MicroRNA-495 suppresses human renal cell carcinoma malignancy by targeting SATB1

Abstract

Deregulated expression of miRNAs is related to progression and initiation of human cancers. Although miR-495 has identified in various tumors, its expression and function in renal cell carcinoma (RCC) is still unknown. In this study, we found that the expression of miR-495 was downregulated in RCC cell lines and tissues. Ectopic expression of miR-495 induced G0/G1 phase arrest and suppressed cell proliferation and migration in RCC cell lines. We further validated SATB1 was a direct target of miR-495 in RCC. In addition, re-expression of SATB1 reversed the miR-495-induced inhibition of cell proliferation and migration. These data suggest that miR-495 functions as a tumor suppressor and may be a promising therapeutic target in RCC in the future.

Keywords: Renal cell carcinoma; SATB1; miR-495; microRNAs.

Figure 1

miR-495 was downregulated in RCC cell lines and tissues (A) qRT-PCR analysis was used to detect the expression of miR-495 in four human renal cell carcinoma cells lines (769-P, 786-O, A498, SN12-PM6) and one normal renal cell line (HK-2). (B) qRT-PCR analysis was used to detect the expression of miR-495 in clinical RCC specimens and normal tissues. (C) The relative expression of miR-495 was downregulated in clinical RCC specimens compared with their corresponding nontumor tissues.

Figure 2

Re-expression of miR-495 suppressed RCC cell proliferation (A) qRT-PCR was performed to measure the miR-495 expression in 786-O cells at 48 hours after miR-495 mimic transfection. (B) Cell cycle assays showed that 786-O cells transfected with miR-495 mimics had an obvious cell cycle arrest at the G0/G1 phase. (C) Re-expression of miR-495 suppressed 786-O cells proliferation using CCK-8 assay.

Figure 3

Overexpression of miR-495 inhibited RCC cell migration. Overexpression of miR-495 repressed the 786-O cells migration.

Figure 4

miR-495 downregulated SATB1 expression by directly targeting its 3’-UTR. A. SATB1 gene harbored a potential miR-495 binding site using a stringent bioinformatics approach. B. Luciferase reporter assay showed that the luciferase reporter activity decreased approximately 67% in the 786-O cells containing the SATB1 WT 3’UTR fragment. C. The ectopic expression of miR-495 suppressed the SATB1 mRNA by using qRT-PCR. D. Overexpression of miR-495 inhibited the SATB1 protein by using Western blot.

Figure 5

Re-expression of SATB1 reversed the miR-495-induced inhibition of cell proliferation and migration. A. The protein expression of SATB1 was measured by using Western blot. B. The data of CCK8 assay showed that re-expression of SATB1 increased the miR-495 overexpressing 786-O cells proliferation. C. Cell cycle assays showed that miR-495 overexpressing 786-O cells transfected with SATB1 vector had an obvious cell cycle at the S phase. D. The migration abilities of miR-495 overexpressing 786-O cells were increased after SATB1 transfection.