Exosomes from human adipose-derived mesenchymal stem cells attenuate localized scleroderma fibrosis by the let-7a-5p/TGF-ßR1/Smad axis.

BACKGROUND: Inflammation and fibrosis of the skin are characteristics of localized scleroderma (LS). Emerging evidence has demonstrated that exosomes from human adipose tissue-derived mesenchymal stem cells (ADSC-Exo) could alleviate skin fibrosis. OBJECTIVE: The impact and potential mechanism of ADSC-Exo on LS fibrosis was examined. METHODS: ADSC-Exo was isolated and identified. The effects of ADSC-Exo on the abilities of proliferation and migration of LS-derived fibroblasts (LSFs) were assessed by CCK-8 and scratch assays, respectively. qRT-PCR, western blot, and immunofluorescence were conducted to detect LSFs stimulated with ADSC-Exo, ADSC-ExoAnti-let-7a-5p, let-7a-5p mimic/TGF-ßR1 shRNA virus, and negative controls. The impact of ADSC-Exo on C57BL/6j LS mice was evaluated by photographic morphology, hematoxylin-eosin (H&E), Masson's trichrome, and immunohistochemical staining. RESULTS: The verified ADSC-Exo limited the proliferation and migration of LSFs and reduced the expression of COL1, COL3, α-SMA, TGF-ßR1, and p-Smad2/ 3 in vitro and in vivo. TGF-ßR1 knockdown and let-7a-5p mimic in LSFs reduced the expression of COL1, COL3, α-SMA, and p-Smad2/3. However, compared with the ADSC-ExoNC group, the dermal thickness was increased, collagen arrangement was disordered, and α-SMA and TGF-ßR1 levels were increased after exposure to ADSC-ExoAnti-let-7a-5p. CONCLUSIONS: In this study, it might show that ADSC-Exo may successfully prevent LSF bioactivity, collagen deposition, and myofibroblast trans-differentiation. Additionally, we confirmed that let-7a-5p in ADSC-Exo could directly target TGF-R1 to control the Smad pathway and reduce fibrosis in LSFs. Our work offered a brand-new therapeutic approach and clarified the unique mechanism for the clinical management of LS.

Targeted inhibition of TGF-ß type I receptor by AZ12601011 protects against kidney fibrosis.

Renal fibrosis, a common feature of chronic kidney disease, causes the progressive loss of renal function, in which TGF-ß1 plays a critical role. In this study, we found that expression levels of TGF-ß1 and its receptor 1 (TGF-ßR1) were both significantly increased in obstructive fibrosis kidneys. AZ12601011 is a small molecular inhibitor of TGF-ßR1; however, its therapeutic potential for renal fibrosis remains unclear. During the experiments, AZ12601011 was applied to various models of renal fibrosis followed by unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) in vivo, in addition to renal tubular epithelial cells (TECs) challenged by hypoxia/reoxygenation (H/R) and TGF-ß1in vitro. Our results revealed that AZ12601011 ameliorated renal injuries and fibrosis shown by PAS, HE, and Masson staining, which was consistent with the decrease in Col-1 and α-SMA expression in the kidneys from UUO and I/R mice. Similarly, in vitro data showed that AZ12601011 inhibited the induction of Col-1 and α-SMA in both TECs treated with TGF-ß1 and H/R. In addition, the results of cellular thermal shift assay (CETSA), molecular docking, and western bolt indicated that AZ12601011 could directly bind to TGF-ßR1 and block activation of the downstream Smad3. Taken together, our findings suggest that AZ12601011 can attenuate renal fibrosis by blocking the TGF-ß/Smad3 signaling pathway and it might serve as a promising clinical candidate in the fight against fibrotic kidney diseases.

