Prothymosin Alpha Expression Research Articles

Circular RNA COL1A1 promotes Warburg effect and tumor growth in nasopharyngeal carcinoma

ObjectiveCircular RNAs (circRNAs), pivotal in the pathogenesis and progression of nasopharyngeal carcinoma (NPC), remain a significant point of investigation for potential therapeutic interventions. Our research was driven by the objective to decipher the roles and underlying mechanisms of hsa_circ_0044569 (circCOL1A1) in governing the malignant phenotypes and the Warburg effect in NPC.MethodsWe systematically collected samples from NPC tissues and normal nasopharyngeal epithelial counterparts. The expression levels of circCOL1A1, microRNA-370-5p (miR-370-5p), and prothymosin alpha (PTMA) were quantitatively determined using quantitative polymerase chain reaction (qPCR) and Western blotting. Transfections in NPC cell lines were conducted using small interfering RNAs (siRNAs) or vectors carrying the pcDNA 3.1 construct for overexpression studies. We interrogated the circCOL1A1/miR-370-5p/PTMA axis's role in cellular functions through a series of assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide for cell viability, colony formation for growth, Transwell assays for migration and invasion, and Western blotting for protein expression profiling. To elucidate the molecular interactions, we employed luciferase reporter assays and RNA immunoprecipitation techniques.ResultsOur investigations revealed that circCOL1A1 was a stable circRNA, highly expressed in both NPC tissues and derived cell lines. A correlation analysis with clinical pathological features demonstrated a significant association between circCOL1A1 expression, lymph node metastasis, and the tumor node metastasis staging system of NPC. Functionally, silencing circCOL1A1 led to substantial suppression of cell proliferation, migration, invasion, and metabolic alterations characteristic of the Warburg effect in NPC cells. At the molecular level, circCOL1A1 appeared to modulate PTMA expression by acting as a competitive endogenous RNA or 'sponge' for miR-370-5p, which in turn promoted the malignant characteristics of NPC cells.ConclusionTo conclude, our findings delineate that circCOL1A1 exerts its oncogenic influence in NPC through the modulation of the miR-370-5p/PTMA signaling axis.

Circular RNA DNAH14 molecular mechanism in an experimental model of hepatocellular carcinoma treated with Cobalt chloride to mimic the hypoxia-like response of transcatheter arterial chemoembolization

Transcatheter arterial chemoembolization (TACE) is the primary local treatment for patients with unresectable hepatocellular carcinoma (HCC). Numerous studies have demonstrated the pivotal role of circular RNAs (circRNAs) in TACE efficacy. This study aimed to investigate the function of circular RNA DNAH14 (circDNAH14) in TACE for HCC and to elucidate its molecular mechanisms. To simulate hypoxia conditions experienced during TACE, HCC cells were treated with cobalt chloride. The expression levels of circDNAH14, microRNA-508-3p (miR-508-3p), and Prothymosin Alpha (PTMA) were modulated via transfection for knockdown or overexpression. Cell Counting Kit-8 and 5-ethynyl-2′-deoxyuridine assays, flow cytometry, and Transwell assays, along with epithelial-mesenchymal transition (EMT) evaluations, were employed to assess cell proliferation, apoptosis, invasion, migration, and EMT. The results indicated that hypoxia treatment downregulated the expression of circDNAH14 and PTMA while upregulating miR-508-3p. Such treatment suppressed HCC cell proliferation, invasion, migration, and EMT, and induced apoptosis. Knockdown of circDNAH14 or PTMA intensified the suppressive effects of hypoxia on the malignant behaviors of HCC cells. Conversely, upregulation of miR-508-3p or PTMA mitigated the effects of circDNAH14 overexpression and knockdown, respectively. Mechanistically, circDNAH14 was found to competitively bind to miR-508-3p, thereby regulating PTMA expression. In vivo, nude mouse xenograft experiments demonstrated that circDNAH14 knockdown augmented the hypoxia-induced suppression of HCC tumor growth. In conclusion, circDNAH14 mitigates the suppressive effects of hypoxia on HCC, both in vitro and in vivo, by competitively binding to miR-508-3p and regulating PTMA expression.

