Transgelin-2 Research Articles

882 MOLECULAR TARGETS REGULATED BY TUMOR SUPPRESSIVE MICRORNA-1 AND MICRORNA-133A IN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012882 MOLECULAR TARGETS REGULATED BY TUMOR SUPPRESSIVE MICRORNA-1 AND MICRORNA-133A IN BLADDER CANCER Takeshi Yamasaki, Hirofumi Yoshino, Hideki Enokida, Takeshi Chiyomaru, Hideo Hidaka, Nijirou Nohata, Naohiko Seki, and Masayuki Nakagawa Takeshi YamasakiTakeshi Yamasaki Kagoshima, Japan More articles by this author , Hirofumi YoshinoHirofumi Yoshino Kagoshima, Japan More articles by this author , Hideki EnokidaHideki Enokida Kagoshima, Japan More articles by this author , Takeshi ChiyomaruTakeshi Chiyomaru Kagoshima, Japan More articles by this author , Hideo HidakaHideo Hidaka Kagoshima, Japan More articles by this author , Nijirou NohataNijirou Nohata Chiba, Japan More articles by this author , Naohiko SekiNaohiko Seki Chiba, Japan More articles by this author , and Masayuki NakagawaMasayuki Nakagawa Kagoshima, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.977AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our expression signatures of human cancer including bladder cancer (BC) revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) were significantly reduced in cancer cells. In human genome, miR-1 and miR-133a located same chromosomal regions (miR-1-2 and miR-133a-1 on 18q11.2, and miR-1-1 and miR-133a-2 on 20q13.33) called cluster. In this study, we investigated the functional significance of miR-1 and miR-133a and identify the novel molecular targets regulated by miR-1 and miR-133a commonly in BC. METHODS Cell proliferation, invasion and apoptosis assay was performed by restoration of mature miR-1 and/or miR-133a in BC cell lines. Genome-wide gene expression analysis was performed to identify the molecular networks of miR-1 and miR-133a by microarray technique. A luciferase reporter assay was used to identify the actual binding site between miR-1 and miR-133a and its candidate target genes. Cell proliferation, invasion and apoptosis assays were performed to investigate the targets genes in BC cell lines. To investigate of expression of target genes, immunohistochemistry was performed by using BC tissue microarray. RESULTS Restoration of miR-1 and/or miR-133a significantly inhibited cell proliferation and invasion in cancer cells (P < 0.05). Apoptosis assay showed that significant apoptotic cells were induced by miR-1 and/or miR-133a transfection (P < 0.05). Genome-wide molecular targets search and luciferase reporter assay showed that Transgelin2 (TAGLN2), prothymosin-alpha (PTMA) and purine nucleoside phosphorylase (PNP) were directly regulated by miR-1 and miR-133a commonly (P <0.0001). Silencing of the target genes studies demonstrated significant inhibition of cell proliferation and invasion, and increase of apoptosis in BC cells (P < 0.05). Immunohistochemistry showed that TAGLN2 and PTMA expression level was significantly higher in BC than normal bladder epitheliums (respectively, P=0.0202 and P =0.0048). CONCLUSIONS miR-1 and miR-133a function as tumor suppressor in BC. TAGLN2, PTMA and PNP were directly regulated by tumor suppressive miR-1 and miR-133a commonly. These genes may function as oncogenes contributed to cell proliferation and invasion in BC. Tumor suppressive miR-1 and miR-133a mediates novel molecular targets provide new insights into the potential mechanisms of BC oncogenesis. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e358-e359 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeshi Yamasaki Kagoshima, Japan More articles by this author Hirofumi Yoshino Kagoshima, Japan More articles by this author Hideki Enokida Kagoshima, Japan More articles by this author Takeshi Chiyomaru Kagoshima, Japan More articles by this author Hideo Hidaka Kagoshima, Japan More articles by this author Nijirou Nohata Chiba, Japan More articles by this author Naohiko Seki Chiba, Japan More articles by this author Masayuki Nakagawa Kagoshima, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Tumor suppressive microRNA-133a regulates novel molecular networks in lung squamous cell carcinoma

