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This study aims to explore the molecular mechanism by which HAS2-AS1 acts as a ceRNA to promote the invasion and migration of glioma cells, which will provide a novel potential target for the targeted therapy of glioma. Gene expression profiles and corresponding clinical data were accessed from the TCGA_LGG and TCGA_GBM databases and then differential analysis was conducted using the "edgeR" package. miRDB, miRTarBase and TargetScan databases were employed to predict target genes and sequentially a ceRNA network was constructed. Quantitative real-time PCR was performed to detect gene expression in glioma cells. Transwell assay was operated to assess cell migratory and invasive abilities. Western blot was conducted to evaluate the protein expression. Dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the targeting relationship between genes. HAS2-AS1 was markedly upregulated in glioma, and the overall survival time of patients with high HAS2-AS1 expression was significantly shorter than that of patients with low one. Silencing HAS2-AS1 inhibited the migration and invasion of glioma cells, while overexpressing HAS2-AS1 produced opposite results. miR-137 was validated as a direct target of and negatively regulated by HAS2-AS1. Further exploration of the downstream target gene indicated that EZH2 competed with HAS2-AS1 to interact with miR-137. Suppressing miR-137 or up-regulating EZH2 reversed the impact of HAS2-AS1 knockdown on glioma cell invasion and migration. HAS2-AS1 regulates EZH2 by sponging miR-137 for the migratory and invasive abilities of glioma cells, which provides a new idea for exploring metastasis mechanism of glioma.

Hepatocellular carcinoma (HCC) is a type of common cancer, often accompanied by tumor recurrence and metastasis after surgery with poor prognosis. Therefore, searching into potential biomarkers that can effectively predict the prognosis and progression of HCC is crucial. In this study, we identified 1,981 differentially expressed genes (DEGs) using mRNA expression profiles from the TCGA-LIHC dataset. Subsequently, weighted gene co-expression network analysis found that the turquoise module closely associated with the pathological grade and clinical stage of HCC was identified. Then, from the key genes in the turquoise module and protein-protein interaction network analysis, 13 hub genes significantly related to the prognosis of HCC were screened. Through co-expression and functional enrichment analyses, these 13 hub genes were found to play an important role in mitosis. Finally, we evaluated the relationship between these hub genes and overall survival and disease-free survival through survival analysis. The result indicated that HCC patients with high hub gene expression had a poorer prognosis than HCC patients with low expression. Receiver operating characteristic curves showed that each hub gene could predict the prognosis of HCC patients. In summary, a total of 13 hub genes were identified that play an important role in the progression of HCC, which can be used as potential biomarkers for HCC patients.

Rationale: The progression of prostate cancer (PCa) to castration-resistant PCa (CRPC) despite continuous androgen deprivation therapy is a major clinical challenge. Over 90% of patients with CRPC exhibit sustained androgen receptor (AR) signaling. KDM4B that removes the repressive mark H3K9me3/2 is a transcriptional activator of AR and has been implicated in the development of CRPC. However, the mechanisms of KDM4B involvement in CRPC remain largely unknown. Here, we sought to demonstrate the molecular pathway mediated by KDM4B in CRPC and to provide proof-of-concept evidence that KDM4B is a potential CRPC target. Methods: CRPC cells (C4-2B or CWR22Rv1) depleted with KDM4B followed by cell proliferation (in vitro and xenograft), microarray, qRT-PCR, Seahorse Flux, and metabolomic analyses were employed to identify the expression and metabolic profiles mediated by KDM4B. Immunoprecipitation was used to determine the KDM4B-c-Myc interaction region. Reporter activity assay and ChIP analysis were used to characterize the KDM4B-c-Myc complex-mediated mechanistic actions. The clinical relevance between KDM4B and c-Myc was determined using UCSC Xena analysis and immunohistochemistry. Results: We showed that KDM4B knockdown impaired CRPC proliferation, switched Warburg to OXPHOS metabolism, and suppressed gene expressions including those targeted by c-Myc. We further demonstrated that KDM4B physically interacted with c-Myc and they were co-recruited to the c-Myc-binding sequence on the promoters of metabolic genes (LDHA, ENO1, and PFK). Importantly, KDM4B and c-Myc synergistically promoted the transactivation of the LDHA promoter in a demethylase-dependent manner. We also provided evidence that KDM4B and c-Myc are co-expressed in PCa tissue and that high expression of both is associated with worse clinical outcome. Conclusions: KDM4B partners with c-Myc and serves as a coactivator of c-Myc to directly enhance c-Myc-mediated metabolism, hence promoting CRPC progression. Targeting KDM4B is thus an alternative therapeutic strategy for advanced prostate cancers driven by c-Myc and AR.

