Down-regulation of miR-27a might inhibit proliferation and drug resistance of gastric cancer cells

<p>Abstract</p> <p>Aims</p> <p>Here we aimed to firstly investigate the role of miR-27a in proliferation and multidrug resistance of gastric cancer cells.</p> <p>Methods</p> <p>The role of miR-27a in gastric cancer cells was detected using MTT assay, soft agar assay, flow cytometry assay, nude mice assay, real-time PCR, western blot and reporter gene assay, etc.</p> <p>Results</p> <p>Down-regulation of miR-27a could inhibit porliferation of gastric cancer cells in vitro and in vivo. Down-regulation of miR-27a could also confer sensitivity of drugs on gastric cancer cells, and might increase accumulation and decrease releasing amount of adriamycin in gastric cancer cells. Down-regulation of miR-27a could significantly decrease the expression of P-glycoprotein and the transcriptional activity of cyclin D1, and up-regulate the expression of p21.</p> <p>Conclusions</p> <p>MiR-27a might play important roles in porliferation and drug resistance of gastric cancer. MiR-27a might be considered as a useful target for cancer therapy.</p

14-3-3θ, AF4 and MLL-AF4 are targets of MiR-27a in t(4;11) acute lymphoblastic leukemia: implication for new targeted therapy options

The t(4;11)(q21;q23) chromosomal translocation results in the KMT2A/AFF1 fusion gene, which encodes the mixed-lineage leukemia (MLL)-AF4 oncogenic chimera, a hallmark of an aggressive form of acute lymphoblastic leukemia (ALL). In t(4;11) ALL, MLL-AF4 recruits the endogenous co-activator AF4 and aberrantly triggers transcription of MLL target genes, including HOXA9 and MEIS1, thereby driving transformation of hematopoietic progenitors. We previously demonstrated that the scaffold protein 14-3-3θ is a direct interactor of AF4 and promotes AF4 binding to the HOXA9 promoter. Notably, 14-3-3θ is a target of MiR-27a, which acts as tumor suppressor in various human leukemias; moreover, expression levels of MiR-27a correlate with relapse free survival in childhood ALL. This PhD thesis aims to assess the potential role of MiR-27a in t(4;11) ALL pathogenesis. In different leukemia cell lines, we found an inverse correlation between levels of MiR-27a and 14-3-3θ, which was particularly relevant in t(4;11) cell lines. In t(4;11) leukemia cells, transient transfection of MiR-27a led to a decrease in 14-3-3θ protein amount and HOXA9, HOXA7 and MEIS1 transcription. Interestingly, our bioinformatic analysis predicted that AFF1-3’UTR, which is shared with KMT2A/AFF1, contains a putative MiR-27a seed sequence. Consistently, MiR-27a overexpression strongly reduced AF4 and MLL-AF4 protein levels, as well as protein level of RUNX1, a known target of MiR-27a with a key role in t(4;11) leukemia context. We therefore cloned AFF1-3’-UTR in an opportune plasmid vector and performed a luciferase reporter assay. The decreased luciferase activity we measured after co-transfection of MiR-27a and the recombinant plasmid proved that AFF1-3’UTR is a direct target of MiR-27a. Accordingly, transfection of anti-MiR-27a enhanced the expression of AF4 protein, in RS4;11 cells. Moreover, ChIP experiments gave direct proof that MiR-27a overexpression impaired MLL-AF4 binding to HOXA9 promoter. Consistently, MiR-27a overexpression decreased viability, proliferation and clonogenicity of t(4;11) cells, whereas enhanced their apoptotic rate. Lastly, we found that relative expression of MiR-27a was significantly lower in 9 patients affected by severe t(4;11) ALL compared with 9 patients affected by t(12;21) leukemia, which has a benign prognosis. Similarly to the t(4;11) cell lines, in our ALL patients we found an inverse relationship between MiR-27a and 14-3-3θ transcript levels. Overall, we demonstrate that MiR-27a has a pivotal role in t(4;11) ALL molecular pathogenesis and therefore it is a promising novel target for the therapy of this aggressive form of leukemia

The role of prohibitin and miR-27a in prostate cancer progression and therapy response

