RUNX2 interacts with SCD1 and activates Wnt/β-catenin signaling pathway to promote the progression of clear cell renal cell carcinoma

Abstract

Background: Previous studies have demonstrated that Runt-associated transcription factor 2 (RUNX2) serves as the main transcription factor for osteoblast differentiation and chondrocyte maturation. RUNX2 is related to a variety of tumors, particularly tumor invasion and metastasis, while the expression and molecular mechanisms of RUNX2 in clear cell renal cell carcinoma (ccRCC) keep to be determined. Stearyl CoA desaturase 1 (SCD1), an endoplasmic reticulum fatty acid desaturase, transfers saturated fatty acids to monounsaturated fatty acids, is expressed highly in numerous malignancies.

Methods: The Cancer Genome Atlas (TCGA) datebase and Western blot was used to analyzed the mRNA and protein levels of the target gene in ccRCC tissues and adjacent tissues. The proliferation ability of ccRCC cells was tested by colony forming and EdU assay. The migration ability of cells was detected by transwell assay. Immunoprecipitation was utilized to detect protein-protein interaction. Cycloheximide chase assay was used to measure the half-life of SCD1 protein.

Results: In this study, the expressions of RUNX2 and SCD1 are increased in ccRCC tissues as well as ccRCC cell lines. Both RUNX2 and SCD1 could promote proliferation and migration in ccRCC cells. Furthermore, RUNX2 could physically interact with SCD1. In addition, the functional degradation and the inactivation of Wnt/β-catenin signaling pathway triggered by the downregulation of RUNX2 could be partly offset by the overexpression of SCD1.

Conclusion: The findings indicate that the RUNX2/SCD1 axis may act as a potential therapeutic target via the Wnt/β-catenin signaling pathway of ccRCC.

Keywords: Wnt/β-catenin; clear cell renal cell carcinoma; progression; runt-related transcription factor 2; stearyl CoA desaturase 1.

FIGURE 1

mRNA expression levels of RUNX2 and SCD1 in RCC from TCGA database. (A, B) RUNX2 was expressed at higher levels in KIRC and KIRP, whereas RUNX2 was downregulated in KICH. SCD1 was highly expressed in KICH, KIRC, and KIRP. (C, D) RUNX2 (left) or SCD1 (right) expression was not correlated with the pathological stage. (E) The mRNA expression of SCD1 was positively correlated to the mRNA expression of RUNX2 which is revealed by the TCGA platform at http://gepia.cancer‐pku.cn. KIRC, clear cell renal cell carcinoma; KICH, chromophobe renal cell carcinoma; KIRP, papillary renal cell carcinoma; red, tumor; blue, normal. p < 0.05 was considered significant, ns, not significant.

FIGURE 2

RUNX2 and SCD1 were expressed at high levels in ccRCC cell lines and ccRCC tissues. (A) RUNX2 and SCD1 protein levels in ccRCC tissues (T) and adjacent normal tissues (N). (B, C) RUNX2 and SCD1 were expressed at a significantly higher level in tumor tissues in comparison with normal tissues. (D) The protein expression of SCD1 was positively correlated to the protein expression of RUNX2 in ccRCC tissues. (E, F) The protein expression of RUNX2 was differently expressed among ccRCC cell lines. p < 0.05 was considered significant, ns, not significant.

FIGURE 3

Downregulation of RUNX2 protein could inhibit proliferation and migration of ccRCC. (A) The protein level of RUNX2 was detected by western blot in 786‐O and ACHN cells transfected with shRNA‐RUNX2. (B) The migration ability of 786‐O and ACHN cells was detected by transwell assay (magnification×20). (C) The proliferation ability of 786‐O and ACHN cells was detected by EdU assay (magnification×200). (D) The colony formation ability of 786‐O and ACHN cells was detected by colony formation assay. p < 0.05 was considered significant, ns, not significant.

