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463 TUMOR SUPPRESSIVE MICRORNA-138 CONTRIBUTES TO INHIBITION OF BOTH CELL MIGRATION AND INVASION THROUGH TARGETING VIMENTIN IN RENAL CELL CARCINOMA
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
rationale for alterations of metabolism in the context of kidney cancer, limited data exists characterizing whether these changes do exist in ccRCC tissues. METHODS: We therefore undertook a dual analysis of both the metabolome (i.e. small molecules) and the transcriptome in order to comprehensively characterize the metabolism of ccRCC which represents the first such analysis for any tumor type. Small molecule analysis was performed via Gas-Chromatography/ Mass Spectrometry (GC-MS) as well as Liquid-Chromatography/ Mass Spectrometry (LCMS). Transcriptional analysis was performed via mRNA microarray analysis and confirmatory real time RT-PCR. RESULTS: First, our data are the first to validate metabolomics as applied to renal cancer. Second, our data demonstrate significant changes in the transcriptional profile of ccRCC at the levels of glycolysis, gluconeogenesis, and oxidative phosphorylation consistent with a glygolytic phenotype. Third, these changes were consistent with our small molecule analysis and therefore demonstrate a correlation between transcription and metabolic pathway utilization. CONCLUSIONS: Collectively, the data support a unifying model of ccRCC metabolism which further reinforces the association between altered metabolism and malignancy. Source of Funding: NIH/NCI Astellas/AUA Foundation Rising Star Award
461 MICRORNA-29 FAMILY AS TUMOR SUPPRESSIVE MICRORNAS IN RENAL CELL CARCINOMA: MICRORNA-29A INHIBITS CELL MIGRATION AND INVASION TARGETING FOCAL ADHESION AND ECM PATHWAYS Tomokazu Yonezawa*, Hideki Enokida, Hirofumi Yoshino, Hideo Hidaka, Takeshi Yamasaki, Toshihiko Itesako, Kaghosima city, Japan; Naohiko Seki, Chiba city, Japan; Masayuki Nakagawa, Kaghosima city, Japan INTRODUCTION AND OBJECTIVES: A growing body of evidence suggests that microRNAs (miRNAs) contribute to human cancer progression, development, and metastasis, including renal cell carcinoma (RCC). Our previous miRNA expression signature revealed that miR-29-family (miR-29a/b/c) was significantly reduced in clinical RCC specimens. The aim of the study was to investigate the functional significance of miR-29-family and to explore the novel molecular pathways and responsibility genes regulated by miR-29a/b/c in RCC. METHODS: Gain-of-function studies were performed to investigate cancer cell proliferation, migration and invasion by mature miRNAs transfection into RCC cell lines (A498 and 786-O). To identify the biological processes or pathways potentially regulated by the miR29-family, we applied genome-wide gene expression analysis and in silico study; TargetScan database and GENECODIS software, which assigned a number of the putative miRNA targets to known pathways in KEGG [http://www.genome.jp/kegg/pathway.html]. The expression levels of miR-29-family target genes were verified using public database of Gene Expression Omnibus (GEO) in clinical RCCs. In these microarray expression data, we examined 78 clinical RCCs and 18 normal kidney tissues. RESULTS: Expression levels of miR-29-family were significantly reduced in clinical RCCs compared to adjacent non-cancerous tissues (P⬍0.001). Restoration of each miR-29-family significantly inhibited cancer cell proliferation, migration, and invasion in the RCC cell lines. The expression and in silico analysis showed that miR-29-family appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in ‘focal adhesion’ and ‘ECM (extra-cellular matrix)-receptor interaction’ signaling pathways (P⬍0.05). Among two pathways, 45 genes were putative targets regulated by miR-29-family (29a/b/c). Gene expression data showed that three genes (COL5A, LAMC1, and VEGFA) were significantly up-regulated in clinical RCCs compared with normal kidney tissues.