Introduction: Prostate cancer is the most commonly diagnosed male cancer in the Western world, with more than 40,000 cases in the UK diagnosed annually. The androgen receptor (AR) is required for the survival and proliferation of the prostate cancer cell and is key to prostate cancer treatment. The levels of both AR itself and its cofactors are in turn modulated by specific microRNAs, hence manipulating key microRNAs that affect androgen signaling is a potential therapeutic strategy for prostate cancer. One such key microRNA is miR-27a, which has a confirmed oncogenic role in gastric cancer and we previously showed to target the AR corepressor Prohibitin. In addition, circulating microRNAs (miRNAs) have been a source of hope as potential new biomarkers for cancer. Results: There is evidence that prohibitin is decreased in the more aggressive prostate cancer cell lines and also in metastatic deposits in clinical cases of prostate cancer. Conversely miR-27a is increased in the more aggressive cell lines and in the metastatic deposits in clinical cases of prostate cancer. Treatment of LNCaP cells with an anti-sense oligomiR inhibitor of miR-27a (ASO-27a) resulted in a decrease in miR-27a levels, an increase in prohibitin levels, and a decrease in cell growth. Conversely when LNCaP cells were induced to express high levels of miR-27a (via stable transfection with a doxycycline-inducible miR-27a expression vector) there was a significant increase in growth. These findings, suggestive of tumour-promoting properties of miR-27a, were supported in an in vivo xenograft model. Mice bearing human prostate tumours and treated systemically with ASO-27a had significantly less tumour growth when compared to those treated with control oligomiR. Surprisingly, we did not observe a direct effect on prohibitin levels in the treated tumours. The genes showing significant differential expression following treatment with ASO-27a were compared with published data sets of gene expression at different clinical stages of prostate cancer tissue. Multiple genes of interest were identified, that were both significantly raised following treatment with ASO-27a and expressed at significantly lower levels in cases of metastatic prostate cancer. In the circulation of men with metastatic prostate cancer I found that miR-27a is generally higher when compared to men with healthy prostates. A targeted panel of miRs was screened in this context, and a subset consisting of miR-21, miR-10b, miR-126, miR-150, miR-378 and miR-93 could differentiate benign from metastatic disease; it would be interesting to explore this 6 miR signature further as potential diagnostic or predictive biomarker test. Conclusion: We have shown that using an anti-sense oligomiR to miR-27a reduces prostate cancer growth both in vitro and in vivo, at least in part via increasing apoptosis, and thus warrants further investigation as a potential future therapy. In addition circulating levels of miRNAs, including miR-27a, may provide clinically useful biomarker information regarding diagnosis, prognosis, or prediction of response to treatment.Open Acces

The association of miR-27a (rs895819) polymorphism with the risk of type 2 diabetes

Background and aims: Type 2 diabetes is an endocrine desease and one of the most common metabolic diseases in the world, and some environmental factors and genetic background are involved in the disease. Genetically, a large number of gene polymorphisms have been identified to be associated with this disease, but few studies have been conducted in polymorphisms miRNA and their correlation with type 2 diabetes. Among miRNAs, miR-27a involves in the predisposition to type 2 diabetes, and mutations can alter the miR-27a function and cause diabetes. The aim of the present study was to investigate the association of miR-27a (rs895819) with the risk of type 2 diabetes. Methods: In the present case-control study, 100 type 2 diabetes patients and 100 healthy individuals were randomly selected. Polymorphism miR-27a (rs895819) was investigated using PCR-RFLP method. To determine the balance or imbalance in two groups and the relationship between polymorphisms and the incidence of type 2 diabetes, Chi-square test was applied. Results: Significant differences in the genotype distribution of miR-27a (rs895819) in patients with type 2 diabetes in comparison to the healthy subjects indicated a P value equal to 0.038 (odds ratio: 2.63, 95% CI: 1.07-6.05). Conclusion: The results suggested that there is an association between miR-27a genetic variants and the incidence of type 2 diabetes. However, further studies are needed to evaluate the significance of genetic variants in different populations

