Regulation of spindle and kinetochore-associated protein 1 by antitumor miR-10a-5p in renal cell carcinoma

Abstract

Analysis of our original microRNA (miRNA) expression signature of patients with advanced renal cell carcinoma (RCC) showed that microRNA-10a-5p (miR-10a-5p) was significantly downregulated in RCC specimens. The aims of the present study were to investigate the antitumor roles of miR-10a-5p and the novel cancer networks regulated by this miRNA in RCC cells. Downregulation of miR-10a-5p was confirmed in RCC tissues and RCC tissues from patients treated with tyrosine kinase inhibitors (TKI). Ectopic expression of miR-10a-5p in RCC cell lines (786-O and A498 cells) inhibited cancer cell migration and invasion. Spindle and kinetochore-associated protein 1 (SKA1) was identified as an antitumor miR-10a-5p target by genome-based approaches, and direct regulation was validated by luciferase reporter assays. Knockdown of SKA1 inhibited cancer cell migration and invasion in RCC cells. Overexpression of SKA1 was observed in RCC tissues and TKI-treated RCC tissues. Moreover, analysis of The Cancer Genome Atlas database demonstrated that low expression of miR-10a-5p and high expression of SKA1 were significantly associated with overall survival in patients with RCC. These findings showed that downregulation of miR-10a-5p and overexpression of the SKA1 axis were highly involved in RCC pathogenesis and resistance to TKI treatment in RCC.

Keywords: miR-10a-5p; MicroRNA; renal cell carcinoma; spindle and kinetochore-associated protein 1; tyrosine kinase inhibitor resistance.

Figure 1

Kaplan‐Meier survival curves based on miR‐10a‐5p expression in patients with clear cell renal cell carcinoma (ccRCC), and schematic representation of the chromosomal location of human miR‐10a. (a) Kaplan‐Meier survival curve for overall survival rate based on miR‐10a‐5p expression in patients with ccRCC from The Cancer Genome Atlas (TCGA) database. (b) miR‐10a is located on human chromosome 17q21.32. Mature microRNAs (miRNAs), miR‐10a‐5p (guide strand) and miR‐10a‐3p (passenger strand), are derived from pre‐miR‐10a.

Figure 2

Expression levels of miR‐10a‐5p in clear cell renal cell carcinoma (ccRCC) clinical specimens and functional significance of miR‐10a‐5p in ccRCC cells. (a) Expression levels of miR‐10a‐5p in ccRCC clinical specimens and cell lines determined using qRT‐PCR. RNU48 was used as an internal control. TKI, tyrosine kinase inhibitor. (b) Cell proliferation was determined by XTT assays 72 h after transfection with 10 nM miR‐10a‐5p. *P < 0.0001, **P < 0.01. (c) Cell migration activity was assessed by wound‐healing assays 48 h after transfection with 10 nM miR‐10a‐5p. *P < 0.0001. (d) Cell invasion activity was characterized by invasion assays 48 h after transfection with 10 nM miR‐10a‐5p. *P < 0.0001.

Figure 3

Identification of miR‐10a‐5p target genes. Flow chart of the strategy for identification of miR‐10a‐5p target genes.

Figure 4

Kaplan‐Meier survival curves based on SKA1 expression in patients with clear cell renal cell carcinoma (ccRCC), and expression levels of SKA1 in ccRCC clinical specimens. (a) Kaplan‐Meier survival curve for overall survival rate based on SKA1 expression in patients with ccRCC. (b) Expression levels of SKA1 in ccRCC clinical specimens and cell lines. GUSB was used as an internal control. TKI, tyrosine kinase inhibitor. (c) Negative correlation between miR‐10a‐5p and SKA1.

Figure 5

Kaplan‐Meier survival curves for overall survival rates based on expression of seven genes, excluding SKA1, in patients with clear cell renal cell carcinoma (ccRCC).