Objective. Protease-activated receptor-2 (PAR2) also known as F2RL1 is a G protein-coupled receptor that intimately correlates with cancer occurrence. DNA methylation turns out a vital mechanism regulating gene expression, while PAR2 promoter methylation is proven to be involved in cancer development. Hence, this study attempted to clarify the molecular mechanism by which PAR2 mediates lung adenocarcinoma (LUAD) progression, via identifying the effect of PAR2 promoter methylation on LUAD cell progression. Methods. Associations of PAR2 promoter methylation with PAR2 gene expression and prognosis of LUAD patients were analyzed via bioinformatics analysis. PAR2 promoter methylation and gene expression at the cellular level were measured using methylation-specific PCR, qRT-PCR, and Western blot assays. DNA methyltransferase inhibitor 5-AzadC was used to treat cells to assess PAR2 gene expression alteration. Cell biological behaviors upon PAR2 overexpression were characterized via MTT, wound healing assay, and Transwell assay. Results. Bioinformatics analysis revealed that PAR2 promoter methylation was negatively related to PAR2 gene expression, while PAR2 promoter hypermethylation and low gene expression indicated favorable LUAD prognosis. Besides, it turned out that PAR2 presented upregulated expression and hypomethylated promoter in LUAD cells. Moreover, PAR2 gene expression was elevated in cells treated with 5-AzadC, and the proliferative, migratory, and invasive capabilities of cells with 5-AzadC or high PAR2 gene expression were all enhanced. Conclusion. In sum, PAR2 promoter hypomethylation potentiates LUAD cell progression, in turn affecting the prognosis of LUAD patients.

Lung cancer is a highly prevalent type of cancer with a poor 5-year survival rate of about 4–17%. Eighty percent lung cancer belongs to non-small-cell lung cancer (NSCLC). For a long time, the treatment of NSCLC has been mostly guided by tumor stage, and there has been no significant difference between the therapy strategy of lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (SCLC), the two major subtypes of NSCLC. In recent years, important molecular differences between LUAD and SCLC are increasingly identified, indicating that targeted therapy will be more and more histologically specific in the future. To investigate the LUAD and SCLC difference on multi-omics scale, we analyzed the methylation and gene expression data together. With the Boruta method to remove irrelevant features and the MCFS (Monte Carlo Feature Selection) method to identify the significantly important features, we identified 113 key methylation features and 23 key gene expression features. HNF1B and TP63 were found to be dysfunctional on both methylation and gene expression levels. The experimentally determined interaction network suggested that TP63 may play an important role in connecting methylation genes and expression genes. Many of the discovered signature genes have been supported by literature. Our results may provide directions of precision diagnosis and therapy of LUAD and SCLC.

Neuropeptide B (NPB) is an endogenous ligand for the orphan G protein-coupled receptors NPBWR1 (GPR7) and NPBWR2 (GPR8). Some reports have investigated the role of NPB in the regulation of feeding, energy metabolism and hormone secretion in many species. However, few papers reported the physiological function of NPB in the pig. In this study, we cloned and sequenced the NPB mRNA from a pig, which was found to consist of 123 bases. NPB mRNA expression was detected in central and peripheral tissues by the quantitative fluorescence method. The results showed that NPB mRNA expression was higher in hippocampus, cerebellum, spinal cord, thymus, tonsil, duodenum, cecum, colon, ovary and testis. The distribution of NPB suggested that it may be involved in the regulation of reproductive functions in the pig. Subsequently, the expression and distribution of NPBWR1 and NPBWR2 were found in Leydig cells and ovarian granular cells. We then investigated the direct effect of NPB on pig reproductive cells in vitro. The results showed that different concentrations of NPB (10-12, 10-10, 10-8 and 10-6 M) promoted the secretion of testosterone in Leydig cells in concentration-dependent manner. Different doses of NPB could promote the secretion of progesterone in ovarian granulosa cells in dose-dependent manner. Low concentrations of NPB (10-8 and 10-10 M) promoted estradiol secretion, but high concentrations of NPB (10-6 M) inhibited its secretion. All the results suggested that the NPB/NPBWR1 or NPBWR2 system may play a role in modulating the reproductive activity in the pig.

Objective Glioma is a common primary malignant brain tumor characterized by high mortality and poor prognosis. The purpose of this study is to explore the molecular mechanism underlying glioma, aiming to provide a new target for the treatment of glioma to improve the prognosis of patients. Methods The differentially expressed genes and regulatory axis affecting the prognosis of glioma were identified with bioinformatics analysis, and the expression of miR-433-3p and SMC4 mRNA was detected with qRT-PCR. The expression of SMC4 and epithelial-mesenchymal transition (EMT)-associated proteins were detected with western blot. The targeting relationship between miR-433-3p and SMC4 was verified with dual-luciferase reporter gene assay. The proliferative ability of glioma cells was detected with CCK-8 assay, while the migration and invasion of glioma cells were detected with Transwell assay. Results We found that the expression of SMC4 was significantly up-regulated in glioma, showing that SMC4 was an unfavorable factor for prognosis and could promote the progression of cancer cells. Its upstream regulator miR-433-3p was significantly down-regulated in glioma, which inhibited the development of cancer cells. Moreover, miR-433-3p could target to inhibit the expression of SMC4. Rescue assay showed that miR-433-3p could affect the development of glioma by regulating the expression of SMC4. Conclusion Our data demonstrate for the first time that SMC4 is a direct target of miR-433-3p, and elucidate the molecular mechanism by which miR-433-3p inhibits the malignant progression of glioma by targeting and down-regulating the expression of SMC4.

