PDL1 targeting by miR-138-5p amplifies anti-tumor immunity and Jurkat cells survival in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) has constituted over 80% of the lung cancer population with a poor prognosis. Over the past decade, immunotherapy has been constructed in the enlargement of immune checkpoint inhibitors as a promising approach for NSCLC treatment. Evading the immune system using the PD-1/PD-L1 axis is an intelligent way for cancers, and T cells cannot respond fully and confront cancer. Recently, the miR-138 was reported as a PD-L1 regulator in NSCLC. However, its inhibitory impact on T-cell exhaustion has not been characterized. The present study aims to impair PD-L1 (B7-H1) expression in Adenocarcinoma cell lines using miR-138-5p and determines how it prevents Jurak cell exhaustion. To gain the purpose, first, 18 highly significant dysregulated miRNAs containing hsa-miR-138 and CD274-mRNA network were detected in NSCLC based on bioinformatics analysis. Moreover, our study revealed a high level of miR-138-5p could make significant changes like PDL1 downregulation, proliferation, and mortality rate in A549/Calu6 cells. We also simulate cancer environmental conditions by culturing Jurak cells and NSCLC cell lines under the influence of stimulator cytokines to show how miR-138-5p survives Jurak cells by targeting PD-L1/PD-1pathway.

Short Chain Fatty Acid Sodium Butyrate Increases miR-21, miR-143 and miR-145 Expression in Human Colorectal Cancer HCT-116 Cell Line.

Background: Sodium butyrate (NaBu) is a short-chain fatty acid; it is one of the histone deacetylase inhibitors, which can alter both genetic and epigenetic expressions. The present study aimed to elucidate the effect of Na-Bu on the expression of miR-21, miR-143, and miR-145 in human colorectal cancer HCT-116 cell lines. Methods: This study was done in Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. HCT-116 cell line was treated with diverse concentrations of NaBu (6.25 mM to 200 mM) at 24, 48, and 72 h. MTT assay was used for assessing the cytotoxicity. Quantitative Real-Time-PCR was performed to investigate the gene expression of miR-21, miR-143, and miR-145. Results: IC50 values were evaluated by MTT assay. IC50 for HCT-116 was 50 mM, 12.5 mM, and 6.25 mM for 24, 48, and 72 h of incubation, respectively. According to the Real-Time-PCR results, 50 mM NaBu after 24 h caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In 48 h, incubation, 12.5 mM NaBu caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 (P<0.05). In treated cells with 6.25 mM NaBu after 72 h of incubation caused a significant up-regulation in the expression of the miR-21, miR-143, and miR-145 compared with untreated cells (P<0.05). Conclusion: The upregulation of miR-21, miR-143, and miR-145 expression are mediated by transcriptional regulation and the activation of this miR promoter is modulated by histone acetylation. The employment of NaBu may represent a promising approach for improving HDACi drug-based therapies for colon cancers.

Distinct power of bone marrow microRNA signatures and tumor suppressor genes for early detection of acute leukemia.

BACKGROUND: Acute leukemia involving lymphocytic and myeloid cells is cancer with a high mortality rate. Swift and timely diagnosis might be a potential approach to improving patient prognosis and survival. The microRNA (miRNA) signatures are emerging nowadays for their promising diagnostic potential. MiRNA levels from bone marrow can be used as prognostic biomarkers. METHODS: The current study was designed to evaluate if the microRNAs and tumor suppressor genes (TSGs) profiling of hematopoietic bone marrow could help in acute leukemia early detection. Also, we assessed the DNA methyltransferase 3A (DNMT3A) expression and its possible epigenetic effects on miRNAs plus TSGs expression levels. The expression levels of ten miRNAs and four TSGs involved in acute lymphocytic leukemia (ALL) as well as acute myeloid leukemia (AML) were quantified in 43 and 40 bone marrow samples of ALL and AML patients in comparison with cancer-free subjects via real-time quantitative PCR (RT-qPCR). The receiver-operating-characteristic (ROC) analysis of miRNAs was performed in the study groups. Further, the correlation between the DNMT3A and TSGs was calculated. RESULTS: Significant differences were detected in the bone marrow expression of miRNAs and TSGs (P < 0.05) between acute leukemia patients and healthy group. ROC analysis confirmed the ability of miR-30a, miR-101, miR-132, miR-129, miR-124, and miR-143 to discriminate both ALL and AML patients with an area under the ROC curve of ≥ 0.80 (P < 0.001) and high accuracy. The correlation between DNMT3A and P15/P16 TSGs revealed that DNMT3A plays a vital role in epigenetic control of TSGs expression. Our findings indicated that the downregulation of bone marrow miRNAs and TSGs was accompanied by acute leukemia development. CONCLUSIONS: The authors conclude that this study could contribute to introducing useful biomarkers for acute leukemia diagnosis.

