Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

BACKGROUND: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. RESULTS: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. CONCLUSIONS: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

Immunotherapy Study on Non-small-Cell Lung Cancer (NSCLC) Combined with Cytotoxic T Cells and miRNA34a.

Immunotherapy has emerged as a promising approach for cancer treatment, and the use of microRNAs (miRNAs) as therapeutic agents has gained significant attention. In this study, we investigated the effectiveness of immunotherapy utilizing miRNA34a and Jurkat T cells in inducing cell death in non-small-cell lung cancer cells, specifically A549 cells. Moreover, we explored the impact of Jurkat T cell activation and miRNA34a delivery using iron oxide nanorods (IONRs) on the killing of cancer cells. A549 cells were cocultured with both activated and inactivated Jurkat T cells, both before and after the delivery of miRNA34a. Surprisingly, our results revealed that even inactive Jurkat T cells were capable of inducing cell death in cancer cells. This unexpected observation suggested the presence of alternative mechanisms by which Jurkat T cells can exert cytotoxic effects on cancer cells. We stimulated Jurkat T cells using anti-CD3/CD28 and analyzed their efficacy in killing A549 compared to that of the inactive Jurkat T cells in conjunction with miRNA34a. Our findings indicated that the activation of Jurkat T cells significantly enhanced their cytotoxic potential against cancer cells compared to their inactive counterparts. The combined treatment of A549 cells with activated Jurkat T cells and miRNA34a demonstrated the highest level of cancer cell death, suggesting a synergistic effect between Jurkat T cell activation and miRNA therapy. Besides the apoptosis mechanism for the Jurkat T cells' cytotoxic effects on A549 cells, we furthermore investigated the ferroptosis pathway, which was found to have an impact on the cancer cell killing due to the presence of miRNA34a and IONRs as the delivery agent inside the cancer cells.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Unlocking the miRNA-34a-5p/TGF-ß and HMGB1/PI3K/Akt/mTOR crosstalk participate in the enhanced cardiac protection of liraglutide against isoproterenol-induced acute myocardial injury rat model.

Liraglutide (LIRA), a drug used to treat type 2 diabetes mellitus that belongs to the glucagon-like peptide-1 class, has recently drawn attention for its potential cardioprotective properties because of its anti-oxidative and anti-inflammatory properties. This current investigation was designed to assess the impact of LIRA on myocardial injury induced by isoproterenol (ISO). The experiment included 24 male Wistar rats in total, and they were divided into four groups: Control, LIRA (200 µg/kg/12 hrs., S.C.), ISO (85 mg/kg, S.C.), and ISO + LIRA. To assess the results, various biochemical and histopathological analyses were carried out. The findings showed elevated serum enzyme levels, a sign of cardiac injury. ISO-treated rats showed an upregulation of oxidative stress and inflammatory biomarkers like MDA, MPO, nitrites, NADPH oxidase, TNF-α, IL-1ß, IL-6, 8-Hydroxyguanosine (8-OHdG), and TGF-ß, as well as altered gene expressions like TLR-1 and miRNA-34a-5p. According to western blotting analysis, protein levels of AKT, PI3K, and mTOR were obviously enhanced. Additionally, ISO-treated samples showed altered tissue morphology, elevated caspase 3, and decreased Bcl2 concentrations. The levels of these dysregulated parameters were significantly normalized by LIRA therapy, demonstrating its cardioprotective function against ISO-induced myocardial injury in rats. This protective mechanism was linked to anti-inflammatory properties, redox balance restoration, and modulation of the miRNA-34a-5p/TGF-ß pathway.

