Doris Schmidt

Identification of novel long non-coding RNAs in clear cell renal cell carcinoma

BackgroundLong non-coding RNAs (lncRNA) play an important role in carcinogenesis; knowledge on lncRNA expression in renal cell carcinoma is rudimental. As a basis for biomarker development, we aimed to explore the lncRNA expression profile in clear cell renal cell carcinoma (ccRCC) tissue.ResultsMicroarray experiments were performed to determine the expression of 32,183 lncRNA transcripts belonging to 17,512 lncRNAs in 15 corresponding normal and malignant renal tissues. Validation was performed using quantitative real-time PCR in 55 ccRCC and 52 normal renal specimens. Computational analysis was performed to determine lncRNA-microRNA (MiRTarget2) and lncRNA-protein (catRAPID omics) interactions. We identified 1,308 dysregulated transcripts (expression change >2-fold; upregulated: 568, downregulated: 740) in ccRCC tissue. Among these, aberrant expression was validated using PCR: lnc-BMP2-2 (mean expression change: 37-fold), lnc-CPN2-1 (13-fold), lnc-FZD1-2 (9-fold), lnc-ITPR2-3 (15-fold), lnc-SLC30A4-1 (15-fold), and lnc-SPAM1-6 (10-fold) were highly overexpressed in ccRCC, whereas lnc-ACACA-1 (135-fold), lnc-FOXG1-2 (19-fold), lnc-LCP2-2 (2-fold), lnc-RP3-368B9 (19-fold), and lnc-TTC34-3 (314-fold) were downregulated. There was no correlation between lncRNA expression with clinical-pathological parameters. Computational analyses revealed that these lncRNAs are involved in RNA-protein networks related to splicing, binding, transport, localization, and processing of RNA. Small interfering RNA (siRNA)-mediated knockdown of lnc-BMP2-2 and lnc-CPN2-1 did not influence cell proliferation.ConclusionsWe identified many novel lncRNA transcripts dysregulated in ccRCC which may be useful for novel diagnostic biomarkers.

Antiproliferative and apoptosis inducing effects of indirubin-3'-monoxime in renal cell cancer cells.

OBJECTIVESnIndirubin-3-monoxime, which is a selective and potent inhibitor of cyclin-dependent kinases (CDKs) has shown preclinical activity in several human cancer cells. This study investigated if indirubin-3-monoxime can induce apoptosis and tumor cell death in 3 human (A498, CAKI-1, CAKI-2) and 1 murine renal cell cancer (RENCA) cell line.nnnMETHODSnThe growth inhibitory and apoptosis induction properties were evaluated by EZ4U, a cytotoxic assay and by flow cytometry of annexin-V/PI staining during treatment with doses ranging from 5.0 to 15.0 μM indirubin-3-monoxime over 72 hours. To further establish the underlying molecular targets of indirubin-3-monoxime, survivin, a major anti-apoptotic protein was additionally determined by intracellular flow cytometry.nnnRESULTSnOur results show that indirubin-3-monoxime induces growth arrest and apoptosis in all renal cell cancer (RCC) cell lines. All RCC lines expressed survivin. However, a clear correlation between apoptosis induction and expression of survivin was not found.nnnCONCLUSIONSnAs treatment of metastatic renal cell cancer (mRCC) remains a challenge, and the need for continuing assessment of novel agents in the treatment of this disease is mandatory. Indirubin-3-monoxime seems to be a candidate for further evaluation.

Identification of novel differentially expressed lncRNA and mRNA transcripts in clear cell renal cell carcinoma by expression profiling.

Clear cell renal cell carcinoma (ccRCC) is a common human malignancy. Despite numerous efforts, there is still no reliable biomarker or combination of biomarkers available for daily practice. Our study was designed to explore the expression profile of messenger RNA (mRNA) and long non-coding RNA (lncRNA) transcripts in ccRCC in order to identify potential diagnostic biomarkers for patients with ccRCC. Total RNA from corresponding normal and malignant tissue of 15 patients with ccRCC was isolated. Expression profiling was performed using a custom Agilent gene expression microarray which allowed the analysis of 34,144 mRNA and 32,183 lncRNA transcripts. We observed that a subset of mRNA (n = 1064; 3.1%) and lncRNA (n = 1308; 4.1%) transcripts are dysregulated (fold change > 2) in ccRCC tissue. The relative higher number of differentially expressed lncRNAs indicates that lncRNA profiling may be better suited for diagnostic purposes; a number of so far unknown RNAs with potential diagnostic interest in ccRCC are identified by our gene expression profiling study. The data are deposited in the Gene Expression Omnibus (GSE61763).

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria.

Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) is among the most common human malignancies.MethodsIn order to provide better understanding of the molecular biology of ccRCC and to identify potential diagnostic/prognostic biomarker and therapeutic targets, we utilized a microarray to profile mRNA expression of corresponding normal and malignant renal tissues. Real-time PCR, Western Blot and immunohistochemistry were applied to study the expression of candidate biomarkers. ccRCC cell lines were treated with sertraline to inhibit the dopamine transporter SLC6A3.ResultsDifferential expression of fourteen mRNAs, yet not studied in ccRCC in depth, was confirmed using qPCR (upregulation: SLC6A3, NPTX2, TNFAIP6, NDUFA4L2, ENPP3, FABP6, SPINK13; downregulation: FXYD4, SLC12A1, KNG1, NPHS2, SLC13A3, GCGR, PLG). Up-/downregulation was also confirmed for FXYD4, KNG1, NPTX2 and SLC12A1 by Western Blot on the protein level. In contrast to the mRNA expression, protein expression of the dopamine transporter SLC6A3 was lower in ccRCC compared to normal renal tissue. Immunohistochemistry indicated that this decrease was due to higher concentrations of SLC6A3 in the proximal tubules. Immunohistochemical analyses further demonstrated that high SLC6A3 expression in ccRCC tissue was correlated with a shorter period of recurrence-free survival following surgery. Treatment of ccRCC cells with the SLC6A3 inhibitor sertraline induced dose-dependent cell-death.ConclusionOur study identified several novel biomarkers with diagnostic potential and further investigations on sertraline as therapeutic agent in ccRCC patients are warranted.

5′-tRNA Halves are Dysregulated in Clear Cell Renal Cell Carcinoma

Purpose: In various malignancies RNA fragments are dysregulated. Our study was designed to determine the expression of 4, 5′‐tRNA halves in the tissue and serum of patients with clear cell renal cell carcinoma. Materials and Methods: Tissue and serum samples of patients with clear cell renal cell carcinoma and nonmalignant disease were collected prospectively in our biobank. We isolated total RNA from 95 clear cell renal cell carcinomas and 50 normal renal tissues as well as serum RNA from 27 patients with clear cell renal cell carcinoma and 13 with nonmalignant urological disease. To specifically determine the expression of 5′‐tRNA halves we dephosphorylated and ligated an adaptor nucleotide to the 3′ end of the tRNA halves. The expression levels of 4, 5′‐tRNA halves (5′‐tRNA‐Arg‐CCT, 5′‐tRNA‐Glu‐CTC, 5′‐tRNA‐Leu‐CAG and 5′‐tRNA‐Lys‐TTT) were then measured by TaqMan® based quantitative reverse transcription‐polymerase chain reaction. Results: All studied 5′‐tRNA halves were down‐regulated in clear cell renal cell carcinoma tissues, indicating a potential role as a tumor suppressor. Furthermore, we noted decreased expression of 5′‐tRNA halves in patients with adverse clinicopathological parameters. All 5′‐tRNA halves were expressed at lower levels in nonorgan confined clear cell renal cell carcinoma. The 5′‐tRNA‐Lys‐TTT halves inversely correlated with ISUP (International Society of Urological Pathology) grade. In patients with clear cell renal cell carcinoma 5′‐tRNA‐Arg‐CCT, 5′‐tRNA‐Glu‐CTC and 5′‐tRNA‐Lys‐TTT halves circulated at lower levels than in control subjects, indicating relevance as noninvasive biomarkers. Conclusions: In patients with clear cell renal cell carcinoma 5′‐tRNA halves have potential as diagnostic and prognostic biomarkers. The 5′‐tRNA halves may act in a tumor suppressive manner, which requires further research to confirm.

Systematic Analysis of the Expression of the Mitochondrial ATP Synthase (Complex V) Subunits in Clear Cell Renal Cell Carcinoma

Mitochondrial dysfunction is common in cancer and the mitochondrial electron transport chain is often affected in carcinogenesis. To date, little is known about the expression of the ATP synthase subunits in clear cell renal cell carcinoma (ccRCC). The NextBio database was used to determine an expression profile of the ATP synthase subunits based on published microarray studies. We observed down-regulation of 23 out of 29 subunits of the ATP synthase. Differential expression was validated exemplarily for 12 genes (ATP5A1, ATP5B, ATPAF1, ATP5C1, ATP5D, ATP5O, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5I, ATP5S; screening cohort ccRCC n = 18 and normal renal tissue n = 10) using real-time PCR. Additional eight genes (ATP5A1, ATP5B, ATPAF1, ATP5F1, ATP5G1, ATP5G2, ATP5G3, ATP5S) were internally validated within an enlarged cohort (ccRCC n = 74; normal renal tissue n = 36). Furthermore, down-regulation of ATP5A1, ATPAF1, ATP5G1/G2/G3 was confirmed on the protein level using Western Blot and immunohistochemistry. We observed that altered expression of ATPAF1 and ATP5G1/G2/G3 was correlated with overall survival in patients with ccRCC. In conclusion, down-regulation of many ATP Synthase subunits occurs in ccRCC and is the basis for the reduced activity of the mitochondrial electron chain. Alteration of the expression of ATP5A1, ATPAF1, and ATP5G1/G2/G3 is characteristic for ccRCC and may be prognostic for ccRCC patients outcome.