Mulberry is a widespread and important economic crop in China. Cutting is a suitable method for large-scale propagation of fine cultivars, but it is limited by the low rate of adventitious root (AR) formation. Currently, we found large variations of R2R3-MYB genes during AR formation in Morus alba. There are a few studies on this issue, and therefore a systematic research is needed to improve the cuttage technique. The Hidden Markov Model (HMM) was built and applied in searching MYB genes. Phylogenetic relationships with subtribe designations and clustering expression of R2R3-MYB proteins were obtained referring to the transcriptome sequencing data in the normal cutting process. In addition, hormone trials with exogenous hormone treatment were performed to observe R2R3-MYB gene expression levels by quantitative real-time PCR (qRT-PCR) along with physiological changes during AR formation. In this study, we first identified 116 R2R3-MYB family genes of 247 MYB genes in mulberry (M. alba). They are clustered into 51 subtribes according to the structures of motifs and phylogenetic classes. On the basis of data analysis, the majority of R2R3-MYB genes play roles as activating transcription factors in the cortex during AR formation, which synchronously participate in many related signal pathways. Our results showed the R2R3-MYB genes have a detailed classification and express differently in the process of cutting, indicating that this gene family plays important roles during AR formation and functions differently in response to the corresponding signal pathways.

Objective The study aimed to explore the changes in mitochondrial DNA (mtDNA) copy number, collagen, and matrix metalloproteinase (MMPs) expression with pelvic organ prolapse (POP) in the uterosacral ligaments of premenopausal women. Materials and methods A group of 56 premenopausal women, all younger than 52 years of age, were enrolled in this study. Uterosacral ligament (UL) biopsies were obtained from uterine specimens taken from 22 women with POP ( n = 22, study group) and 34 myoma patients without POP ( n = 34, control group) during abdominal or vaginal hysterectomy. Quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemistry analysis were applied in the present study. Results The rate of high body mass index (BMI) (> 24 kg/m 2 ) women was significantly higher in the POP group (81.8% vs. 35.3%, p = 0.001 *), and the BMI of the POP women was higher than that of the nonPOP women ( p = 0.029 *). The mtDNA copy number ( p = 0.001 *), collagen III alpha 1 (COL3α1) expression ( p = 0.025 *), and MMP2 expression ( p = 0.047 *) were significantly higher in the POP group when compared with the nonPOP group. The high BMI women had a higher mtDNA copy number ( p = 0.002 *), COL3α1 ( p = 0.028 *) gene expressions compared with the standard BMI women. Conclusion In the premenopausal state, higher BMI may be a stronger associate factor than vaginal birth for the development of POP. The higher mtDNA copy number, COL3α1, and MMP2 gene expressions are highly associated with POP in the UL of premenopausal women.

Mulberry is an important economic crop spread widely in China for long time, and cutting is the most suitable method for good varieties in large scale propagation, which is limited by the low adventitious rooting rate. Currently, the regulation mechanisms in adventitious roots (AR) formation of mulberry (Morus alba L.) are not completely known. A genome-wide gene expression profiling was conducted by Solexa sequencing at pivotal stages. Differentially expressed annotated genes were obtained from the analysis of Gene Orthology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Orthology (KO). Some related genes were selected to confirm the altered expression levels by quantitative real-time PCR (qRT-PCR). By transcript profiling analysis we found differentially expressed genes associated with common pathways including hormone signal transduction, cell cycle, carbohydrate metabolism and energy metabolism that play important roles in AR formation (M. alba L), which were found more obvious in the period of root primordia formation, not initial formation of callus. In addition, the qRT-PCR confirmations agreed well with the results. The period of root primordia formation was verified to be more important during the whole procedure of AR formation in cuttings, mainly regulated by several critical signal pathways of hormones and effected by other genes in cell cycle and energy metabolism.

The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), (3)H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro.

The long noncoding RNA HOTAIR has been reported to be a good biomarker for poor prognosis in a variety of human cancers. However, whether HOTAIR could serve as novel biomarker to predict prognosis in cervical cancer or not is unknown. The aim of the present study was to examine the expression of HOTAIR in cervical cancers and to investigate the relationship between this lncRNA expression levels and existing clinicopathological factors and patient survival. We examined the expression of HOTAIR in 218 cervical cancer tissues and matched 218 adjacent normal tissues using quantitative real-time RT-PCR and analyzed its correlation with the clinical parameters. The results showed that HOTAIR expression in cervical cancer tissues was significantly upregulated compared with the matched nontumorous tissues (P < 0.0001). Increased HOTAIR expression was significantly correlated with FIGO stage (P < 0.0001), lymph node metastasis (P < 0.0001), depth of cervical invasion (P < 0.0001), tumor size (P = 0.006) and age (P = 0.020), but not other clinical characteristics. Moreover, cervical cancer patients with HOTAIR higher expression have shown significantly poorer overall survival (P < 0.0001) and disease-free survival (P < 0.0001) than those with lower HOTAIR expression. Univariate (P < 0.0001, HR = 4.566, 95 % CI 2.122–9.825) and multivariate (P = 0.012, HR = 2.863, 95 % CI 1.263–76.490). Cox regression analyses showed that HOTAIR expression served as an independent predictor for overall survival. our data indicate that high expression of HOTAIR is involved in cervical cancer progression and could be a potential target for diagnosis and gene therapy.