PTMA binds to HMGB1 to regulate mitochondrial oxidative phosphorylation and thus affect the malignant progression of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor of the digestive tract with complex pathogenesis. There is a pressing need to search for ESCC targeted therapy sites and explore its pathogenesis. Prothymosin alpha (PTMA) is abnormally expressed in numerous tumors and has a significant regulatory effect on tumor malignant progression. However, the regulatory role and mechanism of PTMA in ESCC have not yet been reported. We first detected the PTMA expression in ESCC patients, subcutaneous tumor xenograft models of ESCC, and ESCC cells. Subsequently, PTMA expression in ESCC cells was inhibited by cell transfection, and cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) staining, flow cytometry, and Western blot. A dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to detect reactive oxygen species (ROS) level in cells, and MitoSOX fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl carbocyanine iodide (JC-1) staining, mitochondrial complex kit, and Western blot were used to detect the expression of mitochondrial oxidative phosphorylation. Next, the combination between PTMA and high mobility group box 1 (HMGB1) was detected using Co-immunoprecipitation (co-IP) and immunofluorescence (IF) techniques. Finally, the expression of PTMA was inhibited and the expression of HMGB1 was overexpressed in cells via cell transfection, and the regulatory effect of PTMA and HMGB1 binding on mitochondrial oxidative phosphorylation in ESCC was determined through related experiments. The expression of PTMA in ESCC was abnormally elevated. The inhibition of PTMA expression in ESCC cells significantly decreased the activity of ESCC cells and increased their apoptosis. Moreover, interference with PTMA can induce ROS aggregation in ESCC cells by inhibiting mitochondrial oxidative phosphorylation, which may be achieved by binding to HMGB1. PTMA binds to HMGB1 to regulate mitochondrial oxidative phosphorylation, thereby affecting the malignant progression of ESCC.

Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB)

BackgroundEsophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.MethodsIn this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.ResultsIn total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.ConclusionIn this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

PO-116 Increase in prothymosin alpha expression with histological aggressiveness of glioma

IntroductionWhile much progress has been made in melanoma treatment in the recent years, melanoma still remains a generally lethal disease at the metastatic stage. Therefore, discovery of additional critical target genes and pathways, and the development of novel therapeutic approaches remain a priority. Like most cancers, abnormal gene regulatory and epigenetic mechanisms are also linked to melanomas.The transcription factor interferon regulatory factor 4 (IRF4) was discovered as a factor implicated in immunoglobulin expression in B-cells, and was later shown to perform central roles in immune cell development and function. We have previously demonstrated the critical role of IRF4 in a variety of B-cell cancer cells, and identified its mechanisms of action. These and related work suggested IRF4 pathways as therapy targets in cancer. Several studies implicate IRF4 expression and function also in non-immune cells, such as melanocytes. For instance, a number of genome-wide genetic studies associated variation at the IRF4 gene with pigmentation phenotypes and skin cancers. However, despite the observed genetic links and generally high expression, the role of IRF4 in melanoma remains under-studied and poorly understood.Material and methodsWe set out to identify the functions of IRF4 in melanoma cells using genome-wide and cell and molecular biological approaches. We have taken a candidate approach to discover the upstream modulators of IRF4 expression in melanoma cells. In parallel, we have performed localization (ChIP-seq) and transcriptomic (RNA-seq) assays in order to identify the genome-wide targets of IRF4 in melanoma cell lines.Results and discussionsAs upstream regulators of IRF4 expression, we identified a known melanoma master transcription factor and a major signalling pathway with therapeutic targeting options. For downstream targets of IRF4, together with The Cancer Genome Atlas (TCGA) data analyses, our data point to a role of IRF4 in epigenetic regulation of melanoma cells, among other cancer- and development-related pathways. In addition, our preliminary studies implicate IRF4 as a critical factor in melanoma cell proliferation and survival.ConclusionOverall these studies on IRF4 in melanoma cells are complementing mechanistically the available genetic findings in melanoma patient samples, and describing a novel pathway in melanoma cells, with the potential to point to new therapy approaches for melanoma.