Electrochemical miRNA-34a-based biosensor for the diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia type and a leading cause of death and disability in the elderly. Diagnosis is expensive and invasive, urging the development of new, affordable, and less invasive diagnostic tools. The identification of changes in the expression of non-coding RNAs prompts the development of diagnostic tools to detect disease-specific blood biomarkers. Building on this idea, this work reports a novel electrochemical microRNA (miRNA) biosensor for the diagnosis of AD, based on carbon screen-printed electrodes (C-SPEs) modified with two gold nanostructures and a complementary anti-miR-34a oligonucleotide probe. This biosensor showed good target affinity, reflected on a 100 pM to 1 µM linearity range and a limit of detection (LOD) of 39 pM in buffer and 94 aM in serum. Moreover, the biosensor's response was not affected by serum compounds, indicating selectivity for miR-34a. The biosensor also detected miR-34a in the cell culture medium of a common AD model, stimulated with a neurotoxin to increase miR-34a secretion. Overall, the proposed biosensor makes a solid case for the introduction of a novel, inexpensive, and minimally invasive tool for the early diagnosis of AD, based on the detection of a circulating miRNA overexpressed in this pathology.

Possible Role of IL-6R/STAT3/MiRNA-34a Feedback Loop in Osteosarcoma.

OBJECTIVE: Osteosarcoma is considered the most common primary malignant tumor that develops from the primary osteoblasts. MiRNAs are small non-coding RNAs that play a key role in tumorigenesis. The aim of this study was to detect the possible relationship between expression levels of miRNA-34a and levels of Signal transducer and activator of transcription 3 (STAT3) and interleukin-6 receptor (IL-6R) in osteosarcoma and the possible role of this relationship in development of metastases in these patients. METHODS: A total of thirty-six (36) bone samples were included in the study. They were divided into 3 groups: Group (I): Twelve normal bone samples as control group. Group (II): Twelve patients with non-metastatic osteosarcoma. Group (III): Twelve patients with metastatic osteosarcoma. MiRNA-34a expression levels were estimated using qRT-PCR. STAT3 and IL-6R levels were measured by ELISA. RESULTS: Expression level of miRNA-34a was downregulated in osteosarcoma groups compared to control group. STAT3 and IL-6R levels were upregulated in osteosarcoma groups compared to control group. This difference in expression levels was found to be more significant in the metastatic group than the non-metastatic one (P<0.001 each). There was a significant positive correlation between STAT3 and IL-6R (r=0.868, P<0.001), and a significant inverse correlation between IL6 and miRNA-34a (r=-0.993, P<0.001). CONCLUSION: miRNA-34a, STAT3 and IL-6R feedback loop could be a potential target for treatment of osteosarcoma and can be used as prognostic indicator for this disease.

The Upregulation of HLA-G1 and miRNA-34a in Lens Epithelial Cells of Diabetic Retinopathy Patients.

Background: Retinopathy of diabetes is a chronic diabetes mellitus complication affecting retinal vessels, and some ocular complications' molecular mechanisms remain obscure. Objective: To evaluate the expression of HLA-G1, HLA-G5, miRNA-181a, and miRNA-34a in the lens epithelial cells of patients with retinopathy of diabetes. Methods: In a case-control study, 30 diabetic patients with retinopathy, 30 diabetic patients without retinopathy, and 30 cataract patients without diabetes mellitus as the control group were enrolled after a full description with details about the study methods and objectives. The expression of HLA G1, HLA G5, miRNA-181a, and miRNA-34a in lens epithelial cells was assessed by quantitative RT PCR. Moreover, the levels of HLA-G protein in aqueous humor were evaluated by the ELISA method. Results: HLA-G1 expression was significantly upregulated in the retinopathy group (P=0.003). The aqueous humor of diabetic retinopathy patients contained significantly higher levels of HLA-G protein compared with the non-diabetic patients (P=0.001). miRNA-181a was significantly downregulated in the diabetic retinopathy group compared with the patients without diabetes (P=0.001). In addition, miRNA-34a was upregulated in the retinopathy group (P=0.009). Conclusion: Taken together, the present results showed that HLA-G1 and miRNA-34a can be valuable markers for diabetic retinopathy. Our data offers new perspectives for improving the control of inflammation in the lens epithelial cells by considering HLA-G and miRNA.