Analysis of the microRNA (miRNA) expression signature of lung squamous cell carcinoma (lung-SCC) revealed that the expression levels of miR-133a were significantly reduced in cancer tissues compared with normal tissues. In this study, we focused on the functional significance of miR-133a in cancer cell lines derived from lung-SCC and the identification of miR-133a-regulated novel cancer networks in lung-SCC. Restoration of miR-133a expression in PC10 and H157 cell lines resulted in significant inhibition of cell proliferation, suggesting that miR-133a functions as a tumor suppressor. We used genome-wide gene expression analysis to identify the molecular targets of miR-133a regulation. Gene expression data and web-based searching revealed several candidate genes, including transgelin 2 (TAGLN2), actin-related protein2/3 complex, subunit 5, 16kDa (ARPC5), LAG1 homolog, ceramide synthase 2 (LASS2) and glutathione S-transferase pi 1 (GSTP1). ARPC5 and GSTP1 likely represent bona fide targets as their expression is elevated in lung-SCC clinical specimens. Furthermore, transient transfection of miR-133a, repressed ARPC5 and GSTP1 mRNA and protein levels. As cell proliferation was significantly inhibited in lung-SCC cells following RNAi knock down of either gene, ARPC5 and GSTP1 may function as oncogenes in the development of lung-SCC. The identification of a tumor suppressive miRNA and the novel cancer pathways it regulates could provide new insights into potential molecular mechanisms of lung-SCC carcinogenesis.

The functional significance of miR-1 and miR-133a in renal cell carcinoma

PurposeThe aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. MethodsWe checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. ResultsThe expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. ConclusionsOur data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

A novel interplay between oncogenic PFTK1 protein kinase and tumor suppressor TAGLN2 in the control of liver cancer cell motility

The PFTK1 gene encodes a cdc2-related serine/threonine protein kinase that has been shown to confer cell migratory properties in hepatocellular carcinoma (HCC). However, the prognostic value and biological mechanism by which PFTK1 promotes HCC motility remain largely unknown. Here, we showed from tissue microarray that common upregulations of PFTK1 in primary HCC tumors (n=133/180) correlated significantly with early age onset (40 years), advance tumor grading and presence of microvascular invasion (P0.05). To understand downstream phosphorylated substrate(s) of PFTK1, phospho-proteins in PFTK1 expressing and knockdown Hep3B cells were profiled by two-dimensional-polyacrylamide gel electrophoresis mass spectrometric analysis. Protein identification of differential spots revealed β-actin (ACTB) and transgelin2 (TAGLN2) as the two most profound phosphorylated changes affected by PFTK1. We verified the presence of TAGLN2 serine phosphorylation and ACTB tyrosine phosphorylation. Moreover, reduced TAGLN2 and ACTB phosphorylations in PFTK1-suppressed Hep3B corresponded to distinct actin depolymerizations and marked inhibition on cell invasion and motility. Given that TAGLN2 is a tumor suppressor whose function has been ascribed in cancer metastasis, we examined if TAGLN2 is an intermediate substrate in the biological path of PFTK1. We showed in PFTK1-suppressed cells that knockdown of TAGLN2 over-rode the inhibitory effect on cell invasion and motility, and a recovery on actin polymerization was evident. Interestingly, we also found that unphosphorylated TAGLN2 in PFTK1-suppressed cells elicited strong actin-binding ability, a mechanism that possibly halts the actin cytoskeleton dynamics. Site-directed mutagenesis of TAGLN2 suggested that PFTK1 regulates the actin-binding affinity of TAGLN2 through the S83 and S163 residues, which if mutated can significantly affect HCC cell motility. Taken together, our data propose a novel, oncogene-tumor suppressor interplay, where oncogenic PFTK1 confers HCC cell motility through inactivating the actin-binding motile suppressing function of TAGLN2 via phosphorylation.

The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer

Background:On the base of the microRNA (miRNA) expression signature of bladder cancer (BC), we found that miR-1 and miR-133a were significantly downregulated in BC. In this study, we focussed on the functional significance of miR-1 and miR-133a in BC cell lines and identified a molecular network of these miRNAs.Methods and results:We investigated the miRNA expression signature of BC clinical specimens and identified several downregulated miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370). MiR-1 and miR-133a showed potential role of tumour suppressors by functional analyses of BC cells such as cell proliferation, apoptosis, migration, and invasion assays. Molecular target searches of these miRNAs showed that transgelin 2 (TAGLN2) was directly regulated by both miR-1 and miR-133a. Silencing of TAGLN2 study demonstrated significant inhibitions of cell proliferation and increase of apoptosis in BC cell lines. The immunohistochemistry showed a positive correlation between TAGLN2 expression and tumour grade in clinical BC specimens.Conclusions:The downregulation of miR-1 and miR-133a was a frequent event in BC, and these miRNAs were recognised as tumour suppressive. TAGLN2 may be a target of both miRNAs and had a potential oncogenic function. Therefore, novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.

MiR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.