Protein–protein interactions constitute the regulatory network that coordinates diverse cellular functions. Co-immunoprecipitation (co-IP) is a widely used and effective technique to study protein–protein interactions in living cells. However, the time and cost for the preparation of a highly specific antibody is the major disadvantage associated with this technique. In the present study, a co-IP system was developed to detect protein–protein interactions based on an improved protoplast transient expression system by using commercially available antibodies. This co-IP system eliminates the need for specific antibody preparation and transgenic plant production. Leaf sheaths of rice green seedlings were used for the protoplast transient expression system which demonstrated high transformation and co-transformation efficiencies of plasmids. The transient expression system developed by this study is suitable for subcellular localization and protein detection. This work provides a rapid, reliable, and cost-effective system to study transient gene expression, protein subcellular localization, and characterization of protein–protein interactions in vivo.

The aim of this study was to investigate the effect of hyaluronic acid (HA) on the expression of osteopontin (OPN) and CD44 mRNA of fibroblast-like synoviocytes (FLS) in the patients with osteoarthritis (OA) of the knee. FLS were obtained from the knees of 10 patients with OA. Real-time quantitative polymerase chain reaction (Q-PCR) was used to assess the expression of the OPN mRNA and CD44 mRNA. The relative OPN mRNA expression in HA group (6.47 ± 2.30-fold) was significantly higher than in the control group (P = 0.045, P0.05), while in HYL group (0.65 ± 0.21-fold) it was lower than in the control group (P = 0.037, P0.05), and the difference in OPN mRNA expression between HA group and HYL group also showed statistically significant (P = 0.001, P0.05); however, there was no significant difference between each group of the relative CD44 mRNA expression (P0.05). Our study showed that HA can upregulate OPN mRNA expression in OA fibroblast-like synoviocytes, and the high expression of OPN mRNA in OA may be a result of increased HA level of OA synovitis; however, HA cannot affect the CD44 mRNA expression in OA fibroblast-like synoviocytes, and the high expression of CD44 mRNA in OA may be not a result of increased HA level of OA synovitis.

Huntington disease (HD) is a degenerative disorder caused by expanded CAG repeats in exon 1 of the huntingtin gene (HTT). Patients with late-stage HD are known to have abnormal auditory processing, but the peripheral auditory functions of HD patients have yet to be thoroughly assessed. In this study, 19 HD patients (aged 40–59 years) were assessed for hearing impairment using pure-tone audiometry and assessment of auditory brainstem responses (ABRs). PTA thresholds were markedly elevated in HD patients. Consistent with this, elevated ABR thresholds were also detected in two mouse models of HD. Hearing loss thus appears to be an authentic symptom of HD. Immunohistochemical analyses demonstrated the presence of mutant huntingtin that formed intranuclear inclusions in the organ of Corti of HD mice, which might interfere with normal auditory function. Quantitative RT-PCR and Western blot analyses further revealed reduced expression of brain creatine kinase (CKB), a major enzyme responsible for ATP regeneration via the phosphocreatine–creatine kinase (PCr-CK) system, in the cochlea of HD mice. Treatment with creatine supplements ameliorated the hearing impairment of HD mice, suggesting that the impaired PCr-CK system in the cochlea of HD mice may contribute to their hearing impairment. These data also suggest that creatine may be useful for treating the hearing abnormalities of patients with HD.

The RNA helicase Vasa is a member of the DEAD box protein family that plays an indispensable role in germ cell determination in eukaryotes such as in Drosophila and Xenopus species. In this study, the grass carp homologue of the Drosophila vasa gene, Civasa (Ctenopharyngodon idella vasa) was obtained using degenerate primers in RT-PCR and 5′ and 3′ rapid amplification of cDNA end polymerase chain reaction from the grass carp ovary SMART cDNA. This cDNA sequence encodes a 670 amino acid residue protein that contains eight consensus regions for the DEAD box protein family, 9 arginine–glycine repeats and 9 arginine–glycine–glycine repeats, a common character of known Vasa homologues. CiVasa shows high identity to that of zebrafish and other animals, suggesting Vasa is highly conserved through evolution. RT-PCR analysis reveals that in grass carp tissues, both ovaries and testes contain large amounts of vasa gene transcripts whereas no Civasa transcript is detected in somatic tissues examined. The Civasa transcripts are present at a high level from the 2 cell stage to gastrula stages which indicated that Cicasa transcripts are maternally inherited. The predicted protein sequence, localization and conserved pattern of gene expression suggest that Civasa plays an important role in the germ cell determinant and development in grass carp as proposed for other teleost species.