Multifunctional DNA Tetrahedron for Alzheimer's Disease Mitochondria-Targeted Therapy by MicroRNA Regulation.

The principal hallmark of Alzheimer's disease (AD) is neuron mitochondrial dysfunction, whereas mitochondrial miRNAs potentially play important roles. Nevertheless, efficacious mitochondria organelle therapeutic agents for treatment and management of AD are highly advisable. Herein, we report a multifunctional DNA tetrahedron-based mitochondria-targeted therapeutic platform, termed tetrahedral DNA framework-based nanoparticles (TDFNs), which was modified with triphenylphosphine (TPP) for mitochondria-targeting, cholesterol (Chol) for crossing the central nervous system, and functional antisense oligonucleotide (ASO) for both AD diagnosis and gene silencing therapy. After injecting intravenously through the tail vein of 3 × Tg-AD model mice, TDFNs can both easily cross the blood brain barrier and accurately arrive at the mitochondria. The functional ASO could not only be detected via the fluorescence signal for diagnosis but also mediate the apoptosis pathway through knocking miRNA-34a down, leading to recovery of the neuron cells. The superior performance of TDFNs suggests the great potential in mitochondria organelle therapeutics.

The Emerging Role of Circular RNA Homeodomain Interacting Protein Kinase 3 and Circular RNA 0046367 through Wnt/Beta-Catenin Pathway on the Pathogenesis of Nonalcoholic Steatohepatitis in Egyptian Patients.

Background: Non-alcoholic fatty liver disease is a major problem worldwide that needs non-invasive biomarkers for early diagnosis and treatment response assessment. We aimed to assess the correlation between circRNA-HIPK3 and miRNA-29a expression and its role as miRNA-29a sponge, as well as the correlation between circRNA-0046367 and miRNA-34a expression and its role as miRNA-34a sponge and their effect on regulation of the Wnt/ß catenin pathway, which may provide a new target for treatment of non-alcoholic steatohepatitis. Methods: the research was performed on 110 participants: group (I): fifty-five healthy donors served as controls and group (II): fifty-five patients with fatty liver pattern on abdominal ultrasound. Lipid profile and liver functions were assessed. RT-PCR was performed to assess the RNAs: circRNA-HIPK3, circRNA-0046367, miRNA-29a, miRNA-34a and Wnt mRNA gene expression. ELISA was performed to determine ß-catenin protein levels. Results: miRNA-34a and circRNA-HIPK3 expression were significantly greater, while miRNA-29a and circRNA-0046367 expression were significantly less, in patients than in controls. Wnt/ß-catenin regulated by miRNA-29a and miRNA-34a showed a significant decrease that leads to its abnormal effect on lipid metabolism. Conclusions: our results imply that miRNA-29a can be investigated as a target for circRNA-HIPK3, while miRNA-34a can be investigated as a target for circRNA-0046367, and that circRNA-HIPK3 and circRNA-0046367 may have emerging roles that can affect the pathogenesis of nonalcoholic steatohepatitis through the Wnt/ß-catenin pathway and thus be used as therapeutic targets for the disease.