Blood SSR1: A Possible Biomarker for Early Prediction of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease associated with age. Early diagnosis of PD is key to preventing the loss of dopamine neurons. Peripheral-blood biomarkers have shown their value in recent years because of their easy access and long-term monitoring advantages. However, few peripheral-blood biomarkers have proven useful. This study aims to explore potential peripheral-blood biomarkers for the early diagnosis of PD. Three substantia nigra (SN) transcriptome datasets from the Gene Expression Omnibus (GEO) database were divided into a training cohort and a test cohort. We constructed a protein-protein interaction (PPI) network and a weighted gene co-expression network analysis (WGCNA) network, found their overlapping differentially expressed genes and studied them as the key genes. Analysis of the peripheral-blood transcriptome datasets of PD patients from GEO showed that three key genes were upregulated in PD over healthy participants. Analysis of the relationship between their expression and survival and analysis of their brain expression suggested that these key genes could become biomarkers. Then, animal models were studied to validate the expression of the key genes, and only SSR1 (the signal sequence receptor subunit1) was significantly upregulated in both animal models in peripheral blood. Correlation analysis and logistic regression analysis were used to analyze the correlation between brain dopaminergic neurons and SSR1 expression, and it was found that SSR1 expression was negatively correlated with dopaminergic neuron survival. The upregulation of SSR1 expression in peripheral blood was also found to precede the abnormal behavior of animals. In addition, the application of artificial intelligence technology further showed the value of SSR1 in clinical PD prediction. The three classifiers all showed that SSR1 had high predictability for PD. The classifier with the best prediction accuracy was selected through AUC and MCC to construct a prediction model. In short, this research not only provides potential biomarkers for the early diagnosis of PD but also establishes a possible artificial intelligence model for predicting PD.

Estradiol Ameliorates Acute Kidney Ischemia-Reperfusion Injury by Inhibiting the TGF-ßRI-SMAD Pathway.

Renal ischemia-reperfusion injury (IRI) is less extensive in females than males in both animals and humans; however, this protection diminishes after menopause, suggesting that estrogen plays a pivotal role in IRI, but the underlying mechanism remains largely unknown. Our study found that 45 min of warm ischemia was sufficient to induce significant pathological changes without causing death in model animals. Compared with male rats, female rats exhibited less extensive apoptosis, kidney injury, and fibrosis; these effects were worsened in ovariectomized (OVX) rats and ameliorated upon estradiol (E2) supplementation. Furthermore, the levels of TGF-ßRI, but not TGF-ßRII or TGF-ß1, were significantly increased in OVX rats, accompanied by phosphorylated SMAD2/3 activation. Interestingly, the alteration trend of the nuclear ERα level was opposite that of TGF-ßRI. Furthermore, dual luciferase reporter and chromatin immunoprecipitation assays showed that ERα could bind to the promoter region of TGF-ßRI and negatively regulate its mRNA expression. Moreover, an in vitro study using NRK-52E cells showed that ERα knockdown blocked E2-mediated protection, while TGF-ßRI knockdown protected cells against hypoxic insult. The findings of this study suggest that renal IRI is closely related to the TGF-ßRI-SMAD pathway in females and that E2 exert its protective effect via the ERα-mediated transcriptional inhibition of TGF-ßRI expression.

PBX2-Mediated circTLK1 Activates JAK/STAT Signaling to Promote Gliomagenesis via miR-452-5p/SSR1 Axis.

Glioma is considered one of the most lethal brain tumors, as the aggressive blood vessel formation leads to high morbidity and mortality rates. However, the mechanisms underlying the initiation and progression of glioma remain unclear. Here, we aimed to reveal the role of circTLK1 in glioma development. Our results revealed that circTLK1 is highly expressed in glioma tumor tissues and glioma cell lines. We then conducted a series of experiments that showed that circTLK1 was involved in the progression of gliomas. Mechanistically, investigation of the factors downstream of circTLK1 revealed that circTLK1 activated JAK/STAT signaling in glioma cells. Furthermore, AGO2-RIP, RNA-pull down, and luciferase reporter gene assays led to the identification of the novel circTLK1/miR-452-5p/SSR1 axis. Moreover, we investigated the upstream regulator of circTLK1 and found that circTLK1 expression in glioma cells could be regulated by the transcriptional factor PBX2. Taken together, our findings show that circTLK1 mediated by PBX2 activates JAK/STAT signaling to promote glioma progression through the miR-452-5p/SSR1 pathway. These results provide new insights into glioma diagnosis and therapy.