FIGURE 4

Upregulation of RUNX2 protein could promote proliferation and migration of ccRCC. (A) The protein level of RUNX2 was detected by western blot in CAKI‐1 and OS‐RC‐2 cells transfected with LV‐RUNX2. (B) The migration ability of CAKI‐1 and OS‐RC‐2 cells was detected by transwell assay (magnification×20). (C) The proliferation ability of CAKI‐1 and OS‐RC‐2 cells was detected by EdU assay (magnification×200). (D) The colony formation ability of CAKI‐1 and OS‐RC‐2 cells was detected by colony formation assay. p < 0.05 was considered significant, ns, not significant.

FIGURE 5

Wnt/β‐catenin pathway was activated by the overexpression of RUNX2. (A) The expression of β‐catenin was detected by western blot in 786‐O and ACHN cells transfected with shRNA‐RUNX2. (B) The expression of β‐catenin was detected by western blot in CAKI‐1 and OS‐RC‐2 cells transfected with LV‐RUNX2. (C) RUNX2 and β‐catenin protein levels in ccRCC tissues (T) and adjacent normal tissues (N). (D) The protein expression of β‐catenin was positively correlated to the protein expression of RUNX2 in ccRCC tissues. p < 0.05 was considered significant, ns, not significant.

FIGURE 6

RUNX2 regulated the SCD1 protein expression in ccRCC cells. (A) The mRNA level of RUNX2 and SCD1 was determined by RT‐qPCR in 786‐O and ACHN cells transfected with shRNA‐RUNX2. (B) The protein level of RUNX2 and SCD1 was determined by western blot in 786‐O and ACHN cells transfected with shRNA‐RUNX2. (C) The mRNA level of RUNX2 and SCD1 was determined by RT‐qPCR in CAKI‐1 and OS‐RC‐2 cells transfected with LV‐RUNX2. (D) The protein level of RUNX2 and SCD1 was determined by western blot in CAKI‐1 and OS‐RC‐2 cells transfected with LV‐RUNX2. (E) The fold enrichment of RUNX2 on SCD1 promotor in 786‐O and ACHN cells with high RUNX2 expression. (F) The physical interaction between RUNX2 and SCD1 in 786‐O and ACHN cells was examined by co‐immunoprecipitation assay. p < 0.05 was considered significant, ns, not significant.

FIGURE 7

The decline of cell ability in proliferation and migration caused by RUNX2 knockdown was partially reversed with SCD1 overexpression. (A) Cells transfected with shRNA‐Ctrl and shRNA‐RUNX2 were transfected with LV‐Ctrl or LV‐SCD1, and the mRNA expression of RUNX2 and SCD1was determined by RT‐qPCR. (B) The protein expression of β‐catenin, RUNX2, and SCD1 was detected by Western blot. (C) The migration ability of 786‐O and ACHN cells was examined by transwell assay (magnification × 20). (D) The proliferation ability of 786‐O and ACHN cells was determined by EdU assay (magnification × 200). (E) The colony formation ability of 786‐O and ACHN cells was determined by colony formation assay. p < 0.05 was considered significant, ns, not significant.

FIGURE 8

The elevated potential of cell proliferation and migration caused by RUNX2 overexpression was partially restored by the downregulation of SCD1. (A) Cells transfected with LV‐Ctrl and Lv‐RUNX2 were transfected with shRNA‐Ctrl or shRNA‐SCD1, and the mRNA expression of RUNX2 and SCD1was determined by RT‐qPCR. (B) The protein expression of β‐catenin, RUNX2, and SCD1 was detected by Western blot. (C) The migration ability of CAKI‐1 and OS‐RC‐2 cells was examined by transwell assay (magnification × 20). (D) The proliferation ability of CAKI‐1 and OS‐RC‐2 cells was determined by EdU assay (magnification × 200). (E) The colony formation ability of CAKI‐1 and OS‐RC‐2 cells was determined by colony formation assay. p < 0.05 was considered significant, ns, not significant.