Dendrobium officinale Kimura & Migo polysaccharide inhibits hyperglycaemia-induced kidney fibrosis via the miRNA-34a-5p/SIRT1 signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Fibrosis is a fundamental change occurring in impaired renal function and plays an important role in the progression of diabetic kidney disease (DKD). Dendrobium officinale Kimura & Migo polysaccharide (DOP), a primary active component of Dendrobium officinale Kimura & Migo, is reported to act on reducing blood glucose, suppressing inflammation. However, the anti-fibrosis effect of DOP in the treatment of DKD is still unclear. AIM OF THE STUDY: To explore the therapeutic effect of DOP on renal fibrosis in DKD. MATERIALS AND METHODS: We used db/db mice as a DKD model and administered DOP by oral gavage. The expression of miRNA-34a-5p, SIRT1, and fibrosis molecules (TGF-ß, CTGF, and a-SMA) were detected in renal tissue. Human renal tubular epithelium cells (HK-2) were cultured with 5.5 mM glucose (LG) or 25 mM glucose (HG), and intervened with 100-400 µg/ml DOP. The changes of the above indicators were observed in vitro. RESULTS: MiRNA-34a-5p was mainly localised in the nucleus and increased expression in the DKD mice. Inhibition or excitation of miRNA-34a-5p is involved in renal fibrosis by regulating SIRT1. DOP could depress the miRNA-34a-5p/SIRT1 signalling pathway to relieve renal fibrosis. Moreover, DOP has outstanding results in the treatment of DKD through hypoglycaemic action and weight reduction. CONCLUSIONS: DOP plays a protective role in arresting or slowing the progression of fibrosis, which may provide a novel clinical treatment strategy for DKD.

Corrigendum: Resveratrol attenuates allergic asthma and associated inflammation in the lungs through regulation of miRNA-34a that targets FoxP3 in mice.

[This corrects the article DOI: 10.3389/fimmu.2018.02992.].

Significance of MiRNA-34a and MiRNA-192 as a risk factor for nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: NAFLD is one of the fast-growing health problems that affects up to 25% of people worldwide. Numerous miRNAs have been clarified as important regulators of liver pathophysiology, including NAFLD. Thus, we investigated the expression of the MiRNA-34a and MiRNA-192 as diagnostic markers for NAFLD. PATIENTS AND METHODS: Blood samples were collected from NAFLD cases and healthy controls. The expression profile of both studied miRNAs was detected via real-time PCR analysis. RESULTS: The present study showed that both studied miRNAs were upregulated in NAFLD patients compared to controls. Interestingly, miRNA-34a and MiRNA-192 are upregulated in NAFLD patients with early fibrosis compared to controls [with a fold change of 4.02 ± 11.49 (P = 0.05) and 18.43 ± 47.8 (P = 0.017), respectively]. However, miRNA-34a is downregulated in NAFLD patients with advanced fibrosis compared to controls, with fold expression of 0.65 ± 1.17 (P = 0.831). The area under the receiver operating characteristics (AUROC) for miRNA-34a and miRNA-192 were 0.790 and 0.643, respectively; furthermore, the sensitivities and specificities were 76.7%, 100% for miRNA-34a and 63.3%, and 93.3% for miRNA-192 (P < 0.05). Additionally, MiRNA34a was positively correlated with hypertension and fasting blood sugar, and it also was negatively correlated with hemoglobin level and total leucocyte count (P < 0.05). CONCLUSION: The results obtained indicated that both studied miRNAs could potentially be used as diagnostic biomarkers for the early stage of liver fibrosis in NAFLD cases. Also, miRNA-34a was positively correlated with metabolic disorders associated with NAFLD such as hypertension and diabetes. However, their expression showed no association with advanced fibrosis. Thus, larger cohorts are necessitated to certify the utility of serum MiRNA-34a and MiRNA-192 in monitoring the deterioration of NAFLD.

Comparing the Variants of Iron Oxide Nanoparticle-Mediated Delivery of miRNA34a for Efficiency in Silencing of PD-L1 Genes in Cancer Cells.