Clinical Value for Diagnosis and Prognosis of Signal Sequence Receptor 1 (SSR1) and Its Potential Mechanism in Hepatocellular Carcinoma: A Comprehensive Study Based on High-Throughput Data Analysis.

OBJECTIVE: Hepatocellular Carcinoma (HCC) has the characteristics of high incidence and poor prognosis. However, the underlying mechanism of HCC has not yet been fully elucidated. This study aims to investigate the potential mechanism and clinical significance of signal sequence receptor (SSR1) in HCC through bioinformatics methods. METHODS: Four online (GEPIA, TIMER, TCGA, and GEO) databases were used to explore the expression level of SSR1 in HCC. The summary receiver operating characteristic (SROC) analysis and standardized mean difference (SMD) calculation were performed further to detect its diagnostic ability and expression level. The Human Protein Atlas (HPA) database was applied to verify the level of SSR1 protein expression. Chi-square test and Fisher's exact test were carried out to determine the clinical relevance of SSR1 expression. KM survival analysis, univariate and multivariate COX regression analyses were employed to explore the prognostic impact of SSR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene set enrichment analysis (GSEA) were implemented to reveal the underlying mechanism of SSR1. Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR) was used to verify the expression of SSR1 in HCC. RESULTS: SSR1 was significantly overexpressed in HCC (SMD=1.25, P=0.03) and had the moderate diagnostic ability (AUC=0.84). SSR1 expression was significantly correlated with T stage, Gender, Pathologic stage (All P<0.05). Patients with high SSR1 expression had shorter overall survival (OS). Univariate and multivariate Cox regression analyses showed that high SSR1 expression was an independent risk factor for poor prognosis. KEGG analysis showed that SSR1-related genes were enriched in the cell cycle, DNA replication, and TGF-beta signaling pathway. GSEA analysis also shows that the high expression of SSR1 is related to the activation of the above three signal pathways. qRT-PCR showed that the SSR1 expression in HCC was significantly higher than the Peri-carcinoma tissue (PHCC) and the corresponding normal liver tissue. CONCLUSION: SSR1 expression was significantly up-regulated, and it had the potential as a biomarker for the diagnosis and prognosis of HCC. It was very likely to participate in the occurrence and development of HCC by regulating the cell cycle. In summary, our study comprehensively analyzed the clinical value of SSR1 and also conducted a preliminary study on its potential mechanism, which will provide inspiration for the in-depth study of SSR1 in HCC.

Circular RNA hsa_circ_0026552 inhibits the proliferation, migration and invasion of trophoblast cells via the miR­331­3p/TGF­ßR1 axis in pre­eclampsia.

Globally, pre­eclampsia (PE) is a gestational disorder that causes increased morbidity of the fetus and mortality induced by pregnancy. Despite various studies, the understanding of the causes or mechanism of the development of PE remains elusive. Thus, the present study aimed to investigate the role of circular (circ)RNA hsa_circ_0026552 (hsa_circ_0026552) in the development of PE and its mechanism of regulation. hsa_circ_0026552 differential expression in PE tissue data and clinical samples were analyzed and it was observed that hsa_circ_0026552 is highly upregulated in PE samples. Furthermore, miR­331­3p was detected as an hsa_circ_0026552 target miRNA and TGF­ßR1 gene as a target of miR­331­3p. These results were confirmed using various assays, including dual­luciferase reporter, reverse transcription­quantitative PCR and RNA pull­down assay. It was observed that miR­331­3p expression was negatively correlated to hsa_circ_0026552 relative expression, while TGF­ßR1 expression was positively correlated to hsa_circ_0026552 expression evaluated by Pearson's correlation test. The functional experiments, including Cell Counting Kit­8, colony formation and Transwell assay, showed that silencing hsa_circ_0026552 could significantly strengthen the proliferation, migration and invasion of the trophoblastic HTR­8/SVneo cells, but the subsequent overexpression of hsa_circ_0026552 reversed this. Mechanistically, it was concluded that hsa_circ_0026552 acts as a miR­331­3p sponge to upregulate TGF­ßR1 expression in trophoblasts and is involved significantly in PE development and progression in pregnant women. The circRNA hsa_circ_0026552 could be a novel therapeutic target and prognostic biomarker for PE.