Abstract 5297: A novel role of cdc-family gene PFTK1 in the control of liver cancer cell motility

Abstract Cancer metastasis remains a major cause of cancer morbidity and mortality in individuals diagnosed with hepatocellular carcinoma (HCC). We previously reported on regional chromosome 7q21-q22 gains in close association with HCC progression, and discerned a candidate proto-oncogene PFTK1 within this region by array-based CGH mapping. The PFTK1 protein, PFTAIRE protein kinase 1, is a novel member of the Cdc2-related serine/threonine protein kinases. Our earlier investigations by ectopic expression and gene knockdown of PFTK1 confirmed a functional role for the PFTAIRE protein kinase in the motile phenotype of HCC cells, yet the biological basis remained to be determined. The aims of this study are therefore to establish the clinicopathologic significance of PFTK1 in HCC, and to define the PFTK1-modulated mechanisms in the control of HCC cell motility. Recent Tissue Microarray Analysis (TMA) on 180 paired primary HCC and their adjacent non-tumoral liver suggested common up-regulated PFTK1 compared to non-malignant counterpart (76.1%; p&amp;lt;0.0001). Correlative analysis further indicated PFTK1 over-expression in association with advanced tumor grading (P&amp;lt;0.0001) and the presence of microvascular invasion (P=0.05). By 2D-PAGE coupled with mass spectrometry, comparative proteomic profiling for phosphorylated proteins in PFTK1-suppressed HCC cells highlighted 5 differentially down-regulated spots, which included β-actin (ACTB), transgelin2 (TAGLN2), heat shock protein 70, mitochondrial aldehyde dehydrogenase and 14-3-3 gamma protein. Western blot analysis further verified a consistent reduction on ACTB phosphorylation and the serine phosphorylation of the TAGLN2 protein in the absence of PFTK1 protein kinase. Immunofluorescence analysis for cytoskeletal organizations indicated marked reduction on the actin stress fibers in PFTK1 knockdown cells. In conclusion, our results suggested that PFTK1 can affect the actin cytoskeletal organization and thus a motile phenotype in HCC cells through phosphorylation of ACTB and TAGLN2 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5297.

Identification of transgelin‐2 as a biomarker of colorectal cancer by laser capture microdissection and quantitative proteome analysis

To search for potential protein markers of colorectal cancer (CRC), the changes in protein expression levels between microdissected tumor cells and normal mucosa epithelia were analyzed by an acetylation stable isotopic labeling method coupled with linear quadrupole ion trap fourier transform mass spectrometry (LTQ-FTMS). In total, 137 proteins were up-regulated or down-regulated significantly in cancer by at least two-fold. Based on gene ontology analysis, the largest part of differential proteins were unknown for both subcellular localization and biological process. In particular, the significant up-regulation of transgelin-2 (TAGLN2) in CRC was validated by Western blot analysis and further evaluated by immunohistochemistry in paired tumor and normal mucosa samples from 120 consecutive CRC patients, 20 adenomas, and eight synchronous hepatic metastases of CRC. TAGLN2 expression was frequently observed in cancer cells, precancerous lesions, and hepatic metastases, whereas in normal epithelia expression was rarely observed. The overexpression of TAGLN2 was associated with lymph node and distant metastasis, advanced clinical stage (P < 0.001), and shorter overall survival in CRCs. Cox regression analysis indicated that high tumor-TAGLN2 expression represents an independent prognostic factor. Consequently, over-expression of TAGLN2 may serve as a new biomarker for predicting progression and prognosis of CRC.

Tissue Proteomics Reveals Differential and Compartment-Specific Expression of the Homologs Transgelin and Transgelin-2 in Lung Adenocarcinoma and Its Stroma

Discovery of tissue-specific biomarkers for human cancer is crucial for early diagnosis and molecular understanding of the disease. To overcome the limitations posed by the large dynamic concentration range and compositional complexity of tissue biomacromolecules, we applied heparin affinity fractionation for proteomic enrichment. Comparing the proteomes of five paired samples of normal lung and pulmonary adenocarcinoma tissue by 2-D difference gel electrophoresis, 14 spots were found to be differentially expressed. From these candidate spots, three proteins overexpressed in cancer were identified by mass spectrometry as transgelin (TAGLN, SM22-alpha, WS3-10), transgelin-2 (TAGLN2), and cyclophilin A (PPIA). Quantitative RT-PCR indicated that both TAGLN2 and PPIA were upregulated at the transcriptional level. Differential protein expression levels were validated by Western blot analysis using an independent set of 10 paired lung adenocarcinoma samples. Using immunohistochemistry on human tissue sections, we discovered that overexpression of TAGLN was strictly localized to the tumor-induced reactive myofibroblastic stromal tissue compartment, whereas overexpression of TAGLN2 was exclusively localized to the neoplastic glandular compartment. Thus, the highly homologous protein pair TAGLN and TAGLN2 displayed mutually exclusive, compartment-specific cell type expression regulation in tumor stroma vs neoplastic epithelial cells. Our data further suggest that TAGLN may be a marker of active stromal remodeling in the vicinity of invasive carcinomas. It may shed light on mechanisms of tumor-stroma interaction and could be useful for early diagnosis, treatment guidance, and treatment response monitoring.

