Expression of Toll-Like Receptors in the Lung Tissue of Mouse Fetuses Generated by in vitro Embryo Culture and Embryo Transfer.

Mouse fetuses generated by in vitro embryo culture and embryo transfer exhibit impaired lung development, altered composition of pulmonary epithelial cells associated with downregulation of several genes involved in lung development and toll-like receptor (TLR) signaling pathway. The aims of the present study were to determine the expression of all TLRs and to examine if the expression of TLRs, along with genes involved in TLR signaling pathway, is altered in the lung tissue of mouse fetuses generated through embryo culture and embryo transfer. Two experimental (EGs) and one control (CG) group were included in the study. Embryos cultured at 5% CO2-95% air for 95 h or less than 24 h were transferred to pseudo-pregnant females to obtain fetuses comprising EGin vitro (n = 18) and EGin vivo (n = 18), respectively. Fetuses obtained from naturally ovulating females on day 18 of pregnancy served as the CG (n = 18). Western blot and immunohistochemistry were used to determine the expression of TLR proteins. The expression of transcripts encoding TLRs, and the genes involved in TLR signaling pathway (Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos), was determined using qRT-PCR. While all TLRs were expressed by cells lining the bronchial/bronchiolar epithelium of lung tissues in all groups, some of the TLRs were expressed in a specific pattern. When compared to CG, the expression of transcripts encoding TLR-2, -3, -4, -5, -7, -8, -9, -12, -13, Lbp, Pik3r1, Pik3cb, Nfkbia, and Fos was significantly downregulated in both EGs. It appears that stress imposed on embryos at preimplantation stages of development is associated with downregulation of TLRs, along with some of the genes involved in TLR signaling pathway, in the lung tissue during the perinatal period. It remains to be determined if downregulation of TLRs, along with the genes involved in TLR signaling pathway, has any functional consequences in the adult lung tissue.

Computational and Bioinformatics Methods for MicroRNA Gene Prediction.

MicroRNAs (miRNAs) are 20-24-nucleotide-long noncoding RNAs that bind to the untranslated region (3' UTR) of their target mRNAs. The importance of miRNAs in medicine has grown rapidly in the 20 years since the discovery of them. As the regulatory function of miRNAs on biological processes was discovered, they were advocated to play a role in the underlying mechanisms of human pathogenesis. Functional studies have confirmed that miRNAs are promising in preclinical development through deregulation of genes targeted by miRNAs in many cancer cases. In this chapter, we summarize the miRNAs identified for some specific types of cancer and their functions. Besides, miRNAs function as cancer biomarker and their benefits to diagnosis and treatment of cancer are also discussed.

Effects of low-density pulsed ultrasound treatment on transforming growth factor-beta, collagen level, histology, biomechanics, and function in repaired rat tendons.

OBJECTIVES: The aim of this study is to compare the effects of low-density pulsed ultrasound (LIPUS) treatment on growth factors/collagen production, histological, biomechanical, and function of rats with Achilles tendon injury. MATERIALS AND METHODS: A total of 44 Wistar Albino rats were used in the study between April 2017 and June 2018. The rats were randomized to two treatment groups. Group 1 (n=6) received LIPUS treatment (0.3 Watt/cm2; 1 MHz, 1:5 pulse mode) and Group 2 (n=6)received sham ultrasound (US) treatment following Achilles tendon surgery. Transforming growth factor-beta 1 (TGF-ß1) and collagen gene expression levels were evaluated using polymerase chain reaction. The histological evaluation was performed with the Bonar scoring system. The tensile strength was measured by biomechanical testing and the function was evaluated with the Achilles Functional Index (AFI). RESULTS: Although TGF-ß1 expression and tensile strength evaluation showed a tendency to improve in favor of the LIPUS group, no statistically significant difference was found (p=0.065 and p=0.053, respectively). The COL3 gene expression in the LIPUS group and the COL1 expression in the sham US group were significantly higher. Bonar scores and AFI scores showed a statistically significant improvement in the LIPUS group, compared to the sham US group. CONCLUSION: Our study results show that LIPUS yields positive effects on tendon histology and functional status in repaired Achilles tendon in rats.

Relation of ADRB3, GEF, ROCK2 gene polymorphisms to clinical findings in overactive bladder.

PURPOSE: Adrenergic and cholinergic pathways play an important role in contraction-relaxation harmony in human bladder. Functional changes in any proteins in these pathways may result in overactive bladder. We aimed to investigate whether single gene polymorphisms affecting adrenergic and cholinergic pathways are associated with OAB syndrome. METHODS: 60 patients with idiopathic OAB and 60 healthy controls were included in the study. A validated OAB-V8 questionnaire was given to all patients. Polymorphisms of ADRB3, ROCK2, and GEF gene were detected by PCR from whole blood samples. Genotypic structures of patients and controls were compared. The relationship between genotypic structures and OAB symptom scores were investigated. RESULTS: We found no significant difference in the genotype and allele frequencies between the patients and controls for all three SNP. While there was no relationship between ADRB3 and GEF gene polymorphisms and OAB scores in OAB patients, the OAB score in heterozygous polymorphic individuals was significantly higher than in homozygous polymorphic individuals in the ROCK2 gene (p = 0.039). CONCLUSION: The polymorphisms of the ADRB3, ROCK2, and GEF genes were present in both OAB group and healthy controls, but were not associated with OAB syndrome.

Exploring biomarkers in the overactive bladder: Alterations in miRNA levels of a panel of genes in patients with OAB.