Epidermal growth factor receptor intron 1 polymorphism and microsatellite instability in sporadic colorectal cancer.

Epidermal growth factor receptor (EGFR) expression is commonly upregulated in sporadic colorectal cancer (CRC) and its high expression is associated with poor prognosis in patients with CRC. CA-SSR1 is a dinucleotide CA repeat of the EGFR gene that can modulate EGFR transcription and is a potential target of the mismatch repair machinery in tumours with microsatellite instability (MSI). In the present study, 160 sporadic colon cancer samples were analysed for EGFR CA-SSR1 polymorphism and MSI status. Additionally, EGFR mRNA and protein expression levels in the tumour centre and in the invasive tumour front, compared with those in adjacent normal tissue samples, were evaluated in 80 tumour samples. An inverse association was identified between EGFR mRNA levels and the sum of repeats in both alleles of the CA-SSR1 polymorphism in normal tissues. Changes in CA-SSR1 were detected in the tumour centre as well as in the invasive tumour front and metastases in all MSI high (MSI-H) tumours. Analysis of EGFR expression at the mRNA and protein levels according to MSI status revealed lower EGFR mRNA and protein expression in MSI-H tumours than microsatellite-stable (MSS) tumours. Furthermore, higher EGFR levels in the invasive tumour front compared with in the tumour centre in MSS tumours were identified, suggesting a role of EGFR in tumour progression and higher invasive potential of MSS than MSI-H tumours.

SSR1 is involved in maintaining the function of mitochondria electron transport chain and iron homeostasis upon proline treatment in Arabidopsis.

Although increasing intracellular proline under stressed condition could help the plants survive, treating plant with high level of proline under normal condition could be inhibitory to plant growth. Among other possible mechanisms, proline-induced mitochondrial reactive oxygen species (ROS) production due to electron overflow in mitochondria electron transport chain (mETC) caused by elevated proline degradation may contribute to the proline toxicity. However, direct evidences are still elusive. Here, we reported a functional characterization of SSR1, encoding a protein localized in mitochondria matrix, in maintaining the function of mETC through analyzing the proline hypersensitive phenotype of an Arabidopsis mutant ssr1-1 with a truncated SSR1 protein. Our analysis demonstrated that upon proline treatment, there were higher mitochondrial ROS, lower ATP content, reduced activity of mETC complex I and II, and reduced iron content in ssr1-1, in comparison to the wild type. Therefore, SSR1 is involved in maintaining normal capacity of mETC in transporting electrons in a way that related to iron homeostasis. Our results also supported that normal mETC activity is required for alleviating the proline toxicity.

In Ovo and In Silico Evaluation of the Anti-Angiogenic Potential of Syringin.