PANC-1 cancer stem-like cell death with silybin encapsulated in polymersomes and deregulation of stemness-related miRNAs and their potential targets.

OBJECTIVES: Cancer stem cells (CSCs) have powerful self-renewal ability and tumor recurrence. Pancreatic ductal adenocarcinoma is a malignancy with high mortality rate and ˃5% survival. Silybin has anticancer and hepatoprotective properties. We loaded silybin in PEG400-OA (SPNs) and evaluated its cytotoxic effects on PANC-1 cells and PANC-1 CSCs. MATERIALS AND METHODS: Spheroids from PANC-1 cells were obtained by the hanging drop method. Anti-proliferative and apoptotic functions of SPNs were evaluated in spheroids and non-spheroids with MTT, DNA fragmentation, PI and PI/AnnexinV assays. The expression of CD markers was assessed with flow cytometry. QRT-PCR was used to evaluate the expression of some miRNAs and targets. RESULTS: IC50 of SPNs was identified to be 50 µg/ml, 45 µg/ml, and 42µg/ml, respectively after 24 hr, 48 hr, and 72 hr in PANC-1 treated cells. PI staining and PI/AnnexinV assay showed that ~20%, ~60%, and ~80%, of cells treated with 30, 50, and 60 µg/ml of SPNs were in sub-G1 and apoptosis phase, respectively. DNA degradation was confirmed after SPNS stimulation. CD24, CD44, and CD133 expression decreased after SPNs treatment both in PANC-1 spheroid cells and PANC-1 cancer cell line. Under-expression of onco-miRs (miR-21, miR-155, and miR-221), over-expression of several apoptotic potential targets of oncomiRs (Bax, Casp-9, and P53), over-expression of tumor suppressive-miRs (let-7b, miR-34a, and miR-126), and under-expression of Bcl-2 was found in SPNs-treated cells. CONCLUSION: We suggest that silybin encapsulated in polymersomes (SPNs) may be useful as a complementary agent for destroying both pancreatic cancer cells and pancreatic CSCs along with chemotherapeutic agents.

Involved microRNAs in alternative polyadenylation intervene in breast cancer via regulation of cleavage factor "CFIm25".

Cleavage factor "CFIm25", as a key repressor at proximal poly (A) site, negatively correlates to cell proliferation and tumorigenicity in various cancers. Hence, understanding CFIm25 mechanism of action in breast cancer would be a great benefit. To this aim four steps were designed. First, potential miRNAs that target 3'-UTR of CFIm25 mRNA, retrieved from Targetscan web server. Second, screened miRNAs were profiled in 100 breast cancer and 100 normal adjacent samples. Third, miRNAs that their expression was inversely correlated to the CFIm25, overexpressed in MDA-MB-231 cell line, and their effect on proliferation and migration monitored via MTT and wound healing assays, respectively. Fourth, interaction of miRNAs of interest with 3'-UTR of CFIm25 confirmed via luciferase assay and western blot. Our results indicate that CFIm25 considerably down-regulates in human breast cancer tissue. qRT-PCR assay, luciferase test, and western blotting confirm that CFIm25 itself could be directly regulated by oncomiRs such as miR-23, -24, -27, -135, -182 and -374. Besides, according to MTT and wound healing assays of cell lines, CFIm25 knockdown intensifies cell growth, proliferation and migration. Our results also confirm indirect impact of CFIm25 on regulation of mRNA's 3'-UTR length, which then control corresponding miRNAs' action. miRNAs directly control CFIm25 expression level, which then tunes expression of the oncogenes and tumor proliferation. Therefore, regulation of CFIm25 expression level via miRNAs is expected to improve treatment responses in breast cancer.