PO-119 Ferritin heavy subunit enhances apoptosis of non-small cell lung cancer cells through modulation of a miR-125B/P53 axis

IntroductionGliomas are the most common primary brain tumours accounting for 2.5% of the total cancer deaths worldwide. c-Myc is a proto-oncogene, it plays an important role in initiation and progression of gliomas. It upregulates expression of nuclear onco-protein prothymosin alpha (PTMA). PTMA is a transcription factor with a spectrum of functions ranging from cell cycle regulator to immunomodulatory function depending upon its distribution. Its overexpression is reported in malignancies of breast, liver, lung and bladder.Material and methodsThis study was conducted on patients who underwent elective surgery for glioma (n=176). The histological grading and MIB1 labelling index was recorded from surgical pathology reports. Expression levels of PTMA in these tissues were assessed by immunohistochemistry.Results and discussionsOur study population consisted of 30 (17%) Grade I, 56 (31.8%) Grade II, 45 (25.6%) Grade III and 45 (25.6%) Grade IV glioma cases. PTMA expression was significantly associated with the histological grades of glioma (p<0.0001, Kruskal-Wallis test) and proliferative capacity assessed by MIB1 labelling index (p=0.0125, Kruskal-Wallis test). PTMA expression was not associated with age, sex and the site of the glioma. Though c-Myc expression was not done in this study previous studies showed its expression was associated with the histological grades of glioma.ConclusionOur study demonstrates the involvement of PTMA overexpression in the pathogenesis of glioma and its association with histological grading and proliferation. Differential expression of this protein suggests its potential utility in histological grading and prognosis of glioma.

Identification of the β-catenin/JNK/prothymosin-alpha axis as a novel target of sorafenib in hepatocellular carcinoma cells.

Sorafenib is a kinase inhibitor used as anticancer drug against various human tumors, including advanced hepatocellular carcinoma (HCC). β-Catenin and prothymosin alpha (PTMA) are overexpressed in HCC and other tumors. Previous studies have shown that PTMA expression modulates the response of HCC cells to sorafenib. However, the underlying mechanism of PTMA activity in this context remains unclear. We show here that sorafenib inhibits both β-catenin and PTMA in a dose-dependent manner. Silencing β-catenin reduces PTMA level and sensitizes HCC cells to sorafenib. In contrast, ectopic expression of β-catenin induces PTMA expression and cell resistance to the drug. Sorafenib inhibits PTMA expression at the transcriptional level by inhibiting the β-catenin pathway. Nucleotide deletion analysis of the PTMA gene promoter reveals that a DNA segment lying 1,500-1,600 bp upstream of the PTMA transcription start site represents an AP-1-binding site that is critical for β-catenin modulation of gene transcription in response to sorafenib. In addition, chemical inhibitors that target JNK abrogate β-catenin/AP-1 binding to the endogenous PTMA gene and reduces PTMA transcription and protein expression. Silencing of β-catenin or c-Fos induces similar effects on gene regulation and these are reversed by ectopic expression of β-catenin. Mutations in the PTMA promoter at the predicted β-catenin/AP-1 binding site partly abrogate sorafenib's effects on PTMA transcription. These results indicate that PTMA is induced by the oncoprotein β-catenin and protects HCC cells against sorafenib-induced cell death. The β-catenin/JNK/PTMA axis may thus represent a novel target for chemotherapy against HCC.

Prothymosin alpha expression and localization during the spermatogenesis of Danio rerio.

Prothymosin α (PTMA) is a highly acidic, intrinsically disordered protein, which is widely expressed and conserved throughout evolution; its uncommon features are reflected by its involvement in a variety of processes, including chromatin remodelling, transcriptional regulation, cell proliferation and death, immunity. PTMA has also been implicated in spermatogenesis: during vertebrate germ cell progression in the testis the protein is expressed in meiotic and post-meiotic stages, and it is associated with the acrosome system of the differentiating spermatids in mammals. Then, it finally localizes on the inner acrosomal membrane of the mature spermatozoa, suggesting its possible role in both the maturation and function of the gametes. In the present work we studied PTMA expression during the spermatogenesis of the adult zebrafish, a species in which two paralogs have been described. Our data show that ptma transcripts are expressed in the testis, and localize in meiotic and post-meiotic germ cells, namely spermatocytes and spermatids. Consistently, the protein is expressed in spermatocytes, spermatids, and spermatozoa: its initial perinuclear distribution is extended to the chromatin region during cell division and, in haploid phases, to the cytoplasm of the developing and final gametes. The nuclear localization in the acrosome-lacking spermatozoa suggests a role for PTMA in chromatin remodelling during gamete differentiation. These data further provide a compelling starting point for the study of PTMA functions during vertebrate fertilization.

