miR-206 as a prognostic and sensitivity biomarker for platinum chemotherapy in epithelial ovarian cancer.

BACKGROUND: Drug resistance is a major obstacle to successful chemotherapy for epithelial ovarian cancer (EOC). We found a subset of miRNAs associated with the response to first-line platinum-based chemotherapy in EOC by microarray, and miR-206 was one of the most significant miRNAs. The purposes of this study were to evaluate the prognostic and platinum-resistance predictive value of miR-206 in EOC patients and to investigate the functional roles of miR-206 in regulating the platinum resistance of EOC and the underlying mechanism. METHODS: MiRNA expression profiling in EOC specimens was performed using a TaqMan miRNA array. miR-206 expression was confirmed by quantitative real-time PCR (qRT-PCR) analysis. Overexpression of miR-206 in EOC cell lines was achieved by the stable transfection of a recombinant plasmid. In vitro assays of cisplatin cytotoxicity, cell cycle distribution, apoptosis, transwell invasion and cell scratching were employed. Connexin 43 (Cx43) expression was detected by Western blotting. Murine xenograft models were used to determine the effects of miR-206 on platinum resistance in vivo. RESULTS: miR-206 expression was increased in primary platinum-resistant EOC. High miR-206 expression was related to poor prognosis in EOC patients who received platinum-based chemotherapy and predicted chemoresistance to platinum treatment. Overexpression of miR-206 in cisplatin-sensitive EOC cell lines significantly increased cell viability, migration and invasion in the presence of cisplatin and decreased cisplatin-induced apoptosis. Cx43, a target gene of miR-206, was negatively regulated by miR-206 in EOC cell lines and significantly related to better prognosis in patients who received platinum-based chemotherapy (KmPlot). miR-206 had high expression and Cx43 had low expression in platinum-sensitive EOC cell lines compared with resistant ones. In vivo murine xenograft models showed that miR-206 profoundly promoted the chemoresistance of EOC to cisplatin treatment. CONCLUSION: miR-206 was highly expressed in primary platinum-resistant EOCs and functionally promoted platinum resistance in part by downregulating Cx43 expression, thereby providing a useful biomarker for prognostic and platinum-resistance prediction.

MiR-770-5p inhibits cisplatin chemoresistance in human ovarian cancer by targeting ERCC2.

In this study, we examined the role of the miRNA miR-770-5p in cisplatin chemotherapy resistance in ovarian cancer (OVC) patients. miR-770-5p expression was reduced in platinum-resistant patients. Using a 6.128-fold in expression as the cutoff value, miR-770-5p expression served as a prognostic biomarker and predicted the response to cisplatin treatment and survival among OVC patients. Overexpression of miR-770-5p in vitro reduced survival in chemoresistant cell lines after cisplatin treatment. ERCC2, a target gene of miR-770-5p that participates in the NER system, was negatively regulated by miR-770-5p. siRNA-mediated silencing of ERCC2 reversed the inhibition of apoptosis resulting from miR-770-5p downreglation in A2780S cells. A comet assay confirmed that this restoration of cisplatin chemosensitivity was due to the inhibition of DNA repair. These findings suggest that endogenous miR-770-5p may function as an anti-oncogene and promote chemosensitivity in OVC, at least in part by downregulating ERCC2. miR-770-5p may therefore be a useful biomarker for predicting chemosensitivity to cisplatin in OVC patients and improve the selection of effective, more personalized, treatment strategies.

Expression of miR-136 is associated with the primary cisplatin resistance of human epithelial ovarian cancer.