Apoptosis induction and proliferation inhibition by silibinin encapsulated in nanoparticles in MIA PaCa-2 cancer cells and deregulation of some miRNAs.

OBJECTIVES: Silibinin, as an herbal compound, has anti-cancer activity. Because of low solubility of silibinin in water and body fluids, it was encapsulated in polymersome nanoparticles and its effects were evaluated on pancreatic cancer cells and cancer stem cells. MATERIALS AND METHODS: MIA PaCa-2 pancreatic cancer cells were treated with different doses of silibinin encapsulated in polymersome nanoparticles (SPNs). Stemness of MIA PaCa-2 cells was evaluated by hanging drop technique and CD133, CD24, and CD44 staining. The effects of SPNs on cell cycle, apoptosis and the expression of several genes and miRNAs were investigated. RESULTS: IC50 of SPNs was determined to be 40 µg/ml after 24 hr. Our analysis showed that >98% of MIA PaCa-2 cells expressed three stem cell markers. FACS analysis showed a decrease in these markers in SPNs-treated cells. PI/AnnexinV staining revealed that 40 µg/ml and 50 µg/ml of SPNs increased apoptosis up to ~40% and >80% of treated cells, respectively. Upregulation of miR-34a, miR-126, and miR-let7b and downregulation of miR-155, miR-222 and miR-21 was observed in SPNs-treated cells. In addition, downregulation of some genes involved in proliferation or migration such as AKT3, MASPINE, and SERPINEA12, and upregulation of apoptotic genes were observed in treated cells. CONCLUSION: Our results suggested that SPNs induced apoptosis and inhibited migration and proliferation in pancreatic cells and cancer stem cells through suppression of some onco-miRs and induction of some tumor suppressive miRs, as well as their targets.

Combination of low intensity electromagnetic field with chondrogenic agent induces chondrogenesis in mesenchymal stem cells with minimal hypertrophic side effects.

Background: There are different methods to develop in vitro neo-chondral tissues from adipose-derived stem cells (ADSCs). Application of electromagnetic field (EMF) on ADSCs is one of popular approaches, which results in chondrogenesis. If chondrogenic impact of EMF on ADSCs is supposed to be generalized as a protocol in translational medicine field, possible emergence of early or late hypertrophic maturation, mineralization and inflammatory side effects in chondrogenically differentiating ADSCs should be considered.Methods: The advent of chondrogenic and hypertrophic markers by differentiated cells under standard, platelet-rich plasma (PRP)-based or EMF treatments were monitored. Along with monitoring the expressions of chondrogenic markers, inflammatory and hypertrophic markers, VEGF/TNFα secretion, calcium deposition and ALP activity were evaluated.Results: Accordingly, treatment with %5 PRP results in higher GAG production, enhanced SOX9 transcription, lowered TNFα and VEGF secretions compared to other treatments. Although PRP up-regulates miR-146a and miR-199a in early and late stages of chondrogenesis, respectively, application of EMF + PRP down regulates miR-101 and -145 while up-regulates miR-140 and SOX9 expression.Conclusion: Comparing our results with previous reports suggests that presented EMF-ELF in this study with f = 50 Hz, EMF intensity of less than 30 mT, and 5% PRP (v/v), would facilitate chondrogenesis via mesenchymal stem cells with minor inflammation and hypertrophic maturation.Abbreviations: MSCs: mesenchymal stem cells; TGFß: transforming growth factor-beta; PRP: platelet-rich plasma; ELF-EMF: extremely low-frequency electromagnetic fields; GAGs: glycosaminoglycans; ADSCs: adipose-derived stem cells; VEGF: vascular endothelial growth factor; TNFα: tumor necrosis factor alpha; ALP: alkaline phosphatase.

Comparative impact of platelet rich plasma and transforming growth factor-ß on chondrogenic differentiation of human adipose derived stem cells.