Isoginkgetin inhibits lung cancer cell progression through miR-27a-5p/APEX1 axis

Purpose: To determine the influence of isoginkgetin on the progression of pulmonary carcinogenesis. Methods: A549 cells exposed to isoginkgetin were transfected with miR-27a-5p mimetic and suppressor, as well as short hairpin RNA against apurinic/apyrimidinic endo-deoxyribonuclease 1 (sh-APEX1) for 24 h. Then, the proliferative, migration and invasive potential of A549 cells were determined using CCK-8, wound healing and Transwell assays, respectively. The association between miR-27a-5p and APEX1 was determined by dual-luciferase reporter assay. Results: Isoginkgetin significantly suppressed A549 cell proliferative, migration and invasive activities (p &lt; 0.05). Moreover, isoginkgetin significantly increased miR-27a-5p expression. Furthermore, miR-27a-5p suppressor annulled the influence of isoginkgetin on lung cancer progression. More importantly, the inhibitor directly targeted APEX1, and negatively modulated APEX1 expression (p &lt; 0.05). However, APEX1 knockdown annulled the enhancing effect of miR-27a-5p inhibitor on A549 cell viability, migration and invasion (p &lt; 0.05). Conclusion: Isoginkgetin exerts an anti-lung cancer effect via miR-27a-5p/APEX1 axis. Thus, it is a potential therapy for the management of lung cancer; however, clinical studies are required to validate these findings of this study

Upregulation of miR-23a∼27a∼24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer

Hsa-miR-27a-3p overexpression in men with nonobstructive azoospermia: A case-control study

Background: The role of KDM3A and its downstream genes in male fertility has been approved in animal models. Additionally, the expression shrinkage of KDM3A is significantly correlated with human azoospermia phenotype. Aberrant expression of micro-RNAs could mislead spermatogenesis and mostly lead to diverse phenotypes of male infertility. Objective: The aim of this study was to evaluate the expression level of hsa-miR-27a- 3p in azoospermic men to reveal its possible association with infertility. Materials and Methods: This case-control study was conducted on 30 azoospermic men, of whom, 19 had non obstructive azoospermia (NOA) and 11 obstructive azoospermia (OA) according to the pathological examinations. Comprehensive bioinformatics investigations were performed securely and hsa-miR-27a-3p was selected afterward. Reverse Transcriptase-quantitative polymerase chain reaction (RTqPCR) method was used and statistical analysis was performed to compare the expression level of hsa-miR-27a-3p in both OA and NOA individuals. Results: In silico analysis suggested hsa-miR-27a-3p, with its potential binding ability to target KDM3A transcripts. The expression analysis of candidate hsa-miR-27a-3p indicated its significant overexpression in NOA men. Conclusion: The hsa-miR-27a-3p was overexpressed in NOA men compared to OA-control individuals. As a consequence, the overexpressed micro-RNA could downregulate directly KDM3A and indirectly TNP1 and PRM1. Therefore, spermatogenesis could be misled and male infertility could be developed. Key words: hsa-miR-27a-3p, Male infertility, KDM3A

miR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability

Background The brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined. Methods Using a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored. Results Our results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ ß-catenin signaling, a key pathway required for the BBB maintenance. Conclusion For the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function

Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.</p> <p>Methods</p> <p>Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.</p> <p>Results</p> <p>IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.</p> <p>Conclusion</p> <p>This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.</p

Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation.

Endogenous long-chain metabolites of vitamin E (LCMs) mediate immune functions by targeting 5-lipoxygenase (5-LOX) and increasing the systemic concentrations of resolvin E3, a specialized proresolving lipid mediator. SAR studies on semisynthesized analogues highlight α-amplexichromanol (27a), which allosterically inhibits 5-LOX, being considerably more potent than endogenous LCMs in human primary immune cells and blood. Other enzymes within lipid mediator biosynthesis were not substantially inhibited, except for microsomal prostaglandin E2 synthase-1. Compound 27a is metabolized by sulfation and β-oxidation in human liver-on-chips and exhibits superior metabolic stability in mice over LCMs. Pharmacokinetic studies show distribution of 27a from plasma to the inflamed peritoneal cavity and lung. In parallel, 5-LOX-derived leukotriene levels decrease, and the inflammatory reaction is suppressed in reconstructed human epidermis, murine peritonitis, and experimental asthma in mice. Our study highlights 27a as an orally active, LCM-inspired drug candidate that limits inflammation with superior potency and metabolic stability to the endogenous lead