Figure 6

Expression of SKA1 in clinical clear cell renal cell carcinoma (ccRCC) specimens using a tissue microarray and autopsy tissues. Representative immunohistochemical staining for SKA1 in a ccRCC tissue microarray (cat. no. KD806; US Biomax, Inc., Rockville, MD, USA) and autopsy specimens after tyrosine kinase inhibitor (TKI) treatment (Patient D, Table 2). SKA1 was strongly expressed in ccRCC tissues. (a) Normal kidney. (b) ccRCC tissues. (c) ccRCC autopsy tissues after TKI treatment.

Figure 7

Direct regulation of SKA1 by miR‐10a‐5p in clear cell renal cell carcinoma (ccRCC) cells. (a) SKA1 mRNA expression was evaluated using qRT‐PCR in 786‐O and A498 cells 48 h after transfection with miR‐10a‐5p. GAPDH was used as an internal control. *P < 0.0001. (b) SKA1 protein expression was evaluated by western blotting in 786‐O and A498 cells 72 h after transfection with miR‐10a‐5p. GAPDH was used as a loading control. (c) miR‐10a‐5p binding site in the 3′‐UTR of SKA1 mRNA. Dual luciferase reporter assays in 786‐O using vectors encoding the putative miR‐10a‐5p target site of SKA1 3′‐UTR (positions 28‐35). Data were normalized by expression ratios of Renilla/firefly luciferase activities. *P < 0.0001.

Figure 8

Effects of SKA1 silencing on clear cell renal cell carcinoma (ccRCC) cell lines. (a) SKA1 mRNA expression was evaluated using qRT‐PCR analysis of 786‐O and A498 cells 48 h after transfection with si‐SKA1‐1 or si‐SKA1‐2. GAPDH was used as an internal control. *P < 0.0001. (b) SKA1 protein expression was evaluated by western blotting analysis of 786‐O and A498 cells 72 h after transfection with miR‐10a‐5p. GAPDH was used as a loading control. (c) Cell proliferation was determined using XTT assays 72 h after transfection with 10 nM si‐SKA1‐1 or si‐SKA1‐2. *P < 0.0001. (d) Cell migration activity was assessed by wound‐healing assays 48 h after transfection with 10 nM si‐SKA1‐1 or si‐SKA1‐2. *P < 0.0001. (e) Cell invasion activity was characterized by invasion assays 48 h after transfection with 10 nM si‐SKA1‐1 or si‐SKA1‐2. *P < 0.0001.

Figure 9

Effects of cotransfection of SKA1/miR‐10a‐5p in 786‐O cells. (a) SKA1 protein expression was evaluated by western blotting analysis of 786‐O cells 72 h after reverse transfection with miR‐10a‐5p and 48 h after forward transfection with the SKA1 vector. GAPDH was used as a loading control. (b) Cell proliferation was determined using XTT assays 72 h after reverse transfection with miR‐10a‐5p and 48 h after forward transfection with the SKA1 vector. **P < 0.01. (c) Cell migration activity was assessed by wound‐healing assays 48 h after reverse transfection with miR‐10a‐5p and 24 h after forward transfection with the SKA1 vector. *P < 0.0001. (d) Cell invasion activity was characterized by invasion assays 48 h after reverse transfection with miR‐10a‐5p and 48 h after forward transfection with SKA1 vector. *P < 0.0001.

Figure 10

Kaplan‐Meier survival curve based on SKA1 expression in patients with clear cell renal cell carcinoma (ccRCC), and expression levels of SKA1 according to TNM stage, T stage, and histological grade. (a) Kaplan‐Meier survival curves for disease‐free survival rate based on SKA1 expression in patients with ccRCC. (b‐d) Expression levels of SKA1 were significantly increased in cases of advanced TNM stage, advanced T stage, and advanced histological grade. *P < 0.01, **P < 0.001, ***P < 0.0001.

Figure 11

Effects of the gene encoding SKA1 protein on downstream signaling. Knockdown of SKA1 and restoration of miR‐10a‐5p in 786‐O cells reduced the phosphorylation of ERK1/2, AKT, FAK and SRC. GAPDH was used as a loading control.