Introduction: Transforming growth factor-beta (TGF-ß) is known as standard chondrogenic differentiation agent, even though it comes with undesirable side effects such as early hypertrophic maturation, mineralization, and secretion of inflammatory/angiogenic factors. On the other hand, platelet-rich plasma (PRP) is found to have a chondrogenic impact on mesenchymal stem cell proliferation and differentiation, with no considerable side effects. Therefore, we compared chondrogenic impact of TGF-ß and PRP on adipose-derived stem cells (ADSCs), to see if PRP could be introduced as an alternative to TGF-ß. Methods: Differentiation of ADSCs was monitored using a couple of methods including glycosaminoglycan production, miRNAs expression, vascular endothelial growth factor (VEGF)/tumor necrosis factor alpha (TNFα) secretion, alkaline phosphatase (ALP) and calcium content assays. Results: Accordingly, the treatment of differentiating cells with 5% (v/v) PRP resulted in higher glycosaminoglycan production, enhanced SOX9 transcription, and lowered TNFα and VEGF secretion compared to the control and TGF-ß groups. Besides, the application of PRP to the media up-regulated miR-146a and miR-199a in early and late stages of chondrogenesis, respectively. Conclusion: PRP induces in vitro chondrogenesis, as well as TGF-ß with lesser inflammatory and hypertrophic side effects.

Maternal supplementation with fish oil modulates inflammation-related MicroRNAs and genes in suckling lambs.

Dietary n-3 long-chain fatty acids (n-3 LCFA) have been shown to modify lipid metabolism and immune function. The objective of this study was to evaluate the effect of periparturient fish oil (FO) supplementation on the inflammation and metabolic health of ewes and their lambs at a molecular level. Prepartum ewes were fed control diet (CON, n = 12) or CON supplemented with 2% DM of calcium soap of FO (n = 12) from 28 days before until 21 days after parturition. The ewes were evaluated for plasma metabolites and milk composition. The experiment was followed by analyzing the relative transcript abundance of circulating microRNAs (miRNAs) in plasma and targeted miRNA/mRNA expression in peripheral blood mononuclear cells (PBMCs) in both ewes and lambs. FO treatment decreased prepartum feed intake (1812 ± 35 vs 1674 ± 33 g/day, P < 0.01), whereas the influence on plasma metabolites was negligible. Dietary FO supplementation decreased milk fat percentage (8.82 ± 0.49 vs 7.03 ± 0.45, P = 0.02) and reduced milk n-6/n-3 (P < 0.05). Also, it altered the expression of plasma-circulating miRNAs in both ewe and lamb (P < 0.05). Furthermore, maternal nutrition of FO downregulated the relative expression of miR-33a and miR-146b and transcript abundance of genes IL-1ß (0.41-fold) and NF-κB (0.25-fold) in lambs' PBMC. In conclusion, results showed that FO supplementation starting antepartum affects milk composition and circulating miRNA in dams and the inflammatory markers in lambs delivered by the supplemented ewes. These may provide a strategy to maintain immune balance during gestation and develop the immune system in lambs.

The effect of miR-579 on the PI3K/AKT pathway in human glioblastoma PTEN mutant cell lines.

Glioblastoma multiform (GBM) is a type of aggressive brain cancer with limited success in standard treatment. MicroRNAs are one of the most beneficial tools for diagnosis, prognosis, and treatment of cancer. This study aimed to investigate the effect of miR-579 on cellular behaviors and expression of PI3K/AKT signaling pathway in GBM cell lines. In the present study, miR-579 was overexpressed in U251 and A-172 cell lines by using lentil vector, and its effect on cellular behavior such as proliferation and migration was investigated by the cell cycle, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V, colony formation, Transwell and wound healing assays. MiR-579 predicted target genes (AKT1, Rheb, PDK1, and a few others) were also evaluated by real-time polymerase chain reaction or luciferase assay and Western blot analysis. Our results represented that overexpression of miR-579 could inhibit proliferation, migration, cell cycle and also promoted the apoptosis of GBM cell lines. The luciferase reporter assay showed miR-579 directly targets the 3 UTR of mTOR, Rheb, and PDK1 and repressed their expressions. Furthermore, the Western blot analysis showed that miR-579 could downregulate the AKT1 and Rheb protein expression. Overall, our findings propose that miR-579 functions as a novel tumor suppressor gene in GBM by regulating the PI3K/AKT signaling pathway and may serve as a therapeutic target for clinical therapy of glioblastoma multiform.

Overexpression of miR-133 decrease primary endothelial cells proliferation and migration via FGFR1 targeting.