Expression of prothymosin α predicts early recurrence and poor prognosis of hepatocellular carcinoma

Prothymosin alpha (PTMA) is a nuclear oncoprotein-transcription factor essential for cell cycle progression and proliferation. PTMA was overexpressed in several human malignancies including hepatocellular carcinoma (HCC). However, the prognostic significance of PTMA protein expression in HCC remains unclear. In the present study, we evaluated PTMA protein expression by immunohistochemistry in order to elucidate the prognostic roles of PTMA in HCC patients. By immunohistochemistry, we investigated the expression of PTMA protein in tumor tissue from 226 HCC patients who underwent curative hepatectomy. Univariate and multivariate analyses were performed to evaluate its predictive value for tumor recurrence and survival of patients. The median follow-up period was 120 months. PTMA expression was observed in 162 (71.7%) of the 226 HCC patients and was significantly associated with higher Edmondson grade, microvascular invasion, intrahepatic metastasis, higher American Joint Committee on Cancer (AJCC) T-stage, and lower albumin level. PTMA expression was an independent predictor of early recurrence (P=0.001). PTMA expression showed an unfavorable influence on recurrence-free survival (RFS) (P<0.001). Subgroup analysis showed that among patients with tumor size ≤5.0 cm (140 patients), patients at AJCC T-stage 1 (95 patients) and patients with alpha-fetoprotein ≤20 ng/mL (83 patients), the differences in RFS between PTMA-positive and PTMA-negative groups were also statistically significant (P=0.017, P=0.002 and P=0.002, respectively). In addition, PTMA expression was an independent predictor of shorter RFS (P=0.011). PTMA expression showed an unfavorable influence on overall survival (P=0.014), but was not an independent predictor of shorter overall survival (P=0.161). PTMA protein expression might be a novel predictor of early recurrence and RFS in HCC patients, even those at early stage or with alpha-fetoprotein-negative after curative hepatectomy. PTMA could be used as an immunohistochemical biomarker to detect patients with a high risk of recurrence.

Oncogenic c-Myc and prothymosin-alpha protect hepatocellular carcinoma cells against sorafenib-induced apoptosis

Prothymosin alpha (PTMA) is overexpressed in various human tumors, including hepatocellular carcinoma (HCC). The significance of PTMA overexpression and its underlying mechanism remain unclear. We show here that silencing PTMA sensitizes HCC cells to the kinase inhibitor sorafenib. In contrast, ectopic expression of PTMA induces cell resistance to the drug. While inhibitors targeting JNK, ERK or PI3K reduce PTMA expression, only ERK activation is suppressed by sorafenib. In addition, inhibition of ERK produces a dramatic decrease in both endogenous PTMA level and promoter activation. Ectopic expression of active MKK1/2 considerably induces PTMA expression. We also identify a sorafenib-responsive segment lying 1000–1500-bp upstream of the PTMA transcription start site and observe that it is controlled by c-Myc and ERK. Mutation in the PTMA promoter at the predicted c-Myc binding site and silencing of c-Myc both abrogate sorafenib's effect on PTMA transcription. We also find that silencing PTMA potentiates Bax translocation to mitochondria in response to sorafenib and this is associated with increased cytochrome c release from mitochondria and enhanced caspase-9 activation. These results indicate that PTMA is positively regulated by the oncoprotein c-Myc and protects HCC cells against sorafenib-induced cell death, thus identifying PTMA as a new target for chemotherapy against HCC.