The blocking of programmed death-ligand 1 (PD-L1) in tumor cells represents a powerful strategy in cancer immunotherapy. Using viral vectors to deliver the cargo for inactivating the PD-L1 gene could be associated with host cell genotoxicity and concomitant immune attack. To develop an alternative safe gene delivery method, we designed a unique combination for miRNA34a delivery using a transgene carrier in the form of iron oxide magnetic nanoparticles (IONPs) via magnetofection to downregulate PD-L1 expression in cancer cells. We synthesized IONPs of multiple shapes (IONRs (iron oxide nanorods), IONSs (iron oxide nanospheres), and ITOHs (iron oxide truncated octahedrons)), surface-functionalized with polyethyleneimine (PEI) using the ligand exchange method, as gene delivery systems. Under the guidance of an external magnetic field, PEI@IONPs loaded with plasmid DNA (DNA/PEI@IONPs) encoding GFP showed high transfection efficiency at different weight ratios and time points in A549 and MDA-MB-231 cells. Additionally, the DNA/PEI@IONPs with miRNA34a inserts under a static magnetic field resulted in significant knockdown of the PD-L1 gene, as demonstrated via immunoblotting of the PD-L1 protein. Among the three shapes of IONPs, IONRs showed the highest PD-L1 knockdown efficiency. The genetic expression of miRNA34a was also studied using qPCR and it showed high expression of miRNA in cells treated with PEI@IONRs. Flow cytometry and a live/dead assay confirmed apoptosis after transfection with miRNA34a. To conclude, in this paper, a promising transgene carrier with low cost, negligible cytotoxicity, and high transfection efficiency has been successfully established for miRNA gene delivery in the context of cancer immunotherapy.

The miRNA-34a/Sirt1/p53 pathway in a rat model of lens regeneration.

Background: There are many molecular factors involved in Wolffian and corneal lens regeneration, but few in lens regeneration by lens epithelial cells (LECs) in mammals. Silent information regulator 1 (Sirt1) has a variety of physiological functions, such as a transport hub, and is involved in pathological conditions. We studied the expression of the microRNA (miRNA)-34a/Sirt1/tumor protein p53 (p53) pathway in a rat model of lens regeneration. Methods: We performed extracapsular lens extraction in 42 healthy female Sprague-Dawley rats. Slit lamp observation was performed at 3, 7, 14, 21, 30, 60 and 90 days postoperatively, and the rats were killed humanely by cervical dislocation at 30, 60 and 90 days postoperatively to remove the eyeballs. We performed semiquantitative immunofluorescence analysis of Sirt1, p53, alpha-smooth muscle actin (α-SMA) and fibronectin (fn), and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) to detect the relative expressions of miRNA-34a, Sirt1, p53, aquaporin 0 (AQP 0), γA-crystallin, and beaded filament structural protein 1 (BFSP1) mRNA in the lens and posterior capsule. Results: The posterior capsule wrinkled at 3 days and it increased at 7 days. At 14 days, pearl-like opacification appeared under the capsule, with increasing shrinkage. Greater mass-like proliferators in size and number accumulated under the capsule and at the equator after 21 days. A regenerated lens developed in the central depression of the capsule at 30 days, slightly protruding from it. Despite being thickened at 60 days, the central depression persisted, with a smaller change at 90 days than at 60 days. Although the relative mRNA expression of miRNA-34a and p53 in the lens and posterior capsule decreased over time (P=0.000), that of Sirt1 increased (P<0.01). α-SMA was uniformly expressed in the crystals and gradually decreased, while fn expression gradually increased. Conclusions: miRNA-34a expression decreased and Sirt1 expression increased during lens regeneration. Furthermore, p53 expression decreased, thus reducing apoptosis. Therefore, Sirt1 acted as a key factor in the pathway, and played a protective role in lens regeneration.

LGR4, a G Protein-Coupled Receptor With a Systemic Role: From Development to Metabolic Regulation.