Angiogenesis is one of the essential hallmarks of cancer that is controlled by the balance between positive and negative regulators. FGFR1 signaling is crucial for the execution of bFGF-induced proliferation, migration, and tube formation of endothelial cells (ECs) and onset of angiogenesis on tumors. The purpose of this study is to identify whether or not miR-133 regulates FGFR1 expression and accordingly hypothesize if it plays a crucial role in modulating bFGF/FGFR1 activity in ECs and blocking tumor angiogenesis through targeting FGFR1. The influences of miR-133 overexpression on bFGF stimulated endothelial cells were assessed by cell growth curve, MTT assaying, tube formation, and migration assays. Forced expression of miR-133 caused significant reductions in bFGF-induced proliferation and migratory ability of ECs. MiR-133 Expression was negatively correlated with both mRNA and protein levels of FGFR1 in the transfected ECs isolated from peripheral blood. Moreover, overexpression of miR-133 drastically reduced the rate of cell division and disturbed capillary network formation of transfected ECs. These findings suggest that miR-133 plays an important function in bFGF-induced angiogenesis processes in ECs and provides a rationale for new therapeutic approaches to suppress tumor angiogenesis and cancer.

Targeting histone demethylases KDM5A and KDM5B in AML cancer cells: A comparative view.

Epigenetic modifications play an important role in initiation and progression of cancers including acute myeloid leukemia. Among different epigenetic modifiers, lysine specific demethylases have been noticed as potential therapeutic targets. KDM5 family of histone demethylases which removes methyl marks from lysine residues of H3, are frequently found in the promoter region of transcriptionally active genes resulting in repression of expression. Here we have compared the effects of KDM5A and KDM5B downregulation on HL-60 cell line behavior. KDM5A/5B knockdown resulted in lower viability of HL-60 cells in addition to modified cell cycle distribution and sub-G1 accumulation. Induction of apoptosis was observed in both knockdown cells. But in spite of similarity in their role, downregulation of KDM5A showed more efficient anti-leukemic effects in comparison to KDM5B. Cells showed higher accumulation in sub-G1 and apoptosis occurred significantly higher and also earlier after KDM5A reduction. Expression analysis confirmed almost 5 and 4 fold increased expression for bax and caspase-3 after downregulation of KDM5A in comparison to KDM5B. Due to the present study we propose KDM5A as a potential target for therapeutic aspects of acute myeloid leukemia although further investigations are needed.

Functional SNP in microRNA-491-5p binding site of MMP9 3'-UTR affects cancer susceptibility.

MicroRNAs (miRNA) are small RNA molecules that negatively regulate gene expression through base pairing interactions between 3'-UTR of the target mRNAs and seed sequence of miRNA. Any changes in the recognition site could destroy binding sites or modify binding affinity, resulting in evasion from miRNA regulation. A putative binding site for miR-491-5p resides in 3'-UTR of MMP9, and a genetic variant (rs1056628 A → C) is present in this region. The role of MMP9 over expression well marked in various cancers. However, whether rs1056628 SNP in miR-491-5p binding site of MMP9 3'-UTR could abrogate its post-transcriptional regulation and affect cancer susceptibility remains largely unknown. To test this, the rs1056628 SNP was genotyped in 300 cases of lung, gastric and breast cancers and 200 age- and sex-matched healthy controls. The results showed that compared with the AA genotype, C was a risk genotype for all three cancers development and was also associated with gastric and breast cancers metastasis and invasion. Based on the base pairing analysis and secondary structure evaluation of MMP9 mRNA and miR-491-5p, we found that miR-491-5p had a higher binding affinity for A genotype than the C genotype. The Luciferase activity of MMP9 3'-UTR indicates differential regulation of two genetic variations of MMP9. Overexpression of miR-491-5p decreased MMP9 mRNA level in cell lines of gastric, breast and lung cancers and thus leads to decreasing of the invasion ability. Therefore, for the first time we imply that the C variant of MMP9 contributes to the likelihood of gastric, breast and lung cancers susceptibility via a novel mechanism of subtle gene regulation through miRNA binding capacity.

Survival Improvement in Human Retinal Pigment Epithelial Cells via Fas Receptor Targeting by miR-374a.

