The Functional Role And Mechanism Of VHL-Dependent Dicer Deregulation In Clear Cell Renal Cell Carcinoma

Objective:Renal cell carcinoma (RCC) is one of the most common malignancies in urology, and clear cell RCC (ccRCC) represents the main pathologic subtype. Previous studies demonstrate that von Hippel-Lindau (VHL)/hypoxia-inducible factor (HIF) pathway and microRNA (miRNA) play important roles in development and progression of ccRCC, and this study aims to explore the crosstalk between these two. The function and mechanism of this crosstalk are also determined.Materials and Methods:1. The genomic DNA of 182 ccRCC tissue samples were tested for VHL gene status by VHL exon sequencing and VHL promotor methylation analysis, and miRNA microarray analysis were performed on 8 wild-type VHL ccRCC tissue samples and 8 VHL-deficient ccRCC tissue samples, and the microarray results were validated by quantitative real-time PCR (qRT-PCR). The expression levels of the key enzymes of miRNA post-transcriptional biogenesis including Dicer, Drosha, Exportin-5 and DGCR8 were detected by qRT-PCR and Western blot in 36 wild-type VHL ccRCC tissue samples and 36 VHL-deficient ccRCC tissue samples, and Dicer expression levels were also detected by qRT-PCR in the corresponding adjacent normal renal tissues. Dicer expression levels were detected in ccRCC cells after either VHL overexpression or VHL knockdown.2. The correlation between Dicer and HIF mRNA expression levels in ccRCC tissues were detected by linear regression analysis, and the expression levels of HIF and its downstream genes were detected in ccRCC cells after both Dicer overexpression and Dicer knockdown. Based on microarray and qRT-PCR results, bioinformatic analysis was performed to explore Dicer-mediated miRNAs potentially targeting HIF-2a. Then qRT-PCR, Western blot and luciferase assay were performed to validate whether the candidate miRNA could directly target HIF-2a.3. The biologic function of Dicer in VHL-deficient ccRCC and whether it functions through HIF-2a were detected by colony formation assay, tube formation assay and xenograft tumor growth assay.146 VHL-deficient ccRCC patients were followed up and the association between Dicer expression and clinical prognosis were determined by Kaplan-Meier analysis and COX regression analysis.Results:Microarray and qRT-PCR results showed an overall miRNA downregulation in VHL-deficient ccRCC tissue samples compared with wild-type VHL ccRCC tissue samples, and the precursors of the downregulated miRNAs were increased. Among the key enzymes of miRNA post-transcriptional biogenesis, only Dicer expression levels were downregulated in VHL-deficient ccRCC tissue samples compared with wild-type VHL ccRCC tissue samples. Meanwhile, Dicer expression levels were only downregulated in VHL-deficient ccRCC tissue samples compared with their corresponding adjacent normal renal tissues, whereas this Dicer expression difference were not detected in wild-type VHL ccRCC tissue samples and their corresponding adjacent normal renal tissues. VHL can positively regulate Dicer expressions in ccRCC cells.2. Linear regression analysis showed that Dicer mRNA expressions were inversely correlated with HIF-2α mRNA expressions, and not correlated with HIF-1α mRNA expressions. Dicer can negatively regulate HIF-2α and its downstream genes expressions in VHL-deficient ccRCC cells, and HIF-2α was a direct target of Dicer-mediated miR-182-5p.3. Colony formation assay and tube formation assay confirmed that Dicer can suppress proliferation and angiogenesis of VHL-deficient ccRCC cells in vitro, and xenograft tumor growth assay confirmed that Dicer can suppress tumor growth of VHL-deficient ccRCC cells in vivo as well. Moreover, the tumor-suppressive effect of Dicer was through downregulating HIF-2α. Kaplan-Meier survival analysis showed that the post-operative survival rate in Low Dicer group was poorer than that in High Dicer group in VHL-deficient ccRCC patients, and COX regression analysis showed that reduced Dicer mRNA levels served as an independent prognostic factor for poor survival.Conclusions:Dicer is positively regulated by VHL and plays an important role in suppressing the tumorigenic phenotype by reducing expression of HIF-2α and its downstream genes in VHL-deficient ccRCCs. Reduced Dicer expression in VHL-deficient ccRCCs is an independent prognostic factor for poor clinical outcome. Therefore, Dicer may potentially serve as a risk stratification marker and therapeutic target for managing ccRCC, and provide a new thought for diagnosis and treatment of this disease.