MicroRNAs (miRNAs) are involved in regulating the response of cancer cells to various therapeutic interventions, yet their involvement in the chemoresistance of human epithelial ovarian cancer is not fully understood. We found that miR-136 was significantly downregulated in specimens from patients with chemoresistant epithelial ovarian cancer. In the present study, we aimed to clarify the role of miR-136 in regulating the chemoresistance of ovarian cancer. Thirty-four tumor bank specimens and 2 well-established human ovarian cancer cell lines, C13 and OV2008, were used. We found that miR-136 expression was significantly reduced in primary platinum-resistant patients and the ovarian cancer OVC cell line. Enforced expression of miR-136 decreased the chemoresistance to cisplatin in OVC cells through inhibition of cell survival. In addition, we found no association between miR-136 and migration or invasion potential in the ovarian cancer cell lines. However, in the platinum-resistant C13 cell line, the overexpression of miR-136 markedly promoted an apoptotic response to cisplatin. Furthermore, the levels of adducts corrected with their extent of DNA damage/repair, in terms of the percentage of DNA in comet tails, tail length, tail moment (TM), and olive tail moment (OTM), revealed that miR-136 is essential for the repair of cisplatin-induced DNA damage. Our findings suggest that miR-136 may function as an anti-oncogene and deficiency of miR-136 expression in ovarian cancer can induce chemoresistance at least in part by downregulating apoptosis and promoting the repair of cisplatin-induced DNA damage. Thus, miR-136 may provide a biomarker for predicting the chemosensitivity to cisplatin in patients with epithelial ovarian cancer.

Expression of miR-224-5p is associated with the original cisplatin resistance of ovarian papillary serous carcinoma.

Chemoresistance is a major challenge to successful chemotherapy of ovarian cancer, which represents the leading cause of mortality from gynecologic malignancies. We demonstrated that overexpression of miR-224-5p in ovarian cancer patients is associated with platinum-based chemoresistance using miRNA microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR) validation in vivo, as well as in 4 human ovarian cancer cell lines (C13/OV2008; A2780CP/A2780S) in vitro. In the present study, we aimed to clarify the role of miR-224-5p in regulating the chemoresistance of ovarian cancer. By using the sensitive miRNA transient transfection, we demonstrated expression and bioactivity of miR-224-5p in ovarian cancer cell lines. It is of note that enforced expression of miR-224-5p enhanced chemoresistance to cisplatin in ovarian cancer cells through apoptosis reversion. We predicted and identified the PRKCD gene as one of the targets of miR-224-5p in mediating the primary chemoresistance of ovarian cancer patients. We showed reciprocal expression of miR-224-5p and PRKCD by quantitative analysis in complete response and incomplete response patients in vivo, and 2 pairs of cisplatin resistance and sensitive cell lines in vitro, after either miR-224-5p overexpression or knockdown transfection. Additionally, miR-224-5p and PRKCD can serve as novel predictors and prognostic biomarkers for ovarian papillary serous carcinoma (OPSC) patient response to overall disease-specific survival. Our findings suggest that miR-224-5p may function as an oncogene and induce platinum resistance in OPSC at least in part by downregulating PRKCD, thereby providing a biomarker for predicting chemosensitivity to cisplatin in patients with ovarian cancer.

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma.

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan-Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan-Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P < 0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.

miRNA expression pattern associated with prognosis in elderly patients with advanced OPSC and OCC.

The long-term survival for elderly patients with advanced ovarian papillary serous carcinoma (OPSC) does not exceed 30%, and the incidence and prognosis rise continuously after menopause. The aim of this study was to identify the differences in key miRNAs and their potential regulators through miRNA microarray analysis, functional target prediction, and clinical outcome between the elderly patients with advanced OPSC and ovarian clear cell carcinoma (OCC) who all suffered poor prognosis, to identify the pathogenetic basis, and to improve the understanding of the molecular basis of advanced OPCS in elderly patients. Through microarray analysis, we found 52 unique miRNAs with significant fold­change in expression levels, of which 9 were upregulated, whereas 43 were downregulated in OCC patients compared to elderly OPSC patients with advanced stage. Among these prediction miRNAs, miR-30a, miR-30e and miR-505 were found to have some common cancer-related targets. Lower expression of these three miRNAs of advanced OPSC in elderly patients, all associated with significantly poorer survival rate, strongly suggesting that they could be critical oncogenes and take important roles in OPSC etiology in elderly patients with advantaged stage. Functional analyses support the above hypothesis. Their targets, ATF3, STMN1 and MYC suggest that OPSC also has aggressive biological behavior when presented with advanced stage, and support the epidemiology results that incidence and mortality of advanced OPSC increases continuously. miR-30a, miR-30e and miR-505 may be important pathogenetic factors for OPSC in elderly patients with advanced stage. Age could be regarded as a continuous covariate in this process. This may improve the understanding of molecular underpinnings of advanced OPSC in elderly patients, and provide improved diagnostic, prognostic and therapeutic approaches.

