microRNA-210-3p depletion by CRISPR/Cas9 promoted tumorigenesis through revival of TWIST1 in renal cell carcinoma

Abstract

Previous studies showed that five miRNAs (miR-885-5p, miR-1274, miR-210-3p, miR-224 and miR-1290) were upregulated the most in clear cell renal cell carcinoma (ccRCC). Our focus was to understand from a clinical standpoint the functional consequences of upregulating miR-210-3p. Towards this, we utilized the CRISPR/Cas9 gene editing system to deplete miR-210-3p in RCC cell lines (786-o, A498 and Caki2) and characterized the outcomes. We observed that miR-210-3p depletion dramatically increased tumorigenesis, including altering the morphology of A498 and Caki2 cells in a manner characteristic of epithelial-mesenchymal transition (EMT). These results were corroborated by in vivo xenograft studies, which showed enhanced growth of tumors from miR-210-3p-depleted A498 cells. We identified Twist-related protein 1 (TWIST1) as a key target of miR-210-3p. Analysis of the ccRCC patient data in The Cancer Genome Atlas database showed a negative correlation between miR-210-3p and TWIST1 expression. High TWIST1 and low miR-210-3p expression associated with poorer overall and disease-free survival as compared to low TWIST1 and high miR-210-3p expression. These findings suggest that renal cell carcinoma progression is promoted by TWIST1 suppression mediated by miR-210-3p.

PHGDH as a Key Enzyme for Serine Biosynthesis in HIF2α-Targeting Therapy for Renal Cell Carcinoma

“…All cell lines were tested and found negative for mycoplasma (e-Myco Mycoplasma PCR Detection Kit; iNtRON, Korea). These cell lines were incubated as previously described (15). Gavage feeding of sunitinib (40 or 25 mg/kg, five times a week; biorbyt, CA, USA) was performed in mice.…”

“…Gavage feeding of sunitinib (40 or 25 mg/kg, five times a week; biorbyt, CA, USA) was performed in mice. The tumour sizes and weights were measured and calculated as previously described (15). The chemical structures of sunitinib is in Supplementary Figure 1A.…”

“…The sgRNAs targeting HIF2α were designed as previously described (15). The sgRNA sequence, 5′-GCTGATTGCCAGTCGCATGA-3′ or 5′-CAAGGCCTCCATCATGCGAC-3′, which targeted the exon of HIF2α, was cloned into lentiCRISPR v2.…”

“…For PHGDH overexpression in cells, we used pLJM5-WT PHGDH, which was a gift from Dr. David Sabatini (Addgene plasmid #83901) (14). We produce lentivirus as previously described (15). …”

miR-216b Post-Transcriptionally Downregulates Oncogene KRAS and Inhibits Cell Proliferation and Invasion in Clear Cell Renal Cell Carcinoma

“…Ding et al found that miR-367 expression was increased in both human ccRCC tissues and cell lines and that ectopic high expression of miR-367 could promote cell proliferation and metastasis by targeting metastasis associated protein 3 (MTA3) in ccRCC [21]. Yoshino H et al reported that miR-210-3p was downregulated in ccRCC, and the deletion of miR-210-3p promoted tumorigenesis through revival of TWIST1 [22].…”