Expression and prognostic value of E2F3 transcription factor in non-small cell lung cancer.

E2F transcription factor 3 (E2F3) plays a vital role in the development of various types of cancer. To verify whether E2F3 is a suitable biomarker for the prognosis of lung cancer, bioinformatics analysis was performed to determine the differential expression level of E2F3 in lung cancer and the surrounding non-tumor tissues, and the results were confirmed in a NSCLC cell line and a tissue microarray (TMA). The relevance of E2F3 in non-small cell lung cancer (NSCLC) was investigated in 19 studies from the Oncomine database and confirmed in The Cancer Genome Atlas database. In the lung cancer cell line A549, the inhibition of E2F3 mRNA expression level led to decreased tumor cell viability and cell migration, which was determined by a Cell Counting Kit-8 and wound healing assays, respectively. Immunohistochemistry analyses of E2F3, Bcl-2, Bax and caspase-3 were performed in the NSCLC TMA (n=50). The assessment of TMA detected the increase of E2F3 protein expression level in the tumor tissues, as compared with that in the non-tumor tissues, which was also correlated with the increase in expression of Bcl-2 in tumors. Analysis of the clinical data from patients with NSCLC revealed that the overexpression of E2F3 was associated with early lymphatic spreading, and poor patient survival time. The OncomiR website was used to predict the E2F3 upstream microRNAs and determine their prognostic value in patients with NSCLC. The results from the present study revealed that E2F3 was overexpressed at both the transcriptional and translational levels in NSCLC tissues, as compared with that in non-tumor tissues. The overexpression of E2F3 was associated with the upregulation of the anti-apoptotic factor, Bcl-2, which may contribute to uncontrolled tumor growth. Thus, E2F3 was shown to have important oncogenic properties in the development of NSCLC, and it may become a potential biomarker for patients with NSCLC.

TGF-ß1-regulated miR-3691-3p targets E2F3 and PRDM1 to inhibit prostate cancer progression.

Transforming growth factor-ß1 (TGF-ß1) acts as a tumor promoter in advanced prostate cancer (PCa). We speculated that microRNAs (miRNAs) that are inhibited by TGF-ß1 might exert anti-tumor effects. To assess this, we identified several miRNAs downregulated by TGF-ß1 in PCa cell lines and selected miR-3691-3p for detailed analysis as a candidate anti-oncogene miRNA. miR-3691-3p was expressed at significantly lower levels in human PCa tissue compared with paired benign prostatic hyperplasia tissue, and its expression level correlated inversely with aggressive clinical pathological features. Overexpression of miR-3691-3p in PCa cell lines inhibited proliferation, migration, and invasion, and promoted apoptosis. The miR-3691-3p target genes E2F transcription factor 3 (E2F3) and PR domain containing 1, with ZNF domain (PRDM1) were upregulated in miR-3691-3p-overexpressing PCa cells, and silencing of E2F3 or PRDM1 suppressed PCa cell proliferation, migration, and invasion. Treatment of mice bearing PCa xenografts with a miR-3691-3p agomir inhibited tumor growth and promoted tumor cell apoptosis. Consistent with the negative regulation of E2F3 and PRDM1 by miR-3691-3p, both proteins were overexpressed in clinical PCa specimens compared with noncancerous prostate tissue. Our results indicate that TGF-ß1-regulated miR-3691-3p acts as an anti-oncogene in PCa by downregulating E2F3 and PRDM1. These results provide novel insights into the mechanisms by which TGF-ß1 contributes to the progression of PCa.

MiR-7e-5p downregulation promotes transformation of low-grade follicular lymphoma to aggressive lymphoma by modulating an immunosuppressive stroma through the upregulation of FasL in M1 macrophages.

