miR-106b-5p promotes renal cell carcinoma aggressiveness and stem-cell-like phenotype by activating Wnt/β-catenin signalling

Abstract

Purpose: To examine the role of miR-106b-5p in regulating the cancer stem-cell-like phenotype in clear cell renal cell carcinomas (ccRCC).

Experimental design: Real-time PCR was performed to evaluate miR-106b-5p levels in ccRCC cell lines and patients specimens. A series of in vivo and in vitro assays were performed to confirm the effect of miR-106b-5p on ccRCC stemness phenotype.

Results: ccRCC cells and tissues expressed more miR-106b-5p than normal controls. Gain- and loss-of-function studies demonstrated that overexpression of miR-106b-5p in ccRCC cells increased the spheres formation ability and the proportion of side population cells. Ectopic expression of miR-106b-5p in ccRCC cells increased tumour growth rates and the number of metastatic colonies in the lungs by using an orthotopic kidney cancer model and a tail vein injection model, respectively. Mechanistic studies revealed that, miR-106b-5p has an activating effect on Wnt/β-catenin signalling. miR-106p-5p overexpression simultaneously targets multiple negative regulators of the Wnt/β-catenin pathway, namely, LZTFL1, SFRP1 and DKK2. In addition, we also confirmed that miR-106b-5p and its targets expression correlates with the overall-survival of ccRCC patients from TCGA.

Conclusions: These findings suggest that miR-106b-5p mediates the constitutive activation of Wnt/β-catenin signalling, likely serving as a potential therapeutic target for ccRCC.

Keywords: miR-106b-5p; renal cell carcinoma; stemness; tumorigenesis; wnt signalling.

Figure 1. miR-106b-5p is overexpressed in ccRCC cell lines and tissues

(A) RT–qPCR analysis of miR-106b-5p expression in ccRCC Cell lines, including an immortalized proximal tubule epithelial cell line HK2 and a panel of seven human ccRCC cell lines. (B) Relative expression of miR-106b-5p in 20 pairs of ccRCC tumour tissues (T) and their corresponding adjacent non-cancerous tissues (ANT). (C) Relative expression of miR-106b-5p expression in the indicated cell lines using real-time PCR. Each bar represents the mean ± s.e.m. derived from three independent experiments. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05).

Figure 2. miR-106b-5p promotes stem cell-like properties in ccRCC cells in vitro

(A) Representative images and quantification of spheres formed by the indicated cells. (B) Hoechst 33342 dye exclusion assay showing that overexpressing miR-106b-5p increased the SP cell proportions in the indicated cells, whereas silencing miR-106b-5p decreased these proportions. (C) RT–qPCR analysis of the expression levels of cancer stemness associated markers, including SOX2, OCT4, ABCG2, CD105, and CXCR4 in miR-106b-5p-overexpressing and miR-106b-5p-silenced cells compared with the corresponding control cells. Each bar represents the mean±s.e.m derived from three independent experiments. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05).

Figure 3. miR-106b-5p enhances the tumorigenicity and metastasis of ccRCC cells in vivo

(A) In total, 3 × 10⁶, 3 × 10⁵ and 3 × 10⁴ Caki-1-vector and Caki-1-miR-106b-5p-transfected cells mixed with Matrigel were implanted in the renal capsules of BALB/c nude mice (n = 5 per group). Representative images of the tumours are shown. (B) Representative image of H&E staining showing orthotopic xenograft tumors in the mouse kidney. (C) Tumour formation frequencies in orthotopic xenograft tumor model for different numbers of the indicated cells are shown (D) The number of nodules was qualified on lungs of BALB/c nude mice (n = 10 per group) 6 weeks after tail vein injection of A498-vector cells and A498-miR-106b-5p. (E) Representative metastatic lesions stained by H&E in the lungs of mice. Arrows denotes the metastatic colonies in the lung tissues. The data are presented as the mean ± s.e.m. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05).

Figure 4. miR-106b-5p activates Wnt/β-catenin signalling

(A) RT–qPCR analysis of the expression of the established downstream targets for the Wnt/β-catenin pathway, including TWIST, MYC, MMP7, CCND1, CD44, BMP4 and FGF18, in the indicated cells. (B) Altered nuclear translocation of β-catenin in response to ectopic miR-106b-5p expression. Nuclear fractions of the indicated cells were analysed by western blot analysis. HDAC1 was used as a loading control. (C) Subcellular β-catenin localization in the indicated cells was assessed by immunofluorescence staining. (D) Luciferase assay of TCF/LEF transcriptional activity in indicated cells. (E) Representative images and quantification of cellular spheres formed by the indicated cells. (F) Tumour formation frequencies for the different numbers of indicated cells. Each bar represents the mean ± s.e.m. derived from three independent experiments. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05, NS: not statistically significant).

Figure 5. miR-106b-5p directly targets multiple negative regulators of the Wnt pathway

(A) Predicted binding sites of miR-106b-5p in the wild type 3′-UTRs of LZTFL1, SFRP1 and DKK2. Mutations in these 3′-UTRs are highlighted in red. (B, C) Western blot and RT–qPCR analyses of LZTFL1, SFRP1 and DKK2 expression in the indicated cells. (D) Luciferase activity of reporters with wild type or mutant 3′-UTRs of LZTFL1, SFRP1 and DKK2 in the indicated cells co-transfected with the indicated oligonucleotides. Each bar represents the mean ± s.e.m. derived from three independent experiments. A two-tailed Student's t-test was used for statistical analysis (*P < 0.05).

Figure 6. Upregulation of miR-106b-5p and downregulation of LZTFL1, SFRP1 and DKK2 is correlated with poor prognosis in ccRCC

(A) Kaplan–Meier analysis of the correlation between the miR-106b-5p level and the OS of 504 patients with ccRCC in the TCGA cohort. (B–D) Kaplan–Meier analysis of the mRNA expression levels of LZTFL1, SFRP1 and DKK2 correlated with the OS of 533 patients with ccRCC in the TCGA cohort.