INTRODUCTION: Cancer is considered as one of the deadliest human diseases today. Angiogenesis, the propagation of new blood vessels from pre-existing vasculature, is a critical step in the progression of cancer as it is essential in the growth and metastasis of tumors. Hence, suppression of angiogenesis is a promising approach in cancer therapy. Syringin, a phenylpropanoid glycoside with a molecular formula of C17H24O9, has been found to exhibit chemopreventive effects. However, its anti-angiogenic activity and the underlying mechanism of action are still unknown. METHODS: In this work, in ovo chorioallantoic membrane (CAM) assay has been conducted to evaluate the effect of syringin on neovascularization. Additionally, reverse molecular docking studies have been performed in order to identify the probable enzyme targets in the angiogenesis pathway. RESULTS: Treatment with syringin showed significant dose-dependent inhibition of blood vessel length and junctions in the CAM of duck eggs; the anti-angiogenic activity of syringin at 100 µM and 200 µM is comparable with 200 µM of the positive control celecoxib. The results of reverse docking studies indicate that syringin binds the strongest to dihydrofolate reductase (DHFR) and, to some extent, with transforming growth factor-beta receptor type 1 (TGF-ßR1), vascular endothelial growth factor receptor 2 (VEGFR2), and matrix metalloproteinase-2 (MMP-2). Furthermore, ADMET models revealed that syringin potentially possesses excellent pharmacokinetic and toxicity profiles. CONCLUSION: This study demonstrates the potential of syringin as an anti-angiogenic agent and elicits further investigations to establish its application in cancer suppression.

LncRNA PVT1 Suppresses the Progression of Renal Fibrosis via Inactivation of TGF-ß Signaling Pathway.

BACKGROUND: Renal fibrosis is a frequent pathway leading to end-stage kidney dysfunction. In addition, renal fibrosis is the ultimate manifestation of chronic kidney diseases (CKD). Long noncoding RNAs (lncRNAs) are known to be involved in occurrence of renal fibrosis, and lncRNA plasmacytoma variant translocation 1 (PVT1) has been reported to act as a key biomarker in renal diseases. However, the role of PVT1 in renal fibrosis remains unclear. MATERIALS AND METHODS: HK-2 cells were treated with TGF-ß1 to mimic renal fibrosis in vitro. Gene and protein expressions in HK-2 cells were measured by qRT-PCR and Western-blot, respectively. ELISA was used to test the level of creatinine (CR) and blood urea nitrogen (BUN) in serum of mice. Additionally, unilateral ureteral obstruction (UUO)-induced renal fibrosis mice model was established to investigate the effect of PVT1 on renal fibrosis in vivo. RESULTS: PVT1 was upregulated in TGF-ß1-treated HK-2 cells. In addition, TGF-ß1-induced upregulation of α-SMA and fibronectin in HK-2 cells was significantly reversed by PVT1 knockdown. Meanwhile, PVT1 bound to miR-181a-5p in HK-2 cells. Moreover, miR-181a-5p directly targeted TGF-ßR1. Furthermore, miR-181a-5p antagonist could significantly reverse the anti-fibrotic effect of PVT1 knockdown. Besides, knockdown of PVT1 notably attenuated the symptom of renal fibrosis in vivo. CONCLUSION: Knockdown of PVT1 significantly inhibited the progression of renal fibrosis in vitro and in vivo. Thus, PVT1 may serve as a potential target for the treatment of renal fibrosis.

Ischemia-reperfusion injury of brain induces endothelial-mesenchymal transition and vascular fibrosis via activating let-7i/TGF-ßR1 double-negative feedback loop.

Let-7i modulates the physical function and inflammation in endothelial cells (ECs). However, whether the let-7i of ECs involves in brain vasculature and ischemic stroke is unknown. Using inducible Cadherin5-Cre lineage-tracking mice, a loxp-RNA-sponge conditional knockdown of let-7 in ECs- induced increase of transforming growth factor-ß receptor type 1 (TGF-ßR1), endothelial-mesenchymal transition (endMT), vascular fibrosis, and opening of the brain-blood barrier (BBB). By this lineage-tracking mice, we found that ECs underwent endMT after transient middle cerebral artery occlusion (MCAO). Through specifically overexpressed let-7i in ECs, we found that it reduced TGF-ßR1, endMT, and vascular fibrosis. Furthermore, this overexpression reduced the infarct volume and leakage of the BBB, and improved the neurological function. Further, the expression of let-7i decreased after MCAO, but was reversed by antagonist of TGF-ßR1 or inhibition of Mek phosphorylation. And the inhibition of Mek attenuated the vascular fibrosis after MCAO. In summary, we concluded that ischemic stroke activates a let-7i/TGF-ßR1 double-negative feedback loop, thereby inducing endMT and vascular fibrosis. These results suggest that endMT is a potential target for the treatment of cerebral vascular fibrosis.