BACKGROUND: In follicular lymphoma (FL), histologic transformation to high-grade FL and diffuse large B-cell lymphoma (DLBCL) is a critical adverse step in disease progression. Activation of the oncogene c-MYC and tumor microenvironment remodeling account for FL progression. A panel of microRNA (miRNA) was downregulated in transformed FL (tFL). METHODS: Differentially expressed miRNAs were systematically compared in 11 lymph nodes from patients at different stages of disease. Expression of miR-7e-5p was analyzed in 46 B-cell lymphomas, including 30 FL tissues and 16 DLBCL tissues. In FL cells, transcriptional regulation of the oncogene c-MYC on its target miR-7e-5p was revealed by Chromatin Immunoprecipitation (ChIP) assay. Exosome, carrying differentially expressed miR-7e-5p was isolated and visualized by transmission electron microscope and fluorescence tracing. The effect of miR-7e-5p on recipient macrophage was determined by target gene quantification, flow cytometry, and TUNEL method in a cocultured system with miR-7e-5p-mimics or inhibitors treatment. Expression of miR-7e-5p targets, macrophage proportions, and clinical parameters were included for correlation analysis. RESULTS: We determined that downregulation of miR-7e-5p, driven by c-MYC overexpression, was associated with poorer prognosis in FL patients. The decreased expression of miR-7e-5p in lymphoma cells led to a reduced exosomal transfer to surrounding macrophages. As a result, the target gene of miR-7e-5p, Fas ligand (FasL), was upregulated and activated the caspase signaling, which led to the apoptosis of M1 macrophages in tumor stroma. Finally, in transformed FL tissues, overexpression of FasL and activation of caspase proteins was detected in tumor stromal macrophages. Downregulation of miR-7e-5p was associated with poorer clinical outcomes. CONCLUSION: Downregulation of exosomal miR-7e-5p induces stromal M1 macrophage apoptosis, which leads to immunosurveillance and transformation of FL.

Bone Marrow Mesenchymal Stem Cells Ameliorate Cisplatin-Induced Renal Fibrosis via miR-146a-5p/Tfdp2 Axis in Renal Tubular Epithelial Cells.

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury (AKI). However, the potential function of MSCs in chronic kidney disease remains elusive. Renal fibrosis is the common endpoint of chronic progressive kidney diseases and causes a considerable health burden worldwide. In this study, the protective effects of bone marrow mesenchymal stem cells (BM-MSCs) were assessed in repeated administration of low-dose cisplatin-induced renal fibrosis mouse model in vivo as well as a TGF-ß1-induced fibrotic model in vitro. Differentially expressed miRNAs in mouse renal tubular epithelial cells (mRTECs) regulated by BM-MSCs were screened by high-throughput sequencing. We found microRNA (miR)-146a-5p was the most significant up-regulated miRNA in mRTECs. In addition, the gene Tfdp2 was identified as one target gene of miR-146a-5p by bioinformatics analysis. The expression of Tfdp2 in the treatment of BM-MSCs on cisplatin-induced renal injury was evaluated by immunohistochemistry analysis. Our results indicate that BM-MSC attenuates cisplatin-induced renal fibrosis by regulating the miR-146a-5p/Tfdp2 axis in mRTECs.

Transforming growth factor-ß1 promotes breast cancer metastasis by downregulating miR-196a-3p expression.

Transforming growth factor-ß1 is considered a key contributor to the progression of breast cancer. MicroRNAs are important factors in the development and progression of many malignancies. In the present study, upon studies of breast cancer cell lines and tissues, we showed that microRNA -196a-3p is decreased by transforming growth factor-ß1 in breast cancer cells and associated with breast cancer progression. We identified neuropilin-2 as a target gene of microRNA -196a-3p and showed that it is regulated by transforming growth factor-ß1. Moreover, transforming growth factor-ß1-mediated inhibition of microRNA -196a-3p and activation of neuropilin-2were required for transforming growth factor-ß1-induced migration and invasion of breast cancer cells. In addition, neuropilin-2 expression was suppressed in breast tumors, particularly in triple-negative breast cancers. Collectively, our findings strongly indicate that microRNA -196a-3p is a predictive biomarker of breast cancer metastasis and patient survival and a potential therapeutic target in metastatic breast cancer.