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CONCLUSIONS: miR-29-family functioned as pivotal suppressor of cell migration and invasion in RCC through targeting ‘focal adhesion’ and ‘ECM’ signaling pathways. Elucidation of tumor suppressive miR-29-family-mediated cancer pathways and putative targets might be provided the novel molecular mechanisms to understand tumor progression or distant metastasis of RCC. Source of Funding: None
462 TUMOR SUPPRESSIVE MICRORNA-218 REGULATES ONCOGENES OF FOCAL ADHESION PATHWAYS IN RENAL CELL CARCINOMA Takeshi Yamasaki*, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Hirofumi Yoshino, Hideo Hidaka, Toshihiko Idesako, Hideki Enokida, Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent microRNA (miRNA) expression signature of renal cell carcinoma (RCC) revealed downregulation of miR-218 in RCC, suggesting that it might be a tumor suppressor. The aim of this study is to investigate the functional significance of miR-218 and to identify miR-218-mediated cancer pathways in RCC. METHODS: We evaluated miR-218 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We performed gain-of-function studies (cell migration and cell invasion assays) by using miR-218 transfectants (TFs). Oligo-microarray analyses of the miR-218 TFs and the RCC specimens were carried out to identify molecular targets of miR-218. According to GENECODIS software, we focused on the ‘focal adhesion’ pathway, in which caveolin-2 (CAV2) was a candidate target gene. A luciferase reporter assay was performed to determine miR-218 binding sites with CAV2 3’UTR. Loss-of-function assays using si-CAV2 were performed to investigate the functional significance of CAV2. The expression of CAV2 mRNA and protein was evaluated by qRT-PCR and immunohistochemistry. Furthermore, to investigate signaling pathways regulated by CAV2, we performed gene expression analysis of si-CAV2 TFs. RESULTS: The expression of miR-218 was significantly reduced in the RCC specimens. Significant inhibitions of cell migration and invasion were observed in the miR-218 TFs. Gene expression analysis showed that 615 genes were downregulated in the miR-218 TFs. The GENECODIS software revealed that miR-218 appeared to regulate 25 pathways (P⬍0.0001). In these pathways, we focused on ten genes in ‘focal adhesion’ and seven genes in ‘tight junction’. CAV2 was the most upregulated gene among the genes related to these pathways by using available data sets (GSE36895 and GSE22541) in gene expression omnibus (GEO). Luciferase reporter assay showed that CAV2 was directly regulated by miR-218. Silencing study of CAV2 demonstrated significant inhibition of cell migration and invasion. The mRNA and protein expression of CAV2 were significantly up-regulated in RCC specimens. Gene expression analysis of si-CAV2 TFs revealed that CAV2 might control 15 pathways including ‘tight junction’ pathway, containing pro-metastatic genes such as claudin-1, AKT3 and R-Ras2. CONCLUSIONS: Tumor suppressive miR-218 contributes to inhibition of cell migration and invasion through regulating focal adhesion pathway, especially CAV2 in RCC. This is the first report that CAV2 contributes to metastatic processes in RCC. Source of Funding: None
463 TUMOR SUPPRESSIVE MICRORNA-138 CONTRIBUTES TO INHIBITION OF BOTH CELL MIGRATION AND INVASION THROUGH TARGETING VIMENTIN IN RENAL CELL CARCINOMA Takeshi Yamasaki*, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Hirofumi Yoshino, Hideo Hidaka, Hideki Enokida, Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent miRNA expression signature of renal cell carcinoma (RCC) revealed that mi-