AIMS: It has been demonstrated that there are abundant stable microRNAs (miRNAs) in plasma, which is potentially disease-specific. Adrenergic and muscarinic pathways play an important role in voiding physiology. Alterations in the levels of miRNAs are thought to influence the regulation of these pathways at the molecular level. The aim of this study was to investigate the relationship of miRNAs with overactive bladder pathogenesis and to provide a new perspective to treatment approaches. METHODS: This study included patients with an overactive bladder (OAB) diagnosis and a healthy control group. All patients completed a validated OAB-V8 questionnaire. The relative expression levels of 12 miRNAs were examined in plasma by PCR. Receiver operating characteristic (ROC) curves were generated to evaluate the diagnostic qualification of miRNAs. RESULTS: The relative expression levels of let-7b-5p, miR-92a-3p, miR-98-5p, miR-142-3p, and miR-200c-3p were significantly upregulated and miR-139-5p was significantly downregulated in patients with OAB and no correlation was determined between the levels of miRNAs with OAB symptom score. Among the miRNAs, miR-98-5p provided the highest diagnostic accuracy alone (area under curve [AUC] = 0.79) in ROC analysis. The combination of miR-98-5p + miR-139-5p was seen to be a good indicator (AUC = 0.839). CONCLUSIONS: These results suggest that alteration of the miRNA levels can be used as auxiliary parameters to explain the pathophysiology of OAB syndrome and could shed light on new treatment options.

NF-κB-Induced Upregulation of miR-548as-3p Increases Invasion of NSCLC by Targeting PTEN.

BACKGROUND: Non-Small Cell Lung Cancer (NSCLC) is an aggressive cancer type due to high metastatic capacity. Nuclear Factor Kappa B (NF-κB) is a consistently active transcription factor in malignant lung cancer cells and has crucial significance in NSCLC progression. It is also implicated in the transcriptional regulation of many genes including microRNAs (miRNAs) that function as tumor suppressor or oncogene. It has been increasingly reported that several miRNAs defined as gene members are induced by NF-κB. The present study aimed to find novel miRNAs that are regulated by NF-κB. METHODS: Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiment and bioinformatic analysis were used to determine NF-κB-dependent miRNAs. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR), luciferase reporter gene assays were carried out to investigate the target genes of miRNAs. To determine biologic activity, transwell invasion and MTT assay were carried out on H1299 NSCLC cell line. miRNA expression level was evaluated in metastatic and non-metastatic tissue samples of NSCLC patients. RESULTS: ChIP-Seq and qRT-PCR experiments showed that miR-548as-3p is transcriptionally regulated by NF- κB in response to Tumor Necrosis Factor-α (TNF-α) treatment. Then, we found that tumor suppressor Phosphatase and Tension homolog (PTEN) is a direct target of miR-548as-3p. Furthermore, miR-548as-3p mediates phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway and NF-κB-implicated genes including Matrix Metalloproteinases 9 (MMP9), Slug and Zeb1. We further showed that miR-548as-3p increased invasiveness of NSCLC cells and was upregulated in metastatic tumor tissues compared to non-metastatic ones. CONCLUSION: All these findings provide a miRNAs-mediated novel mechanism for NF-κB signaling and that miR-548as-3p could be a biomarker for NSCLC metastasis.

TGF-ß-SMAD-miR-520e axis regulates NSCLC metastasis through a TGFBR2-mediated negative-feedback loop.

Transforming growth factor-ß (TGF-ß) pathway plays crucial roles during the carcinogenesis and metastasis. TGF-ß receptor 2 (TGFBR2) is a key molecule for the regulation of TGF-ß pathway and frequently downregulated or lost in several cancer types including non-small cell lung cancer (NSCLC), and TGF-ß pathway is often regulated by negative-feedback mechanisms, but little is known about the mechanism of TGFBR2 downregulation in NSCLC. Here, we found that the expression of miR-520e is upregulated in metastatic tumor tissues compared with non-metastatic ones, and its expression is inversely correlated with that of TGFBR2 in clinical samples. We also discovered that TGF-ß dramatically increased the expression of miR-520e, which targeted and downregulated TGFBR2, and the suppression of miR-520e significantly impaired TGF-ß-induced TGFBR2 downregulation. Chromatin immunoprecipitation-PCR experiments further showed that miR-520e is transcriptionally induced by SMAD2/3 in response to TGF-ß. Our findings reveal a novel negative-feedback mechanism in TGF-ß signaling and the expression level of miR-520e could be a predictive biomarker for NSCLC metastasis.

miR-181b-5p, miR-195-5p and miR-301a-3p are related with treatment resistance in schizophrenia.

The aim of the study was to determine the differences between expression levels of certain miRNAs, as their association with schizophrenia has been well presented in the literature, and to investigate their relation to treatment resistance in schizophrenic patients. Three groups were formed: 1) treatment-resistant group, 2) treatment responsive group and 3) healthy control group. Expression levels of miRNAs from peripheric blood samples were determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We investigated the roles of 29 schizophrenia-related miRNAs in schizophrenia treatment and their potentials to be considered as indicators. Among these miRNAs, only miR-181b-5p, miR-195-5p and miR-301a-3p expressions were found to be significantly different between the treatment-resistant group and the group responding well to the treatment. miRNAs may cause resistance by silencing the receptor genes of the drugs used for schizophrenia treatment. miR-181b-5p, miR-195-5p and miR-301a-3p may be candidate indicators that can be used to reveal resistance against schizophrenia treatment.