Synchronous detection of miRNAs, their targets and downstream proteins in transferred FFPE sections: applications in clinical and basic research.

After discovering new miRNAs, it is often difficult to determine their targets and effects on downstream protein expression. In situ hybridization (ISH) and immunohistochemistry (IHC) are two commonly used methods for clinical diagnosis and basic research. We used an optimized technique that simultaneously detects miRNAs, their binding targets and corresponding proteins on transferred serial formalin fixed paraffin embedded (FFPE) sections from patients. Combined with bioinformatics, this method was used to validate the reciprocal expression of specific miRNAs and targets that were detected by ISH, as well as the expression of downstream proteins that were detected by IHC. A complete analysis was performed using a limited number of transferred serial FFPE sections that had been stored for 1-4 years at room temperature. Some sections had even been previously stained with H&E. We identified a miRNA that regulates epithelial ovarian cancer, along with its candidate target and related downstream protein. These findings were directly validated using sub-cellular components obtained from the same patient sample. In addition, the expression of Nephrin (a podocyte marker) and Stmn1 (a recently identified marker related to glomerular development) were confirmed in transferred FFPE sections of mouse kidney. This procedure may be adapted for clinical diagnosis and basic research, providing a qualitative and efficient method to dissect the detailed spatial expression patterns of miRNA pathways in FFPE tissue, especially in cases where only a small biopsy sample can be obtained.

MicroRNA 376c enhances ovarian cancer cell survival by targeting activin receptor-like kinase 7: implications for chemoresistance.

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in gene regulation. We have previously reported that activin receptor-like kinase 7 (ALK7) and its ligand, Nodal, induce apoptosis in human epithelial ovarian cancer cells. In this study, we examined the regulation of ALK7 by miRNAs and demonstrate that miR-376c targets ALK7. Ectopic expression of miR-376c significantly increased cell proliferation and survival, enhanced spheroid formation and blocked Nodal-induced apoptosis. Interestingly, overexpression of miR-376c blocked cisplatin-induced cell death, whereas anti-miR-376c enhanced the effect of cisplatin. These effects of miR-376c were partially compensated by the overexpression of ALK7. Moreover, in serous carcinoma samples taken from ovarian cancer patients who responded well to chemotherapy, strong ALK7 staining and low miR-376c expression was detected. By contrast, ALK7 expression was weak and miR-376c levels were high in samples from patients who responded poorly to chemotherapy. Finally, treatment with cisplatin led to an increase in expression of mRNA encoding Nodal and ALK7 but a decrease in miR-376c levels. Taken together, these results demonstrate that the Nodal-ALK7 pathway is involved in cisplatin-induced cell death in ovarian cancer cells and that miR-376c enhances proliferation, survival and chemoresistance by targeting, at least in part, ALK7.

Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice.

Rapidly progressive glomerulonephritis (RPGN) is a clinical syndrome characterized by loss of renal function within days to weeks and by glomerular crescents on biopsy. The pathogenesis of this disease is unclear, but circulating factors are believed to have a major role. Here, we show that deletion of the Von Hippel-Lindau gene (Vhlh) from intrinsic glomerular cells of mice is sufficient to initiate a necrotizing crescentic glomerulonephritis and the clinical features that accompany RPGN. Loss of Vhlh leads to stabilization of hypoxia-inducible factor alpha subunits (HIFs). Using gene expression profiling, we identified de novo expression of the HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and humans with RPGN. The course of RPGN is markedly improved in mice treated with a blocking antibody to Cxcr4, whereas overexpression of Cxcr4 alone in podocytes of transgenic mice is sufficient to cause glomerular disease. Collectively, these results indicate an alternative mechanism for the pathogenesis of RPGN and glomerular disease in an animal model and suggest novel molecular pathways for intervention in this disease.