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croRNA-138 (miR-138) expression was significantly reduced in RCC, suggesting that miR-138 was a candidate tumor suppressor. Recent publications showed that miR-138 might regulate genes related to epithelial-mesenchymal transition (EMT) in several cancers. The aim of study is to investigate the functional significance of miR-138 and to identify its target genes in RCC. METHODS: We evaluated miR-138 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We observed morphologic changes in the miR-138-transfected RCC cell lines (A498 and 786-O). We performed gain-of-function studies such as cell migration and cell invasion assays by using miR-138 transfectants (TFs). Gene expression analysis of the miR-138 TFs and RCC clinical specimens by oligo-microarray was carried out to identify molecular targets of miR-138. TargetScan database implied that vimentin (VIM), an EMT-related gene, was a promising candidate target gene of miR-138. We investigated whether VIM was regulated by miR-138 by real-time PCR and Western blotting. Loss-of-function studies using si-VIM were performed to investigate the functional significance of VIM. We evaluated the expression levels of both VIM mRNA and protein by qRT-PCR and immunohistochemistry. RESULTS: The expression levels of miR-138 were significantly reduced in the RCC specimens compared with the normal tissues. Restoration of mature miR-138 in A498 and 786-O caused changes in the bleb-like cell morphology, as characteristics of EMT. Significant inhibitions of cell migration and cell invasion were observed in the miR-138 TFs. The expression levels of VIM expression was suppressed in the miR-138 TFs at both mRNA and protein levels. Lossof-function studies demonstrated that significant inhibition of cell migration and invasion occurred in the si-VIM transfectants. In clinical RCC specimens, the expression level of VIM was significantly upregulated in comparison with adjacent normal tissues. Furthermore, immunohistochemistry showed that VIM expression levels in RCC specimens were significantly higher than those in the normal tissues. CONCLUSIONS: These data suggest that VIM might function as an oncogene and might be regulated by tumor suppressive miR138. Tumor suppressive miR-138-mediated EMT pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis. Source of Funding: None
464 TUMOR-SUPPRESSIVE MIR-135A INHIBITS CANCER CELL PROLIFERATION BY TARGETING THE C-MYC ONCOGENE IN RENAL CELL CARCINOMA Hideo Hidaka*, Hirohumi Yoshino, Hideki Enokida, Takeshi Yamasaki, Toshihiko Itesako, Tomokazu Yonezawa, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent microRNAs (miRNAs) expression signatures of renal cell carcinoma (RCC) revealed that downregulation of miR-135a was frequent event in cancer tissues, suggesting that it may function as a tumor suppressive miRNA. The aim of this study is to investigate the functional significance of miR-135a and to identify miR-135a-mediated molecular pathways in RCC. METHODS: We evaluated miR-135a expression in RCC cell lines (caki2 and A498) and 38 RCC clinical specimens by real-time PCR. To investigate the functional role of miR-135a, we performed gain-of-function studies by using miR-135a transfectants (TFs). Gene expression analysis of the transfectants by oligo-microarray analysis was carried out to identify molecular targets and signaling pathways regulated by miR-135a. The GENECODIS software assigned a number of the putative miRNA-targeted genes to the known pathways in KEGG. In this study, we focused on the c-MYC gene, which belongs to Cell cycle pathway and has a putative miR-135a binding site in its 3⬘fUTR region. A luciferase reporter assay was carried out to determine whether 3⬘fUTR of c-MYC has an actual biding site for miR-135a. To
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
confirm whether miR-135a regulates molecular pathways in RCC, we investigated the expression levels of genes in Cell cycle pathway in 53 RCC specimens and 23 adjacent normal kidney specimens using available data sets (GSE36895 and GSE22541). RESULTS: MiR-135a expression in the RCC cell lines was significantly lower than that in normal kidney tissues. XTT and invasion assays demonstrated that cell viability was significantly inhibited in the miR-135a TFs compared with the control TFs. Cell cycle assay showed that G0/G1 arrest was induced in the miR-135a TFs. Gene expression analysis showed that 570 genes were down-regulated in the miR-135a TFs. These genes were classified into 25 signaling pathways in KEGG pathway