Identificación de diferencias proteómicas en muestras oligozoospérmicas

ObjectivesOligozoospermia is one of the most common findings present in subfertile males, but its aetiology remains unknown in most cases. In this work we have used current proteomic tools to identify the different proteins present in oligozoospermic semen samples and therefore potentially involved in infertility. MethodsWe compared the expression of 75 sperm protein spots in 3 pools of 5 oligozoospermic samples to that of 5 semen donor controls using two-dimensional proteomic analysis. ResultsA total of14 protein spots have been identified with a > 2 fold different amount in the oligozoospermic samples as compared to controls. Proteins detected as increased are, creatine kinase B (CKB), histidine triad nucleotide-binding protein 1 (HINT1), succinyl CoA 3 ketoacid coenzyme A transferase1 precursor (OXCT1), transgelin-2 (TAGLN2), 2 spots corresponding to cytoskeletal actin (ACTB), glutathione S-transferase Mu 3 (GSTM3), annexin A5 (ANXA5), cyclin dependent kinase 5 (CDK5), and proteins detected as decreased are ATP synthase beta chain, mitochondrial precursor (ATP5B), outer dense fibre protein 2 (ODF2), tubulin beta subunit (TUBB2), capping protein (actin filament) muscle Z-line, beta (CAPZB), triosephosphate isomerase 1 (PTI1). DiscussionWe have identified several proteins present in different amounts in oligozoospermic sperm samples as compared to control samples. These proteins could be now be further considered as candidates for the development of diagnostic markers and open up the opportunity to gain further insight into the pathogenic mechanisms involved in oligozoospermia.

Label-free detection of cancer biomarker candidates using surface plasmon resonance imaging

In this work, we present an antibody array for the detection of cancer biomarker candidates by a surface plasmon resonance (SPR) imaging sensor with polarization contrast. Responses from the SPR imaging sensor are shown to be similar to those from a conventional spectroscopy-based SPR sensor. Antibodies are spotted onto a self-assembled monolayer (SAM) composed of oligo(ethylene glycol) (OEG)-containing alkanethiol chains. Detection of two cancer biomarker candidates, activated leukocyte cell adhesion molecule/CD 166 (ALCAM) and transgelin-2 (TAGLN2), is demonstrated. Limits of detection for ALCAM and TAGLN2 are established at 6 ng/mL and 3 ng/mL, respectively, in buffer. No cross-reactivity is observed between immobilized antibodies and nonspecific antigen. Biomarker candidates are also detected in a 10% human serum solution.

Correlation between genomic DNA copy number alterations and transcriptional expression in hepatitis B virus-associated hepatocellular carcinoma

Human hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, in which the genetic mechanisms of oncogenesis are still unclear. To investigate whether the genomic DNA copy number alterations may contribute to primary HCC, the cDNA microarray-based comparative genomic hybridization (CGH) analysis was here performed in 41 primary HCC infected by hepatitis B virus and 12 HCC cell lines. The resulting data showed that, on average, 7.25% of genome-wide DNA copy numbers was significantly altered in those samples (4.61 ± 2.49% gained and 2.64 ± 1.78% lost). Gains involving 1q, 6p, 8q and 9p were frequently observed in these cases; and whilst, losses involving Ip, 16q and 19p occurred in most patients. To address the correlation between the alteration of genomic DNA copy numbers and transcriptional expression, the same cDNA microarray was further applied in 20 HCC specimens and all available cell lines to figure out the gene expression profiles of those samples. Interestingly, the genomic DNA copy number alterations of most genes appeared not to be in generally parallel with the corresponding transcriptional expression. However, the transcriptional deregulation of a few genes, such as osteopontin (SPP1), transgelin 2 (TAGLN2) and PEG10, could be ascribed partially to their genomic aberrations, although the many alternative mechanisms could be involved in the deregulation of these genes. In general, this work would provide new insights into the genetic mechanisms in hepatocarcinogenesis associated with hepatitis B virus through the comprehensive survey on correlation between genomic DNA copy number alterations and transcriptional expression.