Systematic Expression Analysis of Mitochondrial Complex I Identifies NDUFS1 as a Biomarker in Clear-Cell Renal-Cell Carcinoma

Introduction Mitochondrial dysfunction is common in cancer, and the mitochondrial electron transport chain is often affected in carcinogenesis. So far, little is known about the expression of the mitochondrial complex I (NADH:ubiquinone oxidoreductase) subunits in clear‐cell renal‐cell carcinoma (ccRCC). Materials and Methods An expression profile of the mitochondrial complex I subunits was determined using the NextBio database. Subsequently, the expression of selected subunits was experimentally validated on mRNA (quantitative real‐time polymerase chain reaction) and protein (Western blot analysis, immunohistochemistry) level. Results We observed that 7 subunits of the complex I were down‐regulated in at least 3 microarray studies. Deregulated mRNA expression was confirmed for NDUFA3, NDUFA, NDUFB1, NDUFB9, NDUFS1, NDUFS8, and NDUFV1. Low NDUFS1 mRNA expression was a significant and independent adverse predictor of a shorter overall survival in our mRNA cohort and the ccRCC cohort of The Cancer Genome Atlas project. NDUFS1 expression was furthermore analyzed on the protein level, and a distinct down‐regulation was observed in ccRCC as well as in the chromophobe and the sarcomatoid subtype compared to normal renal tissue. Conclusion Expression alterations occur in only a few subunits of the mitochondrial complex I subunits in ccRCC, and altered mRNA and protein expression levels of NDUFS1 may be useful to distinguish between renal‐cell carcinoma and normal renal tissue. Micro‐Abstract Mitochondrial dysfunction is common in cancer. A meta‐analysis of published microarray gene expression studies led to the identification of 7 (of 44) down‐regulated subunits of the mitochondrial complex I in clear‐cell renal‐cell carcinoma. NDUFS1 mRNA and protein expression levels were validated and may serve as diagnostic biomarkers. Altered activity of the electron transport chain is related to altered expression of specific subunits.

YRNA Expression Profiles are Altered in Clear Cell Renal Cell Carcinoma

BACKGROUNDnNoncoding RNAs play an important role in human carcinogenesis. YRNAs, a novel class of noncoding RNAs, have been identified as biomarkers in breast cancer patients.nnnOBJECTIVEnTo test the hypothesis that YRNA expression is dysregulated in clear cell renal cell carcinoma (ccRCC).nnnDESIGN, SETTING, AND PARTICIPANTSnWe first measured the expression of all known YRNAs (hY1, hY3, hY4, and hY5) in a screening cohort (30 ccRCC and 15 normal renal tissues). Subsequently, hY3 and hY4 were validated in an independent cohort (88 ccRCC and 59 normal renal tissues). Finally, hY3 and hY4 levels in serum samples from 30 ccRCC and 15 control individuals were measured. YRNAs were detected using quantitative real-time polymerase chain reaction.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnRelative expression values were analyzed using the Mann-Whitney-U test and Kaplan Meier estimates.nnnRESULTS AND LIMITATIONSnExpression of hY3 and hY4 was increased in ccRCC samples compared with normal renal tissue, whereas hY1 and hY5 levels were similar. Expression levels of hY4 correlated with ccRCC stage and the presence of lymph node metastases. Neither hY3 nor hY4 were circulating at different levels in ccRCC patients and control individuals.nnnCONCLUSIONSnThe expression of hY3 and hY4 is altered in ccRCC and associated with advanced disease.nnnPATIENT SUMMARYnIn this report we studied the expression of noncoding YRNA in clear cell renal cell carcinoma tissue. We observed increased hY3 and hY4 expression levels in cancer tissues. However, expression levels were not different in the serum of patients with cancer or benign disease.

Serum miR-122-5p and miR-206 expression: non-invasive prognostic biomarkers for renal cell carcinoma

BackgroundMicroRNAs (miRNA) play a relevant role in carcinogenesis, cancer progression, invasion, and metastasis. Thus, they can serve as diagnostic/prognostic biomarkers. The knowledge on circulating miRNAs for clear cell renal cell carcinomas (ccRCC) is limited. Our study was designed to identify novel biomarkers for ccRCC patients.ResultsThe serum small RNA expression profile was determined in 18 ccRCC and 8 patients with benign renal tumors (BRT) using small RNA sequencing. We detected 29 differentially expressed miRNAs (17 upregulated and 12 downregulated in ccRCC) in the expression profiling cohort. Based on the expression levels, we next validated serum miR-122-5p, miR-193a-5p, and miR-206 levels in an independent cohort (68 ccRCC, 47 BRT, and 28 healthy individuals) using quantitative real-time PCR. Serum expression levels of miR-122-5p and miR-206 were significantly decreased in ccRCC compared to healthy individuals. Both miRNAs were circulating at similar levels in ccRCC and BRT patients. miR-193a-5p expression levels were not different within the study cohort. High serum miR-122-5p and miR-206 levels were associated with adverse clinicopathological parameters: miR-122-5p levels were correlated with metastatic RCC and grade, and miR-206 with pT-stage and metastasis. Furthermore, high miR-122-5p and miR-206 serum levels were associated with a shorter period of progression-free, cancer-specific, and overall survival in patients with ccRCC.ConclusionWe identified serum miR-122-5p and miR-206 as novel non-invasive prognostic biomarkers for patients with ccRCC.