Deciphering oncogenic drivers: from single genes to integrated pathways.

Technological advances in next-generation sequencing have uncovered a wide spectrum of aberrations in cancer genomes. The extreme diversity in cancer mutations necessitates computational approaches to differentiate between the 'drivers' with vital function in cancer progression and those nonfunctional 'passengers'. Although individual driver mutations are routinely identified, mutational profiles of different tumors are highly heterogeneous. There is growing consensus that pathways rather than single genes are the primary target of mutations. Here we review extant bioinformatics approaches to identifying oncogenic drivers at different mutational levels, highlighting the strategies for discovering driver pathways and networks from cancer mutation data. These approaches will help reduce the mutation complexity, thus providing a simplified picture of cancer.

Amino acid contact energy networks impact protein structure and evolution.

One of the most challenging tasks in structural proteomics is to understand the relationship between protein structure, biological function, and evolution. An understanding of amino acid networks based on protein topology has an important role in the study of this relationship; however, the relationship between network parameters underlying protein topology with structural properties or evolutionary rate is still unknown. To investigate this further, we modeled the three dimensional structure of proteins as amino acid contact energy networks (AACENs) with nodes represented as amino acid residues and edges established according to environment-dependent residue-residue contact energies. Five other types of networks were also constructed to investigate their topological parameters and compare their effect on protein structure and evolution: (1) a random contact network (RCN), (2) a rewiring network with the same degree of distribution as AACEN (RNDD), (3) long-range contact energy networks with and without the backbone connectivity (LCEN_BBs and LCENs), and (4) short range contact energy networks (SCENs). The results indicated that the long-range link percentage and the network clustering coefficient showed a significantly positive and negative correlation, respectively, with protein secondary structure density. In addition, the long-range link percentage and network diameter had a significantly positive and negative correlation, respectively, with evolutionary rate. According to our knowledge, this is the first study to identify the potential role of long-range links and network diameter in protein evolution.

The construction of an amino acid network for understanding protein structure and function.

Amino acid networks (AANs) are undirected networks consisting of amino acid residues and their interactions in three-dimensional protein structures. The analysis of AANs provides novel insight into protein science, and several common amino acid network properties have revealed diverse classes of proteins. In this review, we first summarize methods for the construction and characterization of AANs. We then compare software tools for the construction and analysis of AANs. Finally, we review the application of AANs for understanding protein structure and function, including the identification of functional residues, the prediction of protein folding, analyzing protein stability and protein-protein interactions, and for understanding communication within and between proteins.

Anatomical study of the communicating branches of cords of the brachial plexus and their clinical implications.

The aim of this study was to investigate the occurrence and patterns of the communicating branches of cords of the brachial plexus (BPs). This study was performed with 50 fixed adult cadavers (all 100 sides). The BPs were exposed, the presence of the communicating branches of BPs were determined, measured, and photographed. The communicating branches were identified in 27 sides of the BPs. According to enthesis, the communicating branches between the medial and lateral cords (25 sides) were divided into five types. The most common branches connected the lateral cord with the medial root of the median nerve (16 sides). All the communicating branches between the lateral and medial cords obliquely crossed anterior to the axillary artery and passed below the thoracoacromial artery trunk. The distance of the communicating branch with the origin of thoracoacromial artery trunk was 1.60 ± 0.64 cm. The length, transverse diameter, and anteroposterior diameter of communicating branch were 1.67 ± 0.62 cm, 1.77 ± 0.63 mm, and 1.91 ± 0.34 mm, respectively. These anatomical data about the communicating branches will be helpful for surgeons who perform surgical procedures in the cervical and axillary regions.

Molecular dynamic simulation to explore the molecular basis of Btk-PH domain interaction with Ins(1,3,4,5)P4.

