The Role Of MiR-186-5p In Clear Cell Renal Cell Carcinoma Metastasis And Angiogenesis

Background and objective:Clear cell renal cell carcinoma (ccRCC) is the most lethal urological tumor, which accounts for 2%-3% of adult malignancies in the world. RCC is heterogeneous and comprises several histologic subtypes according to the differences in genetics, biology, and behavior. The most common and aggressive RCC subtype is clear cell RCC (ccRCC) with the highest rates of local invasion, metastasis, mortality, and refractory to current treatments. Recently, advancements in understanding of the VHL gene pathway in ccRCC have produced pharmaceutic outcomes based on specific molecular targets that have changed the treatment landscape for patients with metastatic RCC. Unfortunately, the vast majority of treated patients with mRCC eventually develop progressive disease because of acquired resistance or other reasons. Hence, a better understanding of the mechanisms involved in the pathogenesis of ccRCC and more effective therapeutic approaches are urgently required.MicroRNAs (miRNA), a group of small noncoding RNAs of about 22 nucleotides in length, negatively regulate gene expression, primarily through interaction with the 3’untranslated region (3’UTR) of target mRNAs. With the development of the microarray technique, there are a bunch of microRNAs (miRNA) have been revealed to be dysregulated in human tumor tissue and played as crucial modulators of tumorigenesis and tumor metastasis. But our understanding of their roles in ccRCC is limited.In this study, we selected one miRNA named miR-186-5p which has been reported to suppress the ovarian cancer and bladder cancer but has not been reported in ccRCC and aimed to confirm the role of miR-186-5p in ccRCC metastasis and angiogenesis and then dissect the downstream detail mechanism.Methods and results:1. To confirm the function of miR-186-5p in clear cell renal cell carcinoma metastasis using the bioinformatics software.Analyze from the data of miRNA microarray for metastatic clear cell renal cell carcinoma download from the GEO DataSets, we found that the expression of miR-186-5p in clear cell renal cell carcinoma from patients with distant metastases are lower than in tumors from metastasis-free patients. Importantly, using a database in the TCGA, which included gene expression data from 356 primary clear cell renal cell carcinoma samples, we identified that patients with higher miR-186-5p expression had significantly higher recurrence-free survival and higher overall survival compared to patients with lower miR-186-5p expression.2. To confirm the role of miR-186-5p in ccRCC cell invasion and its downstream target gene in vitro.After manipulating the expression of miR-186-5p in the ccRCC cell line, the transwell invasion assay showed that miR-186-5p could suppress the invasion ability of the ccRCC cells. The gene of CDC42 has been predicted as the downstream target gene of miR-186-5p by the bioinformatics software and was validate by qRT-PCR assay and western blot assay. The dual-luciferase reporter assay showed that the CDC42 was the direct target of miR-186-5p and the rescue assay showed that knocking down CDC42 could partially reverse the miR-186-5p suppressed ccRCC invasion.3. To confirm the role miR-186-5p in ccRCC angiogenesis and its downstream target gene in vitro.After manipulating the expression of miR-186-5p in the ccRCC cell line, the tube formation assay showed that miR-186-5p could suppress the tube formation of the HUVEC cells co-cultured with ccRCC cells. The gene of VEGFA has been predicted as the downstream target gene of miR-186-5p by the bioinformatics software and was validate by qRT-PCR assay and western blot assay. The dual-luciferase reporter assay showed that the VEGFA was the direct target of miR-186-5p and the rescue assay showed that using VEGFA neutralizer could partially reverse the miR-186-5p suppressed ccRCC angiogenesis.Conclusion:In summary, the miR-186-5p serves as a potential biomarker for discriminating metastatic from non-metastatic ccRCC. It also serves as a suppressor of ccRCC cell invasion and metastasis by directly targeting the gene of CDC42. Lastly, the miR-186-5p serves as a suppressor of ccRCC angiogenesis by directly targeting the gene of VEGFA.