Cardiac hypertrophy and failure are characterized by transcriptional reprogramming of gene expression. Adult cardiomyocytes in mice primarily express alpha-myosin heavy chain (alpha-MHC, also known as Myh6), whereas embryonic cardiomyocytes express beta-MHC (also known as Myh7). Cardiac stress triggers adult hearts to undergo hypertrophy and a shift from alpha-MHC to fetal beta-MHC expression. Here we show that Brg1, a chromatin-remodelling protein, has a critical role in regulating cardiac growth, differentiation and gene expression. In embryos, Brg1 promotes myocyte proliferation by maintaining Bmp10 and suppressing p57(kip2) expression. It preserves fetal cardiac differentiation by interacting with histone deacetylase (HDAC) and poly (ADP ribose) polymerase (PARP) to repress alpha-MHC and activate beta-MHC. In adults, Brg1 (also known as Smarca4) is turned off in cardiomyocytes. It is reactivated by cardiac stresses and forms a complex with its embryonic partners, HDAC and PARP, to induce a pathological alpha-MHC to beta-MHC shift. Preventing Brg1 re-expression decreases hypertrophy and reverses this MHC switch. BRG1 is activated in certain patients with hypertrophic cardiomyopathy, its level correlating with disease severity and MHC changes. Our studies show that Brg1 maintains cardiomyocytes in an embryonic state, and demonstrate an epigenetic mechanism by which three classes of chromatin-modifying factors-Brg1, HDAC and PARP-cooperate to control developmental and pathological gene expression.

BACKGROUND Current staging methods are inadequate for predicting the outcome of treatment of non-small-cell lung cancer (NSCLC). We developed a five-gene signature that is closely associated with survival of patients with NSCLC. METHODS We used computer-generated random numbers to assign 185 frozen specimens for microarray analysis, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, or both. We studied gene expression in frozen specimens of lung-cancer tissue from 125 randomly selected patients who had undergone surgical resection of NSCLC and evaluated the association between the level of expression and survival. We used risk scores and decision-tree analysis to develop a gene-expression model for the prediction of the outcome of treatment of NSCLC. For validation, we used randomly assigned specimens from 60 other patients. RESULTS Sixteen genes that correlated with survival among patients with NSCLC were identified by analyzing microarray data and risk scores. We selected five genes (DUSP6, MMD, STAT1, ERBB3, and LCK) for RT-PCR and decision-tree analysis. The five-gene signature was an independent predictor of relapse-free and overall survival. We validated the model with data from an independent cohort of 60 patients with NSCLC and with a set of published microarray data from 86 patients with NSCLC. CONCLUSIONS Our five-gene signature is closely associated with relapse-free and overall survival among patients with NSCLC.

Purpose: Mass spectrometry–based biomarker discovery has clinical benefit. To identify novel biomarkers for urothelial carcinoma, we performed quantitative proteomics on pooled urine pairs from patients with and without urothelial carcinoma. Experimental Design: Shot-gun proteomics using liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling identified 219 candidate proteins. The potential implication of SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) was examined by immunoblotting of the urine (n = 13) and urothelial tumors (n = 32). Additional immunohistochemistry was performed on bladder cancer array (n = 1145) and correlated with tumor aggressiveness. Then, biologic functions and signaling pathways of SH3BGRL3 were explored using stable cell lines. Results: The detectable urine SH3BGRL3 in patients with urothelial carcinoma was positively associated with higher histologic grading and muscle invasiveness of urothelial carcinoma. SH3BGRL3 is expressed in 13.9% (159/1145) of bladder cancer cohort and is positively associated with muscle invasion (P = 0.0028). SH3BGRL3 expression is associated with increased risk of progression in patients with nonmuscle-invasive bladder cancer (P = 0.032). SH3BGRL3 expression is significantly associated with a high level of epidermal growth factor receptor (EGFR) in bladder cancer (P < 0.0001). SH3BGRL3 promotes the epithelial–mesenchymal transition, cell migration, and proliferation of urothelial carcinoma in vitro. SH3BGRL3 interacts with phosphor-EGFR at Y1068, Y1086, and Y1173 through Grb2 by its proline-rich motif, and activates the Akt-associated signaling pathway. Conclusions: Evaluation of SH3BGRL3 expression status or urine content may identify a subset of patients with bladder cancer who may require more intensive treatment. SH3BGRL3 deserves further investigation as a cotargeting candidate for designing EGFR-based cancer therapies. Clin Cancer Res; 21(24); 5601–11. ©2015 AACR.