Vascular endothelial growth factor a signaling in the podocyte-endothelial compartment is required for mesangial cell migration and survival.

The glomerular filtration barrier separates the blood from the urinary space and consists of two major cell types: podocytes and fenestrated endothelial cells. Mesangial cells sit between the capillary loops and provide structural support. Proliferation and loss of mesangial cells both are central findings in a number of renal diseases, including diabetic nephropathy and mesangiolysis, respectively. Using cell-specific gene targeting, it was shown previously that vascular endothelial growth factor A (VEGF-A) production by podocytes is required for glomerular endothelial cell migration, differentiation, and survival. For further investigation of the effect of gene dose and VEGF-A knockdown within the glomerulus, mice that carry one hypomorphic VEGF-A allele and one podocyte-specific null VEGF-A allele (VEGFhypo/loxP,Neph-Cre+/-) were generated; in these mice, the "allelic dose" of VEGF-A is intermediate between glomerular-specific heterozygous and null states. VEGFhypo/loxP,Neph-Cre+/- mice die at 3 wk of age from renal failure. Although endothelial cell defects are observed, striking loss of mesangial cells occurs postnatally. In addition, differentiated mesangial cells cannot be found in glomeruli of podocyte-specific null VEGF-A mice (VEGFloxP/loxP,Cre+/-). Together, these results demonstrate a key role for VEGF-A production in the podocyte for mesangial cell survival and differentiation.

Rapid isolation of glomeruli coupled with gene expression profiling identifies downstream targets in Pod1 knockout mice.

Mouse mutations have provided tremendous insights into the molecular basis of renal and glomerular development. However, genes often play important roles during multiple stages of nephrogenesis, making it difficult to determine the role of a gene in a specific cell lineage such as the podocyte. Conditional gene targeting and chimeric analysis are two possible approaches to dissect the function of genes in specific cell populations. However, these are labor-intensive and costly and require the generation, validation, and analysis of additional transgenic lines. For overcoming these shortcomings and, specifically, for studying the role of gene function in developing glomeruli, a technique to isolate and purify glomeruli from murine embryos was developed. Combined with gene expression profiling, this method was used to identify differentially expressed genes in glomeruli from Pod1 knockout (KO) mice that die in the perinatal period with multiple renal defects. Glomeruli from early developing stages (late S-shape/early capillary loop) onward can be isolated successfully from wild-type and KO kidneys at 18.5 d postcoitus, and RNA can readily be obtained and used for genome-wide microarray analysis. With this approach, 3986 genes that are differently expressed between glomeruli from Pod1 KO and wild-type mice were identified, including a four-fold reduction of alpha 8 integrin mRNA in glomeruli from Pod1 KO mice that was confirmed by immunostaining. This procedure may be adapted to any transgenic strain, providing a rapid and efficient method to dissect the function of specific genes in glomerular development.

Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice.

Congenital defects in genital and/or gonadal development occur in 1 in 1000 humans, but the molecular basis for these defects in most cases remains undefined. We show that the basic helix-loop-helix transcription factor Pod1 (capsulin/epicardin/Tcf21) is essential for normal development of the testes and ovaries, and hence for sexual differentiation. The gonads of Pod1 knockout (KO) mice were markedly hypoplastic, and the urogenital tracts of both XX and XY mice remained indistinguishable throughout embryogenesis. Within Pod1 KO gonads, the number of cells expressing the cholesterol side-chain cleavage enzyme (Scc) was increased markedly. Biochemical and genetic approaches demonstrated that Pod1 transcriptionally represses steroidogenic factor 1 (Sf1/Nr5a1/Ad4BP), an orphan nuclear receptor that regulates the expression of multiple genes (including Scc) that mediate sexual differentiation. Our results establish that Pod1 is essential for gonadal development, and place it in a transcriptional network that orchestrates cell fate decisions in gonadal progenitors.