categories. Luciferase reporter assay showed that c-MYC was directly regulated by miR-135a. In clinical specimens, expression levels of c-MYC and CCND1, which is in a c-MYC downstream, were significantly up-regulated in RCC specimens compared with the normal specimens. CONCLUSIONS: miR-135a was significantly down-regulated in RCC clinical specimens and appeared to function as a tumor suppressor in RCC through inhibition of c-MYC and cell cycle progression. Identification of such tumor-suppressive, miRNA-mediated cancer pathways in human RCC could provide new information on potential therapeutic targets in the treatment of RCC. Source of Funding: None
465 RNA SEQUENCING REVEALS UP-REGULATION OF RUNX1-RUNX1T1 GENE SIGNATURES IN CLEAR CELL RENAL CELL CARCINOMA Zuquan Xiong*, Qiang Ding, Shanghai, China, People’s Republic of; Jianfeng Xu, Winston-Salem, NC; Zujun Fang, Shanghai, China, People’s Republic of INTRODUCTION AND OBJECTIVES: In the past few years, targeted therapies against VHL/HIF pathways, such as Sunitinib and Sorafenib, have been developed against clear cell Renal Cell Carcinoma (ccRCC). However, tumors in all of the patients eventually show resistance to these anti-angiogenesis therapies and none of these drugs can cure the disease. Our objective is to find novel pathways for new therapies. METHODS: We performed whole transcriptome sequencing (RNA-seq) of eight matched tumor and adjacent normal tissues, and applied a gene sets enrichment analysis (GSEA) to identify novel pathways which can be potential targets for diagnosis and treatment. RESULTS: From the RNA-seq data, we found 3514 (17.8%) out of 19776 known genes differentially expressed in tumor samples, including 2054 (10.4%) up-regulated genes and 1460 (7.4%) downregulated genes. And we identified novel gene sets, including the ones involved in chromatin modification and a fusion transcription factor RUNX1-RUNX1T1, which were up-regulated in ccRCC. CONCLUSIONS: Based on the critical role of this fusion transcription factor RUNX1-RUNX1T1 in the etiology of acute leukemia, we hypothesize that up-regulation of RUNX1-RUNX1T1 gene set is important to the etiology of ccRCC.
rationale for alterations of metabolism in the context of kidney cancer, limited data exists characterizing whether these changes do exist in ccRCC tissues. METHODS: We therefore undertook a dual analysis of both the metabolome (i.e. small molecules) and the transcriptome in order to comprehensively characterize the metabolism of ccRCC which represents the first such analysis for any tumor type. Small molecule analysis was performed via Gas-Chromatography/ Mass Spectrometry (GC-MS) as well as Liquid-Chromatography/ Mass Spectrometry (LCMS). Transcriptional analysis was performed via mRNA microarray analysis and confirmatory real time RT-PCR. RESULTS: First, our data are the first to validate metabolomics as applied to renal cancer. Second, our data demonstrate significant changes in the transcriptional profile of ccRCC at the levels of glycolysis, gluconeogenesis, and oxidative phosphorylation consistent with a glygolytic phenotype. Third, these changes were consistent with our small molecule analysis and therefore demonstrate a correlation between transcription and metabolic pathway utilization. CONCLUSIONS: Collectively, the data support a unifying model of ccRCC metabolism which further reinforces the association between altered metabolism and malignancy. Source of Funding: NIH/NCI Astellas/AUA Foundation Rising Star Award