Leucine-rich repeat-containing G protein-coupled receptor 4 (LGR4/GPR48), a member of the GPCR (G protein-coupled receptors) superfamily, subfamily B, is a common intestinal crypt stem cell marker. It binds R-spondins/Norrin as classical ligands and plays a crucial role in Wnt signaling potentiation. Interaction between LGR4 and R-spondins initiates many Wnt-driven developmental processes, e.g., kidney, eye, or reproductive tract formation, as well as intestinal crypt (Paneth) stem cell pool maintenance. Besides the well-described role of LGR4 in development, several novel functions of this receptor have recently been discovered. In this context, LGR4 was indicated to participate in TGFß and NFκB signaling regulation in hematopoietic precursors and intestinal cells, respectively, and found to be a new, alternative receptor for RANKL (Receptor Activator of NF kappa B Ligand) in bone cells. LGR4 inhibits the process of osteoclast differentiation, by antagonizing the interaction between RANK (Receptor Activator of NF kappa B) and its ligand-RANKL. It is also known to trigger anti-inflammatory responses in different tissues (liver, intestine, cardiac cells, and skin), serve as a sensor of the circadian clock in the liver, regulate adipogenesis and energy expenditure in adipose tissue and skeletal muscles, respectively. The extracellular domain of LGR4 (LGR4-ECD) has emerged as a potential new therapeutic for osteoporosis and cancer. LGR4 integrates different signaling pathways and regulates various cellular processes vital for maintaining whole-body homeostasis. Yet, the role of LGR4 in many cell types (e.g. pancreatic beta cells) and diseases (e.g., diabetes) remains to be elucidated. Considering the broad spectrum of LGR4 actions, this review aims to discuss both canonical and novel roles of LGR4, with emphasis on emerging research directions focused on this receptor.

Axl Expression in Renal Mesangial Cells Is Regulated by Sp1, Ap1, MZF1, and Ep300, and the IL-6/miR-34a Pathway.

Axl receptor tyrosine kinase expression in the kidney contributes to a variety of inflammatory renal disease by promoting glomerular proliferation. Axl expression in the kidney is negligible in healthy individuals but upregulated under inflammatory conditions. Little is known about Axl transcriptional regulation. We analyzed the 4.4 kb mouse Axl promoter region and found that many transcription factor (TF)-binding sites and regulatory elements are located within a 600 bp fragment proximal to the translation start site. Among four TFs (Sp1, Ap1, MZF1, and Ep300) identified, Sp1 was the most potent TF that promotes Axl expression. Luciferase assays confirmed the siRNA results and revealed additional mechanisms that regulate Axl expression, including sequences encoding a 5'-UTR mini-intron and potential G-quadruplex forming regions. Deletion of the Axl 5'-UTR mini-intron resulted in a 3.2-fold increases in luciferase activity over the full-length UTR (4.4 kb Axl construct). The addition of TMPyP4, a G-quadruplex stabilizer, resulted in a significantly decreased luciferase activity. Further analysis of the mouse Axl 3'-UTR revealed a miRNA-34a binding site, which inversely regulates Axl expression. The inhibitory role of miRNA-34a in Axl expression was demonstrated in mesangial cells using miRNA-34a mimicry and in primary kidney cells with IL-6 stimulated STAT3 activation. Taken together, Axl expression in mouse kidney is synergistically regulated by multiple factors, including TFs and secondary structures, such as mini-intron and G-quadruplex. A unique IL6/STAT3/miRNA-34a pathway was revealed to be critical in inflammatory renal Axl expression.

Dynamical Analysis of a Boolean Network Model of the Oncogene Role of lncRNA ANRIL and lncRNA UFC1 in Non-Small Cell Lung Cancer.

Long non-coding RNA (lncRNA) such as ANRIL and UFC1 have been verified as oncogenic genes in non-small cell lung cancer (NSCLC). It is well known that the tumor suppressor microRNA-34a (miR-34a) is downregulated in NSCLC. Furthermore, miR-34a induces senescence and apoptosis in breast, glioma, cervical cancer including NSCLC by targeting Myc. Recent evidence suggests that these two lncRNAs act as a miR-34a sponge in corresponding cancers. However, the biological functions between these two non-coding RNAs (ncRNAs) have not yet been studied in NSCLC. Therefore, we present a Boolean model to analyze the gene regulation between these two ncRNAs in NSCLC. We compared our model to several experimental studies involving gain- or loss-of-function genes in NSCLC cells and achieved an excellent agreement. Additionally, we predict three positive circuits involving miR-34a/E2F1/ANRIL, miR-34a/E2F1/UFC1, and miR-34a/Myc/ANRIL. Our circuit- perturbation analysis shows that these circuits are important for regulating cell-fate decisions such as senescence and apoptosis. Thus, our Boolean network permits an explicit cell-fate mechanism associated with NSCLC. Therefore, our results support that ANRIL and/or UFC1 is an attractive target for drug development in tumor growth and aggressive proliferation of NSCLC, and that a valuable outcome can be achieved through the miRNA-34a/Myc pathway.