Pod1 is required in stromal cells for glomerulogenesis.

Pod1 (capsulin/epicardin/Tcf21) is a basic-helix-loop-helix transcription factor that is highly expressed in the mesenchyme of developing organs that include the kidney, lung, gut, and heart. Null Pod1 mice are born but die shortly after birth due to a lack of alveoli in the lungs and cardiac defects. In addition, the kidneys are hypoplastic and demonstrate disrupted branching morphogenesis of the ureteric bud epithelium, a marked reduction in the number of nephrons, a delay in glomerulogenesis, and blood vessel abnormalities. To further dissect the cellular function of Pod1 during kidney development, chimeric mice were generated through aggregations of null Pod1 embryonic stem cells and murine embryos ubiquitously expressing enhanced green fluorescent protein (GFP). Histologic, immunohistochemical, and in situ hybridization analysis of the resulting chimeric offspring demonstrated both cell autonomous and non-cell autonomous roles for Pod1 in the differentiation of specific renal cell lineages that include peritubular interstitial cells and pericytes. Most strikingly, the glomerulogenesis defect was rescued by the presence of wild-type stromal cells, suggesting a non-cell autonomous role for Pod1 in this cell population.

The mouse Kreisler (Krml1/MafB) segmentation gene is required for differentiation of glomerular visceral epithelial cells.

Molecular components of the glomerular filtration mechanism play critical roles in renal diseases. Many of these components are produced during the final stages of differentiation of glomerular visceral epithelial cells, also known as podocytes. While basic domain leucine zipper (bZip) transcription factors of the Maf subfamily have been implicated in cellular differentiation processes, Kreisler (Krml1/MafB), the gene affected in the mouse kreisler (kr) mutation, is known for its role in hindbrain patterning. Here we show that mice homozygous for the kr(enu) mutation develop renal disease and that Kreisler is essential for cellular differentiation of podocytes. Consistent with abnormal podocyte differentiation, kr(enu) homozygotes show proteinuria, and fusion and effacement of podocyte foot processes, which are also observed in the nephrotic syndrome. Kreisler acts during the final stages of glomerular development-the transition between the capillary loop and mature stages-and downstream of the Pod1 basic domain helix-loop-helix transcription factor. The levels of Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome (NPHS2), and Nephrin, the gene mutated in congenital nephrotic syndrome of the Finnish type (NPHS1), are slightly reduced in kr(enu)/kr(enu) podocytes. However, these observations alone are unlikely to account for the aberrant podocyte foot process formation. Thus, Kreisler must regulate other unknown genes required for podocyte function and with possible roles in kidney disease.

Glomerular-specific gene excision in vivo.

Podocytes (glomerular visceral epithelial cells) are highly specialized cells that are found in the renal glomerulus and make up a major portion of the filtration barrier between the blood and urinary spaces. Recently, the identification of a number of genes responsible for both autosomal dominant and recessive forms of human nephrotic syndrome has provided insight into a number of molecules responsible for unique features of the podocyte such as the slit diaphragms. Despite these major advances in our understanding of podocyte biology, the function of many genes expressed in the podocyte remains unknown. Targeted gene disruption using homologous recombination in murine embryonic stem cells (ES cells) is a powerful tool to determine the biologic function of genes in vivo. However, resulting embryonic lethal or pleiotropic phenotypes often preclude the analysis of genes in specific renal cell types. To overcome this problem, a glomerular-specific Cre-recombinase transgenic murine line under the control of the Nphs1 (nephrin) promoter (Neph-Cre) was generated. This article reports successful Cre-mediated excision of a 'floxed' transgene specifically in podocytes in vivo. This murine founder line represents a powerful new tool for the manipulation of the expression of genes in podocytes and will provide valuable insight into podocyte biology in the whole animal.