Oxidative conditions of the eye could contribute to retinal cells loss through activating the Fas-L/Fas pathway. This phenomenon is one of the leading causes of some ocular diseases like age-related macular degeneration (AMD). By targeting proteins at their mRNA level, microRNAs (miRNAs) can regulate gene expression and cell function. The aim of the present study is to investigate Fas targeting by miR-374a and find whether it can inhibit Fas-mediated apoptosis in primary human retinal pigment epithelial (RPE) cells under oxidative stress. So, the primary human RPE cells were transfected with pre-miR-374a pLEX construct using polymeric carrier and were exposed to H2 O2 (200 µM) as an oxidant agent for induction of Fas expression. Fas expression at mRNA and protein level was evaluated by quantitative real-time PCR and Western blot analysis, respectively. These results revealed that miR-374a could prevent Fas upregulation under oxidative conditions. Moreover, Luciferase activity assay confirmed that Fas could be a direct target of miR-374a. The cell viability studies demonstrated that caspase-3 activity was negligible in miR-374a treated cells compared to the controls. Our data suggest miR-374a is a negative regulator of Fas death receptor which is able to enhance the cell survival and protect RPE cells against oxidative conditions. J. Cell. Biochem. 118: 4854-4861, 2017. © 2017 Wiley Periodicals, Inc.

Efficient gene delivery to primary human retinal pigment epithelial cells: The innate and acquired properties of vectors.

The purpose of this study is designing non-viral gene delivery vectors for transfection of the primary human retinal pigment epithelial cells (RPE). In the design process of gene delivery vectors, considering physicochemical properties of vectors alone does not seem to be enough since they interact with constituents of the surrounding environment and hence gain new characteristics. Moreover, due to these interactions, their cargo can be released untimely or undergo degradation before reaching to the target cells. Further, the characteristics of cells itself can also influence the transfection efficacy. For example, the non-dividing property of RPE cells can impede the transfection efficiency which in most studies was ignored by using immortal cell lines. In this study, vectors with different characteristics differing in mixing orders of pDNA, PEI polymer, and PLGA/PEI or PLGA nanoparticles were prepared and characterized. Then, their characteristics and efficacy in gene delivery to RPE cells in the presence of vitreous or fetal bovine serum (FBS) were evaluated. All formulations showed no cytotoxicity and were able to protect pDNA from premature release and degradation in extracellular media. Also, the adsorption of vitreous or serum proteins onto the surface of vectors changed their properties and hence cellular uptake and transfection efficacy.

The Effect of Mir-451 Upregulation on Erythroid Lineage Differentiation of Murine Embryonic Stem Cells.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis. MATERIALS AND METHODS: In this experimental study, murine embryonic stem cells (mESCs) were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors (Gata-1, Klf-1, Epor) and hemoglobin chains (α, ß, γ , ε and ζ) genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119 and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days 14thand 21thafter transduction. RESULTS: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with the induction of transcriptional factor (Gata-1, Klf-1, Epor) and hemoglobin chain (α, ß, γ, ε and ζ) genes in mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells (13.084and 13.327-fold increse, respectively) (P<0.05). Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). CONCLUSION: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells (RBCs) without the presence of any stimulatory cytokines.

MicroRNAs modulating angiogenesis: miR-129-1 and miR-133 act as angio-miR in HUVECs.

The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis. Aberrant angiogenesis leads to enormous pathological conditions such as ischemia and cancer. MicroRNAs (also known as miRNAs or miRs) play key roles in regulation of a range of cellular processes by posttranscriptional suppression of their target genes. Recently, new studies have indicated that miRNAs are involved in certain angiogenic settings and signaling pathways use these non-coding RNAs to promote or suppress angiogenic processes. Herein, VEGFR2 and FGFR1 were identified as miR-129-1 and miR-133 targets using bioinformatic algorithms, respectively. Afterwards, using luciferase reporter assay and gene expression analysis at both mRNA and protein levels, VEGFR2 and FGFR1 were validated as miR-129-1 and miR-133 targets. In addition, we showed that miR-129-1 and miR-133 suppress angiogenesis properties such as proliferation rate, cell viability, and migration activity of human umbilical vein endothelial cells (HUVEC) in vitro. We conclude that these miRNAs can suppress key factors of angiogenesis by directly targeting them. These results have important therapeutic implications for a variety of diseases involving deregulation of angiogenesis, including cancer.

