somatic stem cells and human mesenchymal stem cells both transcriptionally and translationally. In transfected unrestricted somatic stem cells, transcriptions of some osteogenic markers were twice that of the control and translations of osteopontin and osteocalcin were increased from 9 to 39 % and from 0 to 21 %, respectively. Up-regulation of transcribed osteogenic markers in transfected mesenchymal stem cells were 50 times greater than controls while no significant change in translations were observed. Prior to these analyses, the authenticity of stem cells, their osteogenic differentiation and transfection efficiency were verified. Transient modulation of mir-221 therefore suggests a mechanism for rapid induction of osteogenesis as a useful strategy for cell-based therapy.

Cell-based approaches offer a potential therapeutic strategy for appropriate bone manufacturing. Capable of differentiating into multiple cell types especially osteoblasts spontaneously, unrestricted somatic stem cell (USSC) seems to be a suitable candidate. Recent studies have shown the involvement of microRNAs in several biological processes. miRNA microarray profiling was applied in order to identify the osteo-specific miRNA signature. Prior to this analysis, osteogenic commitment of osteoblasts was evaluated by measuring ALPase activity, biomineralization, specific staining and evaluation of some main osteogenic marker genes. To support our findings, various in silico explorations (for both putative targets and signaling pathways) and empirical analyses (miRNA transfections followed by qPCR of osteogenic indicators and ALPase activity measurement) were carried out. The function of GSK-3b inhibitor was also studied to investigate the role of WNT in osteogenesis. Transient modulation of multiple osteo-miRs (such as mir-199b, 1274a, 30b) with common targets (such as BMPR, TCFs, SMADs) as mediators of osteogenic pathways including cell-cell interactions, WNT and TGF-beta pathways, suggests a mechanism for rapid induction of the osteogenesis as an anti-miRNA therapy. The results of this research have identified the miRNA signature which regulates the osteogenesis mechanism in USSC. To conclude, our study reveals more details about the allocation of USSCs into osteogenic lineage through modulatory effect of miRNAs on targets and pathways required for creating a tissue-specific phenotype and may aid in future clinical interventions.

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