MiR-532-5p suppresses renal cancer cell proliferation by disrupting the ETS1-mediated positive feedback loop with the KRAS-NAP1L1/P-ERK axis

Wei Zhai,Wei Xue,Saiyang Li,Yonghui Chen,Chen Xu,Junjie Ma,Jin Zhang,Yiran Huang,Rujian Zhu,Junhua Zheng,Jiayi Zheng,Dongkui Gong,Zhiguo Chen,Butang Li,Chen Chen

doi:10.1038/s41416-018-0196-5

Abstract

BackgroundDespite the fact that miRNAs play pivotal roles in various human malignancies, their molecular mechanisms influencing RCC are poorly understood.MethodsThe expression of miRNAs from RCC and paired normal renal specimens was analysed by a combined computational and experimental approach using two published datasets and qRT-PCR assays. The functional role of these miRNAs was further identified by overexpression and inhibition assays in vivo and in vitro. Western blots, luciferase assays, and chromatin immunoprecipitation were performed to investigate the potential mechanisms of these miRNAs.ResultsBioinformatics analysis and qRT-PCR revealed that miR-532-5p was one of the most heavily downregulated miRNAs. Overexpression of miR-532-5p inhibited RCC cell proliferation, while knockdown of miR-532-5p promoted cell proliferation. Mechanistic analyses indicated that miR-532-5p directly targets KRAS and NAP1L1. Interestingly, ETS1 suppressed the transcription of miR-532-5p by directly binding a special region of its promoter. Moreover, high levels of ETS1, as an oncogene in RCC, were significantly associated with poor survival in a large cohort of RCC specimens.ConclusionsOur work presents a road map for the prediction and validation of a miR-532-5p/KRAS-NAP1L1/P-ERK/ETS1 axis feedback loop regulating cell proliferation, which could potentially provide better therapeutic avenues for treating RCC.

Highlights

To determine the expression levels of miR-532-5p in Renal cell carcinoma (RCC), we analysed the RCC data set from the TCGA database and found that the transcriptional level of miR-532-5p was significantly downregulated in RCC tissue compared with normal renal tissue (Fig. 1c, Table S4)
We further examined the different expression levels of miR-532-5p in four RCC cell lines (786-O, OSRC-2, A498, and SN12-PM6), as well as in a human normal renal tubular epithelial cell line HK-2 and found that the expression of miR-532-5p was dramatically decreased in RCC cell lines compared with HK-2 (Fig. 1f)
The results were averaged from three experiments; error bars indicate ± 1 SD, *p < 0.05, **p < 0.01. d Western blot analysis for KRAS, NAP1L1, T-ERK, P-ERK, and ETS1 protein levels of si-KRAS-2 or si-NAP1L1-2 transfection compared to short interfering RNA (siRNA)-NC transfection in SN12-PM6 and 786-O cell lines. β-actin was used as a loading control. e CCK8 assays of 786O cells transfected with anti-miR-NC/anti-miR-53-5p and siRNA-NC/si-KRAS + si-NAP1L1. f Western blot analysis for KRAS, NAP1L1, T-ERK, PERK, and ETS1 protein levels of 786-O cells transfected with anti-miR-NC/anti-miR-53-5p and siRNA-NC/si-KRAS + si-NAP1L1. β-actin was used as a loading control these results indicate that higher levels of miR-532-5p expression might be predictive of a better prognosis in RCC

Summary

Introduction

Renal cell carcinoma (RCC) is one of the most common and aggressive human malignancies throughout the world, representing 3.7% of all new cancer cases, with about 65,340 new cases and 14,970 deaths estimated for 2018 in the United States alone.[1,2] In most cases, RCC is radiotherapy and chemotherapy resistant and best treated by surgical resection.[3,4,5] tyrosine kinase inhibitor (TKI)-based antiangiogenic therapy is the standard treatment for advanced RCC (aRCC) or metastatic RCC (mRCC), the overall prognosis remains poor due to drug resistance and other reasons.[2,6] methods for earlier diagnosis and more effective therapies for RCC are urgently required.MicroRNAs (miRNAs), a class of small noncoding RNAs with transcripts averaging 22 nucleotides in length, negatively regulate target gene expression through mRNA degradation or translational repression.[7]. METHODS: The expression of miRNAs from RCC and paired normal renal specimens was analysed by a combined computational and experimental approach using two published datasets and qRT-PCR assays. The functional role of these miRNAs was further identified by overexpression and inhibition assays in vivo and in vitro. Luciferase assays, and chromatin immunoprecipitation were performed to investigate the potential mechanisms of these miRNAs. RESULTS: Bioinformatics analysis and qRT-PCR revealed that miR-532-5p was one of the most heavily downregulated miRNAs. Overexpression of miR-532-5p inhibited RCC cell proliferation, while knockdown of miR-532-5p promoted cell proliferation. Mechanistic analyses indicated that miR-532-5p directly targets KRAS and NAP1L1. CONCLUSIONS: Our work presents a road map for the prediction and validation of a miR-532-5p/KRAS-NAP1L1/P-ERK/ETS1 axis feedback loop regulating cell proliferation, which could potentially provide better therapeutic avenues for treating RCC

Methods

Results

Conclusion