MicroRNA-129-1 acts as tumour suppressor and induces cell cycle arrest of GBM cancer cells through targeting IGF2BP3 and MAPK1.

BACKGROUND: MicroRNA-129-1 (miR-129-1) seems to behave as a tumour suppressor since its decreased expression is associated with different tumours such as glioblastoma multiforme (GBM). GBM is the most common form of brain tumours originating from glial cells. The impact of miR-129-1 downregulation on GBM pathogenesis has yet to be elucidated. METHODS: MiR-129-1 was overexpressed in GBM cells, and its effect on proliferation was investigated by cell cycle assay. MiR-129-1 predicted targets (CDK6, IGF1, HDAC2, IGF2BP3 and MAPK1) were also evaluated by western blot and luciferase assay. RESULTS: Restoration of miR-129-1 reduced cell proliferation and induced G1 accumulation, significantly. Several functional assays confirmed IGF2BP3, MAPK1 and CDK6 as targets of miR-129-1. Despite the fact that IGF1 expression can be suppressed by miR-129-1, through 3'-untranslated region complementary sequence, we could not find any association between IGF1 expression and GBM. MiR-129-1 expression inversely correlates with CDK6, IGF2BP3 and MAPK1 in primary clinical samples. CONCLUSION: This is the first study to propose miR129-1 as a negative regulator of IGF2BP3 and MAPK1 and also a cell cycle arrest inducer in GBM cells. Our data suggests miR-129-1 as a potential tumour suppressor and presents a rationale for the use of miR-129-1 as a novel strategy to improve treatment response in GBM.

Pancreatic islet differentiation of human embryonic stem cells by microRNA overexpression.

Development of stem cell-based therapies for the treatment of type 1 diabetes would provide a renewable supply of human ß-cells. Human embryonic stem cells (ESCs) are considered to be one of the stem cell populations with sufficient proliferative capacity to achieve this goal. Currently, differentiation protocols directing ESCs toward a pancreatic fate employ a variety of expensive cytokines and inhibitors. With the known significance of microRNAs in islet development, we present a novel and cost-effective strategy in which miR-375 overexpression promotes pancreatic endocrine differentiation in hESCs in the absence of any extrinsic factors. miR-375 has been shown to be a key regulator of pancreatic development and function in zebrafish, mouse and human. In this study, hESCs were transduced with lentiviral vectors containing human miR-375 precursor and aggregated to form human embryoid bodies (hEBs) for up to 21 days. Morphological assessment, immunocytochemistry and DTZ staining confirmed that miR-375-induced hEBs have similar characteristics to those of mature islets. In addition, the dynamic expression profile of endodermal marker Foxa2 and endocrine-specific genes, including HNF4α, Pdx1, Pax6, Nkx6.1, Glut2 and insulin, were detected by quantitative real-time PCR. Finally, insulin release upon glucose stimulation was detected in our differentiated clusters. The data presented here demonstrate the feasibility of using microRNAs to direct differentiation into the pancreatic lineage. Copyright © 2013 John Wiley & Sons, Ltd.

Transcription factor decoy: a pre-transcriptional approach for gene downregulation purpose in cancer.

Gene therapy as a therapeutic approach has been the dream for many scientists around the globe. Many strategies have been proposed and applied for this purpose, yet the void for a functional safe method is still apparent. Since most of the diseases are caused by undesirable upregulation (oncogenes) or downregulation (tumor suppressor genes) of genes, major gene therapy's techniques affect gene expression. Most of the methods are used in post-transcriptional level such as RNA inhibitory (RNAi) and splice-switching oligonucleotides (SSOs). RNAi blocks messenger RNA (mRNA) translation by mRNA degradation or interruption between attachments of mRNA with ribosomes' subunits. However, one of the novel methods is the usage of transcription factor targeted decoys. DNA decoys are the new generation of functional gene downregulatory oligonucleotides which compete with specific binding sites of transcription factors. Considering the exponential growth of this technique in both in vitro and in vivo studies, in this paper, we aim to line out the description, design, and application of decoys in research and therapy.