Abstract 194: MicroRNA-1 induces apoptosis by targeting prothymosin alpha in nasopharyngeal carcinoma cells

Abstract MiR-1 (microRNA-1) can be used as a positive control and for optimizing transfection conditions in microRNA experiments (e.g., Ambion Pre-miR miRNA Starter Kit, QIAGEN Syn-hsa-miR-1 miScript miRNA mimic positive control). We noticed that transfection of nasopharyngeal carcinoma cells with miR-1 reveals a typical apoptotic process when observed by time-lapse microscopy. Annexin V and TUNEL staining and caspase assay confirm that miR-1 induces nasopharyngeal carcinoma cell apoptosis. MiR-1 transfection of HeLa, Cal-27, KYSE30 and NPC-TW06 cell lines which express low levels of endogenous miR-1 also induces apoptosis. However, miR-1 transfection of cell lines such as SW620, HepG2, HEK-293T, SAS and PC-13 which express high levels of endogenous miR-1 does not induce apoptosis. To determine miR-1 target genes that are involved in apoptosis, we analyzed microRNA and pathway databases, and cDNA expression microarrays from miR-1 transfected cells. Using qRT-PCR analysis and luciferase reporter vector assays, we found that miR-1 directly targets PTMA (prothymosin alpha). We used PTMA siRNA to down regulate PTMA mRNA levels and observed whether PTMA siRNA could induce apoptosis. Knocking down PTMA expression alone did not induce apoptosis but could accelerate apoptotic progression in cells treated with apoptosis inducers. We also found that transfection of miR-1 could up regulated some pro-apoptotic proteins (HTRA, SMAC, p53, TNF-R1, TNF-R2, TRAILR-1, TNF-alpha, TNF-beta). They are not miR-1 directly target genes but involve in miR-1 inducing apoptosis. We conclude that miR-1 can induced apoptosis in low levels of endogenous miR-1 cell lines. This is a novel model of microRNA-induced cell apoptosis. The apoptotic inducers including actinomycin D, camptothecin and etoposide are also the chemotherapeutic drugs in clinical cancer therapy and PTMA siRNA can accelerate apoptotic progression in cells treated with those apoptosis inducers. Therefore PTMA siRNA may have potential applications as an adjuvant in cancer chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 194. doi:1538-7445.AM2012-194

Abstract 1103: Molecular targets regulated by tumor suppressive microRNA-1 and microRNA-133a in bladder cancer

Abstract (Background) Bladder cancer (BC) is the fourth most common tumor diagnosed and the second most common cancer of the genitourinary tract in Japan. Though there have been significant advances in treatment, including surgical technique and adjuvant chemotherapy, approximately 70% of first diagnosed BC recurs after treatment, and 10% to 15% of recurrent tumors proceed to muscle invasion and metastasis. Therefore, it is crucial to find a new strategy for BC diagnosis and treatment based on recent genome analysis in BC oncogenesis. 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, migration 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, migration 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 migration and invasion in cancer cells. Apoptosis assay showed that significant apoptotic cells were induced by miR-1 and/or miR-133a transfection. 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. Silencing of the target genes studies demonstrated significant inhibition of cell proliferation and invasion, and increase of apoptosis in BC cells. Immunohistochemistry showed that TAGLN2 and PTMA expression level was significantly higher in BC than normal bladder epitheliums. (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. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1103. doi:1538-7445.AM2012-1103

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 ...

