Login to Access Video or Poster Abstract: MP60-16
Sources of Funding: none

Introduction

CRISPR/Cas9 technology was introduced as an efficient, powerful and broadly used genome editing tool. The aim of this study was to utilize the CRISPR/Cas9 system to control miRNA expression in cancer research.

Methods

In our miRNA expression signatures in clear cell renal cell carcinoma (ccRCC), we focused on miR-210-3p which was one of the most upregulated miRNAs. We used lenti-CRISPR vector to knock out miR-210-3p with two different single guide RNAs against miR-210-3p. We performed cell function studies and xenograft assay with miR-210-3p- depleted cells. Putative target genes of miR-210-3p was examined in miRNA binding assay. In addition, overall survival between high and low expression of miR-210-3p or the target gene were analyzed by the Kaplan-Meier method.

Results

In cells transfected with sgRNA targeting miR-210-3p itself, more than 98% miR-210-3p knock out efficiency was observed in all three cell lines (786-o, A498 and Caki2). miR-210-3p depletion significantly increased invasive capacity (P < 0.01) in vitro, and dramatically promoted tumorigenesis in xenograft experiments (P = 0.0039), which was unexpected due to the fact that these miRNAs were up-regulated in RCC. We also found that twist family bHLH transcription factor 1 (TWIST1) was identified as a direct target of miR-210-3p based on target analyses (P < 0.05). The Cancer Genome Atlas (TCGA) database of ccRCC showed that there was a negative correlation between miR-210-3p and TWIST1 expression (P < 0.0001). In accordance with the results in vivo and in vitro analyses, TCGA database showed that the low miR-210-3p expression group had poor survival in comparison with the high group, and the high TWIST1 expression group had poor overall (P = 0.00054) and disease-free survival (P = 0.00347) compared to the low expression group significantly.

Conclusions

We utilized the CRISPR/Cas9 system to analyze an upregulated miRNA in cancer. CRISPR/Cas9 successfully suppressed miR-210-3p expression in RCC cells. Moreover, by using CRISPR/Cas9 techniques, we found that tumorigenesis was acquired through enhanced expression of oncogenic TWIST1 due to the suppression of miR-210-3p expression via CRISPR/Cas9 techniques. The introduction of the CRISPR/Cas9 methodologies into studies of miRNAs as well as other non-coding RNAs should provide new possibilities in cancer research.

Funding

none