461 MICRORNA-29 FAMILY AS TUMOR SUPPRESSIVE MICRORNAS IN RENAL CELL CARCINOMA: MICRORNA-29A INHIBITS CELL MIGRATION AND INVASION TARGETING FOCAL ADHESION AND ECM PATHWAYS Tomokazu Yonezawa*, Hideki Enokida, Hirofumi Yoshino, Hideo Hidaka, Takeshi Yamasaki, Toshihiko Itesako, Kaghosima city, Japan; Naohiko Seki, Chiba city, Japan; Masayuki Nakagawa, Kaghosima city, Japan INTRODUCTION AND OBJECTIVES: A growing body of evidence suggests that microRNAs (miRNAs) contribute to human cancer progression, development, and metastasis, including renal cell carcinoma (RCC). Our previous miRNA expression signature revealed that miR-29-family (miR-29a/b/c) was significantly reduced in clinical RCC specimens. The aim of the study was to investigate the functional significance of miR-29-family and to explore the novel molecular pathways and responsibility genes regulated by miR-29a/b/c in RCC. METHODS: Gain-of-function studies were performed to investigate cancer cell proliferation, migration and invasion by mature miRNAs transfection into RCC cell lines (A498 and 786-O). To identify the biological processes or pathways potentially regulated by the miR29-family, we applied genome-wide gene expression analysis and in silico study; TargetScan database and GENECODIS software, which assigned a number of the putative miRNA targets to known pathways in KEGG [http://www.genome.jp/kegg/pathway.html]. The expression levels of miR-29-family target genes were verified using public database of Gene Expression Omnibus (GEO) in clinical RCCs. In these microarray expression data, we examined 78 clinical RCCs and 18 normal kidney tissues. RESULTS: Expression levels of miR-29-family were significantly reduced in clinical RCCs compared to adjacent non-cancerous tissues (P⬍0.001). Restoration of each miR-29-family significantly inhibited cancer cell proliferation, migration, and invasion in the RCC cell lines. The expression and in silico analysis showed that miR-29-family appeared to be an important modulator of tumor cell processes through suppression of many targets, particularly those involved in ‘focal adhesion’ and ‘ECM (extra-cellular matrix)-receptor interaction’ signaling pathways (P⬍0.05). Among two pathways, 45 genes were putative targets regulated by miR-29-family (29a/b/c). Gene expression data showed that three genes (COL5A, LAMC1, and VEGFA) were significantly up-regulated in clinical RCCs compared with normal kidney tissues.
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CONCLUSIONS: miR-29-family functioned as pivotal suppressor of cell migration and invasion in RCC through targeting ‘focal adhesion’ and ‘ECM’ signaling pathways. Elucidation of tumor suppressive miR-29-family-mediated cancer pathways and putative targets might be provided the novel molecular mechanisms to understand tumor progression or distant metastasis of RCC. Source of Funding: None
462 TUMOR SUPPRESSIVE MICRORNA-218 REGULATES ONCOGENES OF FOCAL ADHESION PATHWAYS IN RENAL CELL CARCINOMA Takeshi Yamasaki*, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Hirofumi Yoshino, Hideo Hidaka, Toshihiko Idesako, Hideki Enokida, Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent microRNA (miRNA) expression signature of renal cell carcinoma (RCC) revealed downregulation of miR-218 in RCC, suggesting that it might be a tumor suppressor. The aim of this study is to investigate the functional significance of miR-218 and to identify miR-218-mediated cancer pathways in RCC. METHODS: We evaluated miR-218 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We performed gain-of-function studies (cell migration and cell invasion assays) by using miR-218 transfectants (TFs). Oligo-microarray analyses of the miR-218 TFs and the RCC specimens were carried out to identify molecular targets of miR-218. According to GENECODIS software, we focused on the ‘focal adhesion’ pathway, in which caveolin-2 (CAV2) was a candidate target gene. A luciferase reporter assay was performed to determine miR-218 binding sites with CAV2 3’UTR. Loss-of-function assays using si-CAV2 were performed to investigate the functional significance of CAV2. The expression of CAV2 mRNA and protein was evaluated by qRT-PCR and immunohistochemistry. Furthermore, to investigate signaling pathways regulated by CAV2, we performed gene expression analysis of si-CAV2 TFs. RESULTS: The expression of miR-218 was significantly reduced in the RCC specimens. Significant inhibitions of cell migration and invasion were observed in the miR-218 TFs. Gene expression analysis showed that 615 genes were downregulated in the miR-218 TFs. The GENECODIS software revealed that miR-218 appeared to regulate 25 pathways (P⬍0.0001). In these