Crocin attenuates cisplatin-induced hepatotoxicity via TLR4/NF-κBp50 signaling and BAMBI modulation of TGF-ß activity: Involvement of miRNA-9 and miRNA-29.

TLR4-induced mitigation of the BMP down-regulation and activin membrane bound inhibitor (BAMBI) and the consequent enhancement of the transforming growth factor-beta (TGF-ß) profibrogenic signaling has not yet been studied in cisplatin (CIS)-induced hepatotoxicity. miRNA-9 and29 have been previously reported to modulate TLR4 signaling via either tempering the expression of nuclear factor kappa-B p50 (NF-κB p50) or downregulation of extracellular matrix genes respectively. Hence we aimed to investigate the involvement of TLR4-induced modulation of TGF-ß receptor 1 (TGF-ßR1) signaling as well as the implication of miRNA-9 and 29 in CIS-induced hepatotoxicity. Moreover, we examined the ability of the phytochemical; crocin (CROC); to interact with either TLR4 or TGF-ßR1 through a molecular docking study and subsequently explore its capability to attenuate CIS-induced hepatotoxicity. CROC pretreatment ameliorated the CIS-induced enhancement of TLR4 and TGF-ß signaling and enhanced the expression of BAMBI, miRNA-9 and 29. Accordingly, it may be assumed that the protective effect of CROC against CIS-induce hepatotoxicity is mediated via the crosstalk of TLR4/NF-κBp50 signaling and BAMBI modulation of TGF-ß1 activity in addition to the up-regulation of miRNA-9 and 29. These findings came in alignment with our molecular docking results; emphasizing the molecular antagonistic activity of CROC in both TLR4 and TGF-ßR1.

Invasion and migration of MDA-MB-231 cells are inhibited by block of AhR and NFAT: role of AhR/NFAT1/ß4 integrin signaling.

Benzo[α]pyrene (BaP) can have significant role in the development of breast cancer via aryl hydrocarbon receptor (AhR) activation. AhR activation has been studied in several functions such as survival, migration and invasion of cancer cells. In cancer, integrins contribute to the migration/invasion process and are regulated by nuclear factor of activated T cells (NFAT) and transforming growth factor (TGF) beta pathways. The aim of the present study was to examine the effect of BaP, an activator of AhR and cyclosporine A (CsA), as inhibitor of NFAT on migration and invasion of MDA-MB-231 cells. Furthermore, the effects of BaP and CsA were evaluated regarding the crosstalk of AhR, NFAT1 and TGF-ß receptor 1 signaling. Treatment of MDA-MB-231 with BaP resulted in significantly more live cells in low doses; however, blocking NFAT with CsA decreased the viability of the cells. Activation of AhR by BaP induced invasion as well as migration in MDA-MB-231 cells, which was blocked by AhR antagonist. Unlike BaP, block of NFAT with CsA inhibited cell migration and cell invasion. In these cells, BaP significantly reduced AhR expression while this reduction was reversed by CH-223191; however, CsA treatment lowered the AhR expression only at low dose. The level of ß4 integrin was significantly reduced by CsA at 1 and 2.5 µm. Protein levels of Snail and TGF-ß receptor 1 were not significantly altered by BaP and CsA treatments. Considering these findings, the low AhR expression and high ß4 integrin level following BaP and/or CsA treatments may contribute to the higher invasion/migration in MDA-MB-231 cells.

Blocking TGF-ß type 1 receptor partially reversed skin tissue damage in experimentally induced atopic dermatitis in mice.