Erxian herbal pair enhances bone formation in infected bone nonunion models and attenuates lipopolysaccharide-induced osteoblastinhibition by regulating miRNA-34a-5p.

Bacterium-induced inflammatory responses cause bone nonunion. Although antibiotics suppress infection, bone loss after antibacterial treatment remains a critical challenge. Erxian herbal pair (EHP) has been proven effective in promoting bone formation. Our study aimed to investigate the effect of EHP on bone repair after anti-infection treatment, explore its effect on a lipopolysaccharide (LPS)-induced osteoblast. We evaluated effects of EHP on bone repair with Micro-CT, and morphology detecting. Chemical constituents of EHP and EHP-containing serum (EHP-CS) were identified by UHPLC-Q/TOF-MS. In addition, osteoblast induced by LPS was established and administrated with EHP-CS. Cell proliferationwas assessed by MTT. Target prediction identified SMAD2 as a potential target of miRNA-34a-5p. MiRNA mimic, inhibitor and siRNA were transiently transfected into osteoblasts. The mRNA levels and protein expressions of miRNA-34a-5p, BMP2, Runx2, SMAD2 were assessed. The results showed that the main biocactivity ingredients in EHP-CS were Baohuoside Ι and Orcinol Glucoside. EHP could promote bone remolding after anti-infection therapy and restore the activity of LPS-induced osteoblasts. Moreover, miRNA-34a-5p was dramatically downregulated and SMAD2 was upregulated after LPS stimulation, while EHP resisted the inhibition of LPS by promoting miRNA-34a-5p, ALP, and BMP2 expressions. Whereas downregulation of miRNA-34a-5p reversed these effects. Silencing endogenous SMAD2 expression markedly promoted BMP2 and ALP activity and enhanced osteogenesis. Taken together, EHP restored LPS-induced bone loss by regulating miRNA-34a-5p levels and repressing its target gene SMAD2. EHP might be a potential adjuvant herbal remedy for the treatment of bone nonunion, and miRNA-34a-5p is a novel target for controlling bone and metabolic diseases.

Folate targeted hybrid lipo-polymeric nanoplexes containing docetaxel and miRNA-34a for breast cancer treatment.

In spite of established evidence of the synergistic combination of hydrophobic anticancer molecule and microRNA for breast cancer treatment, their in vivo delivery has not been realized owing to their instability in the biological milieu and varied physicochemical properties. The present work reports folate targeted hybrid lipo-polymeric nanoplexes for co-delivering DTX and miR-34a. These nanoplexes exhibited a mean size of 129.3 nm with complexation efficiency at an 8:1 N/P ratio. The obtained nanoplexes demonstrated higher entrapment efficiency of DTX (94.8%) with a sustained release profile up to 85% till 48 h. Further, an improved transfection efficiency in MDA-MB-231 and 4T1 breast cancer cells was observed with uptake primarily through lipid-raft and clathrin-mediated endocytosis. Further, nanoplexes showed improved cytotoxicity (~3.5-5 folds), apoptosis (~1.6-2.0 folds), and change in expression of apoptotic genes (~4-7 folds) compared to the free treatment group in breast cancer cells. In vivo systemic administration of FA-functionalized DTX and FAM-siRNA-loaded nanoplexes showed an improved area under the curve (AUC) as well as circulation half-life compared to free DTX and naked FAM-labelled siRNA. Acute toxicity studies of the cationic polymer showed no toxicity at a dose equivalent to 10 mg/kg based on the hematological, biochemical, and histopathological examination.