pathways, we focused on ten genes in ‘focal adhesion’ and seven genes in ‘tight junction’. CAV2 was the most upregulated gene among the genes related to these pathways by using available data sets (GSE36895 and GSE22541) in gene expression omnibus (GEO). Luciferase reporter assay showed that CAV2 was directly regulated by miR-218. Silencing study of CAV2 demonstrated significant inhibition of cell migration and invasion. The mRNA and protein expression of CAV2 were significantly up-regulated in RCC specimens. Gene expression analysis of si-CAV2 TFs revealed that CAV2 might control 15 pathways including ‘tight junction’ pathway, containing pro-metastatic genes such as claudin-1, AKT3 and R-Ras2. CONCLUSIONS: Tumor suppressive miR-218 contributes to inhibition of cell migration and invasion through regulating focal adhesion pathway, especially CAV2 in RCC. This is the first report that CAV2 contributes to metastatic processes in RCC. Source of Funding: None
463 TUMOR SUPPRESSIVE MICRORNA-138 CONTRIBUTES TO INHIBITION OF BOTH CELL MIGRATION AND INVASION THROUGH TARGETING VIMENTIN IN RENAL CELL CARCINOMA Takeshi Yamasaki*, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Hirofumi Yoshino, Hideo Hidaka, Hideki Enokida, Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent miRNA expression signature of renal cell carcinoma (RCC) revealed that mi-
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croRNA-138 (miR-138) expression was significantly reduced in RCC, suggesting that miR-138 was a candidate tumor suppressor. Recent publications showed that miR-138 might regulate genes related to epithelial-mesenchymal transition (EMT) in several cancers. The aim of study is to investigate the functional significance of miR-138 and to identify its target genes in RCC. METHODS: We evaluated miR-138 expression in 33 RCC clinical specimens and adjacent normal kidney tissues by stem-loop RT-PCR. We observed morphologic changes in the miR-138-transfected RCC cell lines (A498 and 786-O). We performed gain-of-function studies such as cell migration and cell invasion assays by using miR-138 transfectants (TFs). Gene expression analysis of the miR-138 TFs and RCC clinical specimens by oligo-microarray was carried out to identify molecular targets of miR-138. TargetScan database implied that vimentin (VIM), an EMT-related gene, was a promising candidate target gene of miR-138. We investigated whether VIM was regulated by miR-138 by real-time PCR and Western blotting. Loss-of-function studies using si-VIM were performed to investigate the functional significance of VIM. We evaluated the expression levels of both VIM mRNA and protein by qRT-PCR and immunohistochemistry. RESULTS: The expression levels of miR-138 were significantly reduced in the RCC specimens compared with the normal tissues. Restoration of mature miR-138 in A498 and 786-O caused changes in the bleb-like cell morphology, as characteristics of EMT. Significant inhibitions of cell migration and cell invasion were observed in the miR-138 TFs. The expression levels of VIM expression was suppressed in the miR-138 TFs at both mRNA and protein levels. Lossof-function studies demonstrated that significant inhibition of cell migration and invasion occurred in the si-VIM transfectants. In clinical RCC specimens, the expression level of VIM was significantly upregulated in comparison with adjacent normal tissues. Furthermore, immunohistochemistry showed that VIM expression levels in RCC specimens were significantly higher than those in the normal tissues. CONCLUSIONS: These data suggest that VIM might function as an oncogene and might be regulated by tumor suppressive miR138. Tumor suppressive miR-138-mediated EMT pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis. Source of Funding: None