Bruton's tyrosine kinase contains a pleckstrin homology domain, and it specifically binds inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), which is involved in the maturation of B cells. In this paper, we studied 12 systems including the wild type and 11 mutants, K12R, S14F, K19E, R28C/H, E41K, L11P, F25S, Y40N, and K12R-R28C/H, to investigate any change in the ligand binding site of each mutant. Molecular dynamics simulations combined with the method of molecular mechanics/Poisson-Boltzmann solvent-accessible surface area have been applied to the twelve systems, and reasonable mutant structures and their binding free energies have been obtained as criteria in the final classification. As a result, five structures, K12R, K19E, R28C/H, and E41K mutants, were classified as "functional mutations," whereas L11P, S14F, F25S, and Y40N were grouped into "folding mutations." This rigorous study of the binding affinity of each of the mutants and their classification provides some new insights into the biological function of the Btk-PH domain and related mutation-causing diseases.

Anatomical study and clinical significance of the rami communicantes between cervicothoracic ganglion and brachial plexus.

The aim of this study was to provide a detailed characterization of the rami communicantes between the stellate (or cervicothoraic) ganglion (CTG) and brachial plexus (BP). Rami communicantes of 33 fixed adult cadavers were macroscopically observed, and connection between CTG and spinal nerves and branching was investigated. In all cases, except one, the hibateral medial rami communicantes was found to be positioned symmetrically between the CTG and C7, C8 spinal nerves. Gray rami communicantes arising from the CTG joined C8, C7, C6 nerve roots on 66, 63, and 6 sides, respectively, and branched from the rami communicantes to C7, C6, C5 nerve roots lying on 51, 41, and 2 sides, respectively. Forty-five sides of the branches from rami communicantes derived from CTG to C8 were observed to ascend through the transverse foramina of the C7 nerve. The branches from rami communicantes derived from CTG to C7 to the C6 nerve were observed ascending through the foramen transversarium of the six cervical vertebrae along with the vertebral artery and joining the C6 spinal nerve in 41 sides. Knowledge about the general distribution and individual variations of the rami communicantes between CTG and BP will be useful toward studies involving the inference of sympathetic nerve stimulation of the upper limbs and could be important for surgeons who perform surgical procedures in the cervical region or medical blockade of nerve fibers.

TGF-beta1 signal pathway may contribute to rhabdomyosarcoma development by inhibiting differentiation.

Overexpression of transforming growth factor-beta1 (TGF-beta1) and its downstream molecules in the rhabdomyosarcoma (RMS) RD cell line has been reported previously, but the regulatory role of TGF-beta1 on RMS has not been studied extensively. In the present study, we showed that expression of TGF-beta1 and its downstream molecules type II TGF-beta receptor (TbetaRII) and Smad4 was significantly higher in RMS than in normal skeletal muscle, and there was a significant relationship between TGF-beta1 expression and histological grade. Gene silencing with TGF-beta1 short-hairpin RNA (shRNA)-expressing vectors significantly decreased the growth of RD cells, which was confirmed by caspase-3 (in vitro) and TUNEL (in vivo) assays. Moreover, a proportion of treated rhabdomyosarcoma (RD) cells changed to a round shape from the normal fusiform or polygonal shape and expressed myofilaments. Myogenin is one of the myogenic differentiation genes (MyoD) family of myogenic regulators, and was obviously higher in TGF-beta1-shRNA-treated tumors than it in control at the mRNA and protein level. Immunohistochemical staining with myogenic differentiation markers such as myosin and desmin in subcutaneous RMS tissue showed that TGF-beta1 shRNA increased staining for myosin. These results provide new insight into the biological function of TGF-beta1 in malignant tumors, and imply that the TGF-beta1 signal pathway is a potential therapeutic target for drugs that induce differentiation of RMS.

Regulated expression of pancreatic triglyceride lipase after rat traumatic brain injury.