Editorial Comment

Even representing a small percentage of all urothelial carcinomas, upper urinary tract transitional cell cancer (UUT-TCC) has, interestingly, a much greater incidence in Taiwan than worldwide. 1 Yang M.H. Chen K.K. Yen C.C. et al. Unusually high incidence of upper urinary tract urothelial carcinoma in Taiwan. Urology. 2002; 59: 681-687 Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar The tumor stage has been widely recognized as an important prognostic factor 2 Hall M.C. Womack S. Sagalowsky A.I. et al. Prognostic factors, recurrence, and survival in transitional cell carcinoma of the upper urinary tract: a 30-year experience in 252 patients. Urology. 1998; 52: 594-601 Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar ; however, to date, the definite risk factors for recurrence and survival for patients after radical nephroureterectomy remains unclear. This group of investigators had already reported on the potential of urine prothymosin-α (PTMA) as a specific tumor marker for the detection and monitoring of urothelial bladder cancer. 3 Tzai T. Tsai Y. Shiau A. et al. Urine prothymosin-alpha as novel tumor marker for detection and follow-up of bladder cancer. Urology. 2006; 67: 294-299 Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar The same tumor marker has been also investigated by others in prostate cancer. 4 Suzuki S. Takahashi S. Takahashi S. et al. Expression of prothymosin alpha is correlated with development and progression in human prostate cancers. Prostate. 2005; 66: 463-469 Crossref Scopus (37) Google Scholar Duplicating their previous findings in bladder cancerous tissue, 5 Tsai Y. Jou Y. Lee G. et al. Aberrant prothymosin-alpha expression in human bladder cancer. Urology. 2009; 73: 188-192 Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Tzai et al. 3 Tzai T. Tsai Y. Shiau A. et al. Urine prothymosin-alpha as novel tumor marker for detection and follow-up of bladder cancer. Urology. 2006; 67: 294-299 Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar report on the tissue expression of PTMA in UUT-TCC samples and its correlation with other variables and patient outcome. Despite the rather short follow-up and limited sample size, this study provides some intriguing information. First, PTMA is overexpressed in UUT-TCC cells. Second, no significant association was found between the intensity of PTMA expression and tumor stage or grade. Third, cytoplasmic PTMA-expressing tumors have a more advanced stage compared with nuclear PTMA-expressing tumors. Finally, cytoplasmic PTMA has an independent clinical significance in predicting for tumor recurrence in the residual urinary tract, mostly bladder cancer. How these findings will affect the current treatment of patients with UUT-TCC remains to be established. Additional clinical and translational research in this area is warranted. The role of PTMA in transitional cell carcinogenesis needs to be elucidated further and its potential role as a prognostic marker needs to be validated within larger samples with longer oncologic follow-up. Prognostic Relevance of Prothymosin-α Expression in Human Upper Urinary Tract Transitional Cell CarcinomaUrologyVol. 74Issue 4PreviewTo investigate the prognostic role of prothymosin-α (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC). Full-Text PDF

Prognostic Relevance of Prothymosin-α Expression in Human Upper Urinary Tract Transitional Cell Carcinoma

To investigate the prognostic role of prothymosin-alpha (PTMA) expression in human upper urinary tract transitional cell carcinoma (UUT-TCC). Paraffin-embedded tissues were collected from 91 patients with UUT-TCC and from 15 paired normal renal cortex and 13 paired urothelial walls. The primary antibody for PTMA (2F11) used was validated in 4 human urothelial cancer cell lines before assessing the surgical specimen. Immunohistochemistry was then conducted to determine the expression intensity of PTMA, the calculation of immunostaining density using imaging analysis, and for immunostaining localization. The correlates with clinicopathologic characteristics and patient survival were explored. The expression intensity of PTMA demonstrated a significant enhancement of PTMA expression in UUT-TCCs compared with both paired normal tissues (P = .0002 and P = .0004 for UUT-TCC vs the urothelial wall and vs the renal cortex, respectively). As for the localization of PTMA immunoreactivity, of the 91 tumor specimens, 33 (36.3%) were cytoplasmic PTMA-expressing, 51 (56.0%) were nuclear PTMA-expressing, and 7 (7.7%) were PTMA-negative tumors. On univariate and multivariate analyses, PTMA expression localization was the sole independent prognostic indicator for recurrence-free survival (hazard ratio 4.90, 95% confidence interval 1.73-13.9; P = .003), although pathologic staging was an independent prognostic indicator for both progression-free survival (hazard ratio 22.6, 95% confidence interval 2.56-198; P = .005) and disease-specific overall survival (hazard ratio 5.60, 95% confidence interval 1.48-21.2; P = .011). The limitations of our study included small patient numbers and short follow-up. The results of our study have shown that PTMA is overexpressed in UUT-TCCs and that cytoplasmic PTMA expression can provide significant prognostic information for subsequent tumor recurrence in the residual urinary tract after nephroureterectomy.