464 TUMOR-SUPPRESSIVE MIR-135A INHIBITS CANCER CELL PROLIFERATION BY TARGETING THE C-MYC ONCOGENE IN RENAL CELL CARCINOMA Hideo Hidaka*, Hirohumi Yoshino, Hideki Enokida, Takeshi Yamasaki, Toshihiko Itesako, Tomokazu Yonezawa, Kagoshima, Japan; Naohiko Seki, Chiba, Japan; Masayuki Nakagawa, Kagoshima, Japan INTRODUCTION AND OBJECTIVES: Our recent microRNAs (miRNAs) expression signatures of renal cell carcinoma (RCC) revealed that downregulation of miR-135a was frequent event in cancer tissues, suggesting that it may function as a tumor suppressive miRNA. The aim of this study is to investigate the functional significance of miR-135a and to identify miR-135a-mediated molecular pathways in RCC. METHODS: We evaluated miR-135a expression in RCC cell lines (caki2 and A498) and 38 RCC clinical specimens by real-time PCR. To investigate the functional role of miR-135a, we performed gain-of-function studies by using miR-135a transfectants (TFs). Gene expression analysis of the transfectants by oligo-microarray analysis was carried out to identify molecular targets and signaling pathways regulated by miR-135a. The GENECODIS software assigned a number of the putative miRNA-targeted genes to the known pathways in KEGG. In this study, we focused on the c-MYC gene, which belongs to Cell cycle pathway and has a putative miR-135a binding site in its 3⬘fUTR region. A luciferase reporter assay was carried out to determine whether 3⬘fUTR of c-MYC has an actual biding site for miR-135a. To
Vol. 189, No. 4S, Supplement, Sunday, May 5, 2013
confirm whether miR-135a regulates molecular pathways in RCC, we investigated the expression levels of genes in Cell cycle pathway in 53 RCC specimens and 23 adjacent normal kidney specimens using available data sets (GSE36895 and GSE22541). RESULTS: MiR-135a expression in the RCC cell lines was significantly lower than that in normal kidney tissues. XTT and invasion assays demonstrated that cell viability was significantly inhibited in the miR-135a TFs compared with the control TFs. Cell cycle assay showed that G0/G1 arrest was induced in the miR-135a TFs. Gene expression analysis showed that 570 genes were down-regulated in the miR-135a TFs. These genes were classified into 25 signaling pathways in KEGG pathway categories. Luciferase reporter assay showed that c-MYC was directly regulated by miR-135a. In clinical specimens, expression levels of c-MYC and CCND1, which is in a c-MYC downstream, were significantly up-regulated in RCC specimens compared with the normal specimens. CONCLUSIONS: miR-135a was significantly down-regulated in RCC clinical specimens and appeared to function as a tumor suppressor in RCC through inhibition of c-MYC and cell cycle progression. Identification of such tumor-suppressive, miRNA-mediated cancer pathways in human RCC could provide new information on potential therapeutic targets in the treatment of RCC. Source of Funding: None
465 RNA SEQUENCING REVEALS UP-REGULATION OF RUNX1-RUNX1T1 GENE SIGNATURES IN CLEAR CELL RENAL CELL CARCINOMA Zuquan Xiong*, Qiang Ding, Shanghai, China, People’s Republic of; Jianfeng Xu, Winston-Salem, NC; Zujun Fang, Shanghai, China, People’s Republic of INTRODUCTION AND OBJECTIVES: In the past few years, targeted therapies against VHL/HIF pathways, such as Sunitinib and Sorafenib, have been developed against clear cell Renal Cell Carcinoma (ccRCC). However, tumors in all of the patients eventually show resistance to these anti-angiogenesis therapies and none of these drugs can cure the disease. Our objective is to find novel pathways for new therapies. METHODS: We performed whole transcriptome sequencing (RNA-seq) of eight matched tumor and adjacent normal tissues, and applied a gene sets enrichment analysis (GSEA) to identify novel pathways which can be potential targets for diagnosis and treatment. RESULTS: From the RNA-seq data, we found 3514 (17.8%) out of 19776 known genes differentially expressed in tumor samples, including 2054 (10.4%) up-regulated genes and 1460 (7.4%) downregulated genes. And we identified novel gene sets, including the ones involved in chromatin modification and a fusion transcription factor RUNX1-RUNX1T1, which were up-regulated in ccRCC. CONCLUSIONS: Based on the critical role of this fusion transcription factor RUNX1-RUNX1T1 in the etiology of acute leukemia, we hypothesize that up-regulation of RUNX1-RUNX1T1 gene set is important to the etiology of ccRCC.