Animals with impaired transforming growth factor (TGF)-ß1 signaling developed spontaneous lethal autoimmune inflammationand autoimmune diseases. Moreover, evidence for modified TGF-ß signaling in atopic dermatitis (AD) exists. Therefore, the goal of this study was to determine whether SB-431542, a potent and selective inhibitor of the TGF-ß type 1 receptor (TGF-ßR1), could attenuate such a severe reaction in mice. In addition, the molecular underpinnings the possible protective effects were also investigated. Repeated epicutaneous application of DNCB was performed on the ear and shaved dorsal skin of miceto induce AD-like symptoms and skin lesions. SB-431542 (1 mg/kg) was given by intra-peritoneal injection three times weekly for 3 weeks to assess the anti-pruritic effects. Serum levels of TGF-ß1, TGF-ßR1, latency-associated peptide (LAP), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6 were assessed by ELISA. Moreover, the gene expression of TNF-α, IL-1ß and IL-6 were determined. Apoptotic pathway was evaluated by measuring the activity of caspase-3 and by staining skin sections with anti-caspase-3 antibodies. We found that SB-431542 alleviated DNCB-induced AD-like symptoms as quantified by skin lesion,dermatitisscore, ear thickness and scratching behavior. In parallel, SB-431542 blocked DNCB-induced elevation in serum levels of TNF-α, TGF-ß1, TGF-ßR1, LAP, IL-1ß, IL-6 and IgE. The collective results indicate that SB-431542 partially suppresses DNCB-induced AD in micevia reduction of TGF-ß1 signaling pathway associated with inhibition of inflammation and apoptosis.

Reduced expression of let-7f activates TGF-ß/ALK5 pathway and leads to impaired ischaemia-induced neovascularization after cigarette smoke exposure.

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large-scale miRNA profiling and qRT-PCR analyses, we identified let-7f as a pro-angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let-7f mimic restored ischaemia-induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let-7f mimic could also rescue pro-angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF-ßR1), an important modulator of angiogenesis, is a target of let-7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti-angiogenic factors SMAD2/3 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2/3 and PAI-1 both in vitro and in vivo. Moreover, let-7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let-7f and activation of the anti-angiogenic TGF-ß/ALK5 pathway. Overexpression of let-7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia-induced neovascularization in pathological conditions.

Lack of evidence for prognostic value of epidermal growth factor receptor intron-1 CA repeats for oral carcinomas.

Epidermal growth factor receptor (EGFR) expression is altered in several malignancies, including oral squamous cell carcinoma. A CA-repeat polymorphism in intron-1 (CA-SSR-1) of the EGFR gene is reported to influence EGFR expression and is associated with features of various solid tumors and outcomes of cancer patients. In the present study we evaluated the influence of length and zygosity of CA-SSR-1 on the survival of patients with oral squamous cell carcinoma. The length and zygosity of CA-SSR-1 was obtained through microsatellite analysis in 91 patients with oral cancer, who were treated in the Department of Oral and Maxillofacial Surgery of the University Medical Centre Hamburg Eppendorf, Germany, during the years 1998-2008. Follow up was conducted until 2016. Outcome measures were age, gender, tumor stage, occurrence of metastases, and date of recurrence or death. Statistical analysis was conducted using the chi-square test and the log-rank test. Neither length nor zygosity of the CA-SSR-1 in patients with oral squamous cell carcinoma was significantly correlated with sex, age, tumor size, tumor localization, lymph node involvement, metastasis status, disease-free survival, or overall survival. Length and zygosity of the CA-SSR-1 polymorphism in EGFR is not able to serve as a prognostic biomarker in White European patients with oral squamous cell carcinoma.

Anti-tumor activity of arjunolic acid against Ehrlich Ascites Carcinoma cells in vivo and in vitro through blocking TGF-ß type 1 receptor.