Pancreatic triglyceride lipase (PTL), an enzyme of digestive system, plays very important roles in the digestion and absorption of lipids. However, its distribution and function in the central nervous system (CNS) remains unclear. In the present study, we mainly investigated the expression and cellular localization of PTL during traumatic brain injury (TBI). Western blot and RT-PCR analysis revealed that PTL was present in normal rat brain cortex. It gradually increased, reached a peak at the 3rd day after TBI, and then decreased. Double immunofluorescence staining showed that PTL was co-expressed with neuron, but had a few colocalizations in astrocytes. When TBI occurred in the rat cortex, the expression of PTL gradually increased, reached the peak at the 3rd day after TBI, and then decreased. Importantly, more PTL was colocalized with astrocytes, which is positive for proliferating cell nuclear antigen (PCNA). In addition, Western blot detection showed that the 3rd day post injury was not only the proliferation peak indicated by the elevated expression of PCNA, glial fibrillary acidic protein (GFAP) and cyclin D1, but also the apoptotic peak implied by the alteration of caspase-3 and bcl-2. These data suggested that PTL may be involved in the pathophysiology of TBI and PTL may be complicated after injury, more PTL was colocalized with astrocytes. Importantly, injury-induced expression of PTL was colabelled by proliferating cell nuclear antigen (proliferating cells marker), and the western blot for GFAP, PCNA and cyclin D1, showed that 3 days post injury was the proliferation peak, in coincidence to it, the protein level change of caspase-3 and bcl-2 revealed that the stage was peak of apoptotic too. These data suggested that PTL may be involved in the pathophysiology of TBI and that PTL may be implicated in the proliferation of astrocytes and the recovery of neurological outcomes. But the inherent mechanisms remained unknown. Further studies are needed to confirm the exact role of PTL after brain injury.

Expression of fasciculation and elongation protein zeta-1 (FEZ1) in cultured rat neonatal astrocytes.

Astrocytes play a more important role than simply providing physical support for neurons, however, the function(s) of type 1 and type 2 astrocytes (T1As, T2As), remains unclear. A DNA microarray was used to identify gene expression in cultured T1As and T2As isolated from postnatal day 1 rat cortex. Ninety-nine of the 138 differentially expressed genes were involved in a diverse number of processes. The fasciculation and elongation protein zeta-1 (FEZ1) gene was studied further because it has been suggested that it is not expressed by astrocytes. RT-PCR and Western blots confirmed the microarray data and showed that FEZ1 was present in T1 and T2As and is more highly expressed in T2As. Immunocytochemistry revealed that FEZ1 was located in the astrocytic cytoplasm and cell processes but not the nucleus. The results contribute to a clearer understanding of the two types of astrocytes.

Expression of E-cadherin and nm23 is associated with the clinicopathological factors of human non-small cell lung cancer in China.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, functions as maintenance of epithelial integrity. nm23, encoded by non-metastatic 23 gene, plays a key role in differentiation of many kinds of epithelium. Loss or dysfunction of E-cadherin and nm23 was frequently identified in many types of human cancers and is considered to correlate with invasive/metastatic phenotype. We previously reported that defective expression of E-cadherin might play a role in the development of the malignant phenotype in non-small cell lung cancer (NSCLC) [Q.Y. Fei, H.T. Zhang, X.F. Chen, et al., Defected expression of Ecadherin in non-small cell lung cancer, Lung Cancer 37 (2002) 147-152]. In an attempt to evaluate the significance of E-cadherin and nm23 in human non-small cell lung cancer, we performed mRNA expression and genetic structure analyses of the E-cadherin and nm23 genes in 54 NSCLCs and 46 normal lung tissues. The mRNA expression was determined by semi-quantitative RT-PCR, and genetic structure was examined through PCR-SSCP followed by sequencing. Although no mutation of the E-cadherin and nm23 genes was detected, the results obtained in the present study showed that reduction of E-cadherin and nm23 mRNA expression remarkably correlated with low histological differentiation, increasing stage as well as lymph node metastases (P<0.05). These data provide us with support for the idea that dysfunction of E-cadherin and nm23 has a role in progression of NSCLC and that the examination of E-cadherin and nm23 expression can provide experimental evidence for clinical treatment.