Atrial fibrillation (AF) is associated with structural remodeling in atrial myocytes. Emerging evidence suggests that statin has a protective effect on AF through cholesterol-independent mechanisms. The aim of this study is to investigate whether heme oxygenase-1 (HO-1), a potent antioxidant system, mediates the suppressive effect of statin on atrial tachycardia-induced structural remodeling. Treatment of cultured atrium-derived myocytes (HL-1 cell line) with rosuvastatin enhanced HO-1 expression/activity and attenuated tachypacing-induced oxidative stress and myofibril degradation. Heme oxygenase-1 inhibitors and small-interfering RNA for HO-1 blocked the inhibitory effect of rosuvastatin on tachypacing-stimulated changes, suggesting the crucial role of HO-1 in mediating the effect of rosuvastatin. Time-dependent experiments and loss-of-function study demonstrated that Akt/Nrf2 pathways lay to the up-stream of HO-1 in this signaling cascade. Furthermore, the involvement of Akt/Nrf2/HO-1 pathway in the antioxidant effect of rosuvastatin was documented in an ex vivo tachypacing model. The suppressive effect of statin on atrial tachypacing-induced cellular remodeling is mediated via the activation of Akt/Nrf2/HO-1 signaling, which provides a possible explanation for the protective effect of statin on AF.

Regulated expression systems are invaluable for studying gene function, offer advantages of dosage-dependent and temporally defined gene expression, and limit possible clonal variation when toxic or pleiotropic genes are overexpressed. Previously, establishment of inducible expression systems, such as tetracycline- and ecdysone-inducible systems, required assessment of the inducible characteristics of individual clones by tedious luciferase assays. Taking advantage of a green fluorescent protein (GFP) reporter controlled by tetracycline- or ecdysone-responsive element and fluorescence-activated cell sorting, we propose a simple and efficient strategy to select highly inducible cell lines according to their fluorescence profiles after transiently transfecting the candidate cell pools with a surrogate GFP reporter. We have demonstrated that tetracycline- and ecdysone-inducible systems could be set up in Madin-Darby canine kidney and HEK-293 cells by employing this selection scheme. Importantly, this dual regulatory expression system is applied in studying the complex interplay between two Ras-related small GTPases, Cdc42 and Rac1, on detachment-induced apoptosis. Furthermore, establishment of two tightly regulated expression systems in one target cell line could be of great advantage for dissecting small GTPase Rac1-transduced signaling pathways by using global gene expression approaches such as proteomic assays.

The root of Rehmannia glutinosa (Rehmanniae Radix (RR)) is clinically used as a wound-healing agent in traditional Chinese medicine. Angiogenesis acts crucially in the pathogenesis of chronic wound healing. The present study investigated the angiogenesis effect and its underlying mechanism of RR through zebrafish sprout angiogenesis guided-fractionation.The in vivo angiogenesis effect was studied by analyzing the number of ectopic sprouts formed upon sub-intestinal vessel of transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by fluorescence microscopy. Quantitative real-time PCR gene expression of the zebrafish embryos was further performed using a panel of 30 angiogenesis-associated genes designed for zebrafish sprout angiogenesis. Classical in vitro angiogenesis assays using human microvascular endothelial cells (HMEC-1) was accompanied.We demonstrated that among all RR sub-fractions tested, C1-1 treated-zebrafish embryos possessed the most potent angiogenesis activities (from 190 to 780 ng/ml, p0.001) in sprout formation in the zebrafish model. Quantitative gene expression of the treated embryos demonstrated significant up-regulation in MMP-9 (p0.05), ANGPT1 (p0.05), EGFR (p0.05), EPHB4 (p0.01), and significant down-regulation in Ephrin B2 (p0.05), Flt-1 (p0.05) and Ets-1 (p0.05). C1-1 treatment could also significantly (p0.001-0.05) stimulate HMEC-1 cell migration in scratch assay. Significant increase (p0.05) in mean tubule length was observed in the C1-1-treated HMEC-1 cells in the tubule formation assay.Our zebrafish sprout angiogenesis model-guided fractionation revealed that C1-1 possessed the most potent angiogenesis effect in RR. The design of the panel with 30 tailor-made angiogenesis-associated genes exhibited in zebrafish gene expression analysis showed that C1-1 could trigger differential expression of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played key role in angiogenesis. The pro-angiogenic activity of C1-1 was further confirmed in the translated study in motogenic and tubule-inducing effect using HMEC-1.

The initiation of the immune response is regulated, in part, by the effect of cytokines on the level of expression of major histocompatibility complex (MHC) class II antigens on antigen-presenting cells (APC). The expression of class II antigens on B cell and macrophage APC is induced by IFN-gamma and IL-4, and GM-CSF induces class II expression on macrophages (M phi). Our results show that transforming growth factor-beta (TGF-beta) inhibits IL-4- and GM-CSF-induced Ia gene expression on bone marrow macrophages but enhances IFN-gamma-induced gene expression. Nuclear run-on experiments demonstrated that the inhibitory effects of TGF-beta on GM-CSF- and IL-4-induced Ia antigen expression were primarily posttranscriptional and augmentation of IFN-gamma by TGF-beta was largely transcriptionally regulated.