Aberrant Prothymosin-alpha Expression in Human Bladder Cancer

To investigate the prothymosin-alpha (PTMA) expression in human bladder cancer using tissue microarrays. Two tissue microarray slides of 50 bladder tumors and 42 paired normal adjacent tissues were investigated using immunohistochemical staining. The staining distribution was categorized as negative, nuclear, cytoplasmic, and mixed expression. Quantitative immunoreactivity was measured using image analysis, as represented by the integrated optical density for each tissue core. In 36 of 42 normal adjacent tissues, positive PTMA immunoreactivity could be seen in some nuclei of the normal urothelial cells, but not, or only minimally, in the cytoplasm and underlying submucosal tissues. A statistically significant enhancement of PTMA expression was found in bladder tumors of each grade compared with the normal adjacent tissue (P < .0001 for normal adjacent tissues vs grade 1, 2, or 3 tumors, paired t test). Of 48 transitional cell carcinoma specimens, only 4 (8.3%) were graded as negative and 44 (91.7%) were positive for PTMA expression, including nuclear (n = 8), cytoplasmic (n = 12), and mixed expression (n = 24) patterns. A statistically significant correlation was found between high grade and mixed expression (P = 0.0020, chi(2) test). Increased PTMA expression was found in human bladder cancers compared with the paired normal adjacent bladder tissue. The distribution of PTMA expression was changed in high-grade tumors. The clinical significance of such an aberrant PTMA expression in bladder cancer is worthy of additional investigation.

Apoptotic and proliferating hepatocytes differ in prothymosin α expression and cell localization

Prothymosin alpha is an acidic protein, reported to be involved in cell proliferation and apoptosis, although its precise function in both processes are still unknown. Due to the importance of these processes in the pathogenesis of hepatic diseases and the need to understand the molecular mechanisms underlying these diseases we aimed to investigate the behavior of this protein in liver growth and apoptosis, in two models of hepatocytes in culture. Prothymosin alpha expression varied throughout the hepatocyte cell cycle, according to its progression. Proliferating hepatocytes showed increased expression of the protein, while apoptotic ones showed decreased levels. The subcellular location of prothymosin alpha differed according to the different phases of the cell cycle. Thus, it appeared with a stippled and widely dispersed pattern throughout the nucleus in quiescent and proliferating hepatocytes, while it became cytoplasmic in mitotic and late apoptotic cells. These results are in agreement with the idea that high levels of prothymosin alpha need to be present in the nucleus for proliferation, and programmed cell death requires low levels of prothymosin alpha outside of the nucleus. The differences in prothymosin alpha expression and localization during hepatocyte proliferation and apoptosis suggest that this protein may have a pleiotropic function that depends not only on its availability but also on its various localizations in different subcellular compartments.

Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13-acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.

Expression of prothymosin alpha is correlated with development and progression in human prostate cancers

Our previous study clearly demonstrated that decreased expression of prothymosin alpha (PTMA) was associated with inhibition of rat prostate carcinogenesis by isoflavones. The purpose of the present investigation was to provide a better understanding of the role of PTMA in human prostate cancers. PTMA expression in 68 prostate cancer cases and in prostate cancer cell lines was examined by immunohistochemistry and immunoblotting, and its levels were increased with progression from normal epithelium, through prostatic intraepithelial neoplasia (PIN) to carcinomas, correlating with the Gleason's pattern. All cell lines studied (LNCaP, 22Rv1, DU145, and PC3) showed high PTMA expression compared with prostate epithelial cells (PrEC). Knockdown of PTMA expression in PC3 cells by RNAi resulted in the inhibition of both cell growth and invasion in vitro. The present study clearly demonstrated that PTMA expression is intimately involved in the differentiation and progression of human prostate cancers, and could be a target for therapy and diagnostic purposes.

Prothymosin Alpha Location in the Proliferative Compartments of the Digestive Tract Epithelium.

Prothymosin alpha is a polypeptide whose function is still a matter of debate. The present paper represents the first morphological description of the prothymosin alpha distribution in the human and rat digestive tract tissues using immunohistochemical techniques. In these epithelia prothymosin alpha immunostaining was localized in the nuclei of the proliferating cells, e.g. in the basal layer cells of the oesophagus, cells of the isthmus and the neck of the gastric glands, and crypt cells in the small and large intestines. At the ultrastructural level, prothymosin alpha immunolabelling was evident in the nucleus, being confined to euchromatin. The present data indicates that prothymosin alpha expression in the digestive tract epithelia is associated with proliferating cell populations. In agreement with this hypothesis, we found that the patterns of distribution of prothymosin alpha and proliferating cell nuclear antigen were similar. The distribution of prothymosin immunoreactivity was also similar to that obtained for bromodeoxyuridine when the animal was sacrificed 12hr after injection. The finding that intracellular distribution of the prothymosin alpha in the digestive tract epithelia is identical to that found in other tissues supports the idea that prothymosin alpha must play a universal role in proliferating cells.