We aimed to evaluate therapeutic potential of arjunolic acid (AA), in Terminalia Arjuna bark, on Ehrlich Ascites carcinoma (EAC) in-vivo and in-vitro. EAC was induced in fifty female Swiss albino mice. Two doses of AA was used 100 and 250mg/kg. Arjunulic acid reduced tumor volume and cells count. AA decreased EAC cells viability and increased cell toxicity. Moreover, AA reduced TNF-α, IL-1ß, TGF-ß, TGF-ß type I receptor and latency-associated peptide levels associated with elevated IL-10 in-vivo and in-vitro. In conclusion, AA produced antitumor activity against EAC by increasing cytotoxicity and apoptosis and partially blocking the TGF-ßR1 and affecting inflammatory cytokine levels.

Molecular genetic alterations in egfr CA-SSR-1 microsatellite and egfr copy number changes are associated with aggressiveness in thymoma.

BACKGROUND: The key role of egfr in thymoma pathogenesis has been questioned following the failure in identifying recurrent genetic alterations of egfr coding sequences and relevant egfr amplification rate. We investigated the role of the non-coding egfr CA simple sequence repeat 1 (CA-SSR-1) in a thymoma case series. METHODS: We used sequencing and egfr-fluorescence in situ hybridization (FISH) to genotype 43 thymomas; (I) for polymorphisms and somatic loss of heterozygosity of the non-coding egfr CA-SSR-1 microsatellite and (II) for egfr gene copy number changes. RESULTS: We found two prevalent CA-SSR-1 genotypes: a homozygous 16 CA repeat and a heterozygous genotype, bearing alleles with 16 and 20 CA repeats. The average combined allele length was correlated with tumor subtype: shorter sequences were significantly associated with the more aggressive WHO thymoma subtype group including B2/B3, B3 and B3/C histotypes. Four out of 29 informative cases analysed for somatic CA-SSR-1 loss of heterozygosity showed allelic imbalance (AI), 3/4 with loss of the longer allele. By egfr-FISH analysis, 9 out of 33 cases were FISH positive. Moreover, the two integrated techniques demonstrated that 3 out of 4 CA-SSR-1-AI positive cases with short allele relative prevalence showed significantly low or high chromosome 7 "polysomy"/increased gene copy number by egfr-FISH. CONCLUSIONS: Our molecular and genetic and follow up data indicated that CA-SSR-1-allelic imbalance with short allele relative prevalence significantly correlated with EGFR 3+ immunohistochemical score, increased egfr Gene Copy Number, advanced stage and with relapsing/metastatic behaviour in thymomas.

Renal protective effects of arjunolic acid in a cisplatin-induced nephrotoxicity model.

Cisplatin is the first platinum-containing anti-cancer drugs. Cisplatin notable side effect of nephrotoxicity limits its use in clinic. Meanwhile, arjunolic acid possesses anti-inflammatory properties and plays protective roles against chemically induced organ pathophysiology. This study was conducted to find out whether arjunolic acid could attenuate kidney damage in rats, and to elucidate its possible mechanism of action. Fifty rats were treated with cisplatin (10mg/kg) in the presence/absence of 100 or 250mg/kg arjunolic acid. Arjunolic acid is given 1h after cisplatin. Morphological changes were assessed in kidney sections stained with Hematoxylin/Eosin and Masson Trichrome. Kidney samples were used for measurements of transforming growth factor (TGF)-ß1 and its type 1 receptor (TGF-ßR1), tumor necrosis factor (TNF)-α and interleukin (IL)-1ß by ELISA. Gene expression NFκB was determined by real time-PCR. Kidney tissue apoptosis was assessed by measuring the activities of caspase-3/8/9. The renal protective effect of arjunolic acid was confirmed by approximately normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. Furthermore, arjunolic acid showed dose dependent reduction in cisplatin-induced elevation in renal levels of TGF-ßR1, TGF-ß1, TNF-α, IL-1ß and caspases. These findings demonstrated that arjunolic acid attenuates cisplatin nephrotoxicity either indirectly by enhancing body antioxidant activity or directly through several mechanisms, including inhibition of pro-inflammatory cytokines, blocking activation of TGF-ß1, and anti-apoptotic effects.