The transforming growth factor beta family of peptides have diverse actions on the reproductive tracts of primates and rodents. In this study we report the expression of high levels of mRNA of one member of this superfamily, TGF-beta 2, in the pregnant mouse uterus. Using Northern blot analysis and in situ hybridization techniques, we have examined the pattern of expression of TGF-beta 1, TGF-beta 2 and colony-stimulating factor (CSF-1) in mouse maternal and fetal tissue at specific days of gestation. We report here that TGF-beta 2 is synthesized primarily in maternal decidual and uterine epithelial tissues. We observed a shift in the major site of synthesis from decidua to uterus between days 8.5 and 10.5 of gestation. These data demonstrate that the expression of TGF-beta 2 is differentially regulated in the decidua and uterine epithelial cells at various times during gestation. Small amounts of TGF-beta 2 mRNAs were detected in the fetus, and none was detected in placenta, yolk sac, or amniotic membrane. The uterus is likely the major site of synthesis of the TGF-beta 2 found in mouse amniotic fluid. TGF-beta 1 mRNAs are expressed in the uterus at markedly lower levels when compared to TGF-beta 2 mRNAs in both the decidua and uterus. Our results suggest that there is a unique regulation of TGF-beta 2 during pregnancy which may depend on pregnancy hormone(s) and differentiates it from the other mammalian isoforms of the TGF-beta s. TGF-beta 2 may play an important, albeit unknown, role at the maternal/fetal interface.

Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66∼70% of total cell material of these recombinant strains.

We established Hep3B cells stably-expressing wild-type and mutated AMF/PGI with differing enzymatic activities in order to investigate how AMF/PGI affects TGF-beta-induced apoptosis, and demonstrated that AMF/PGI against TGF-beta-induced apoptosis was correlated with its enzymatic activity. AMF/PGI did not alter TGF-beta-receptor expression nor affect TGF-beta-induced PAI-1 gene promoter or Smad3/4 activity. AMF/PGI induced PI 3-kinase activity, IRS and Akt phosphorylation, which can further regulate BAD phosphorylation. Constitutively-active p110 enhanced AMF/PGI-mediated anti-apoptosis activity, and dominant negative Akt alleviated anti-TGF-beta-induced apoptosis. We also demonstrated that STAT3 is a weak anti-apoptotic agent but has an increased anti-apoptotic effect in cooperation with PI 3-kinase/Akt.

DHEA is known to have anti-proliferative effect. The mechanism is not completely understood. We investigated the mechanism underlying DHEA-induced growth arrest of hepatoma cells. Growth inhibition was associated with increased G6PD activity, and insensitive to reversal by mevalonate. Thus, DHEA does not act via inhibition of G6PD and HMGR. Instead, growth stagnation was accompanied by reduced expression of nucleus-encoded mitochondrial genes; morphological and functional alterations of mitochondria; and depletion of intracellular ATP. Conversely, pyruvate supplementation alleviated DHEA-induced growth inhibition. It is likely that DHEA suppresses cell growth by altering mitochondrial gene expression, morphology and functions.

Multiple xylanolytic enzymes of Streptomyces thermonitrificans NTU-88 were induced by oat-spelt xylan and separated by two-dimensional polyacrylamide and zymogram gels. Nineteen clear spots differed in pI and molecular weight values were found on the zymogram, and only spot one was seen on the corresponding silver-stained gel. These results revealed that multiple xylanases were secreted when S. thermonitrificans NTU-88 was induced and the spot (STXF10), identified as being a glycosyl hydrolase family 10 xylanase, was the predominant one among xylanases. STXF10 showed a tolerance for high temperatures and broad pH ranges and high affinity and hydrolysis efficiency for xylans. Furthermore, it also featured the minor ability to degrade different lignocellulosic substrates. Although S. thermonitrificans NTU-88 possesses multiple xylanases, our results suggest that the major form of xylanase might be selectively and specifically induced depending on the type of substrate to which the microorganism is exposed.

Background The tetracycline-regulated transcriptional silencer (tTS) has been demonstrated to mitigate leaky expression of the tetracycline-inducible promoter under uninduced condition, and, when conjugated with reverse-type tetracycline-controlled transactivator (rtTA), shows great promise for gene therapy. This effect was attributed to the effectiveness of tTS as a repressor of transcription at the tetracycline-regulated promoter. However, we observed an unexpected increase in transactivational activity by rtTA in the presence of tTS under inducible condition. Methods To explore the nature of this co-activational effect of tTS on rtTA, we examined the expression patterns of rtTA by Western blotting analysis of total cellular lysates or an enriched ubiquitinated pool of proteins under various conditions, including the one when proteasomal degradation is inhibited. Results We demonstrate tTS, in addition to its established role as a transcriptional silencer, can enhance rtTA expression level by salvaging rtTA from the ubiquitin-dependent proteasomal degradation pathway. Along with this finding, we also demonstrate that doxycycline, a commonly used tetracycline analogue, inhibits the susceptibility of rtTA to ubiquitin/proteasome-mediated degradation and enhances the expression level of rtTA. Conclusions Taken together, our data establish an unappreciated role of doxycycline and tTS in tetracycline-regulated gene expression and the functionality of rtTA, and should shed light on the design of gene therapy vectors based on tetracycline-controlled transcriptional regulation systems. Copyright © 2004 John Wiley & Sons, Ltd.

Oxidative stress has been implicated in aging and pathogenesis of cancer and degenerative diseases. Cells are equipped with various antioxidant systems, some of which depend on NADPH for ultimate reducing equivalents. Nicotinamide nucleotide transhydrogenase represents an important source of NADPH in mitochondria. The physiological role of transhydrogenase in cell remains elusive. In the present study, we generated stable SK-Hep1 clones which express shRNA against NNT. The growth rate and colony forming ability of NNT knockdown cells were lower than those expressing scrambled shRNA controls. The migration and invasion abilities of the knockdown cells were lower than the control. When transplanted subcutaneously in immunodeficient mice, the NNT knockdown cells did not form tumors readily as the control cells did. The growth slowdown was accompanied by an increase in reactive oxygen species (ROS) regeneration as well as oxygen consumption and energy charge. But there are no differences of mitochondrial mass and membrane potential. In microarray experiment, we found that NNT deficiency decrease many tumorgenetic gene expression, such as DMBT1, MACC1, CABL2 and AHRR. These findings suggest that deficiency in NNT may cause growth cessation as well as increased ROS generation. Abstract

The XR-1 Chinese hamster ovary cell line is impaired in DNA double-strand break repair (DSBR) and inability to support V(D)J recombination of transiently introduced substrates. We now show that XR-1 cells support recombination-activating gene 1- and 2-mediated initiation of V(D)J recombination within a chromosomally integrated substrate, but are highly impaired in ability to complete the process by forming coding and recognition sequence joins. On this basis, we isolated a human cDNA sequence, termed XRCC4 , whose expression confers normal V(D)J recombination ability and significant restoration of DSBR activity to XR-1, clearly demonstrating that this gene product is involved in both processes. The XRCC4 gene maps to the previously identified locus on human chromosome 5, is deleted in XR-1 cells, and encodes a ubiquitously expressed product unrelated to any described protein.

Background Transient receptor potential channel vanilloid subfamily member-1 (TRPV1) may play a role in esophageal perception. TRPV1 mRNA and protein expression were examined in the esophageal mucosa of non-erosive reflux disease (NERD) and erosive esophagitis (EE) patients and correlated to esophageal acid exposure. Methods Seventeen NERD patients, eight EE patients and 10 healthy subjects underwent endoscopy after a 3-week washout from proton pump inhibitors or H2 antagonists. Biopsies, obtained from the distal esophagus, were used for conventional histology, for Western blot analysis and/ or quantitative real-time polymerase chain reaction (qPCR). Overall 13 NERD patients, four EE patients and five controls underwent ambulatory pH-testing. Key Results TRPV1 expression was increased in all NERD and EE patients, as measured by Western blot analysis (0.65 ± 0.07 and 0.8 ± 0.05 VS 0.34 ± 0.04 in controls; P < 0.01) and by qPCR (1.98 ± 0.21 and 2.52 ± 0.46 VS 1.00 ± 0.06; P < 0.01). Neutrophilic infiltration, in the mucosa, was detected only in EE patients. Conclusions & Inferences Non-erosive reflux disease and EE patients presented increased TRPV1 receptors mRNA and protein, although no correlation with acid exposure was demonstrated. Increased TRPV1 in the esophageal mucosa may contribute to symptoms both in NERD and EE patients and possibly account for peripheral mechanisms responsible for esophageal hypersensitivity in NERD patients.

Immunoglobulin gene expression requires that cells combine the correct genetic information by DNA rearrangement and RNA processing to produce mature, translatable mRNAs. The mechanisms that operate to produce IgD heavy chain (δ chain) mRNA are thought to be especially complex1–3. Like other immunoglobulins, IgD exists in membrane and secreted forms4,5. By analogy with results of studies of μ6–10 and γ11,12 gene expression, we expected that δ chains would also be encoded by two different mRNAs, containing common sequence information transcribed from constant region gene segments but different information transcribed from coding sequences located 3′ to the constant region gene segments. Instead, we identified13 more than two δ mRNAs in normal mouse spleen and in two IgD-secreting plasmacytomas14, TEPC 1017 and TEPC 1033. Now we have used 32P-labelled DNA fragments prepared from a δ cDNA clone15 and a δ genomic clone16 to characterize by hybridization these δ RNAs, fractionated on methyl mercury hydroxide agarose gels. We find that normal mouse spleen contains a major 2.9-kilobase (kb) and a minor 2.1-kb RNA encoding membrane-bound δ chains (δm) and that TEPC 1017 and TEPC 1033 contain similar δm RNAs plus a 1.75-kb RNA encoding secreted δ chains (δm). We have also observed less abundant δ RNA species (2.65 and 3.2 kb). Different gene segments or combinations of segments are used in the 3′ termini of the multiple δ RNAs.