Tumor-suppressive microRNA-135a inhibits cancer cell proliferation by targeting the c-MYC oncogene in renal cell carcinoma

Abstract

Recently, many studies have suggested that microRNAs (miRNAs) are involved in cancer cell development, invasion, and metastasis of various types of human cancers. In a previous study, miRNA expression signatures from renal cell carcinoma (RCC) revealed that expression of microRNA-135a (miR-135a) was significantly reduced in cancerous tissues. The aim of this study was to investigate the functional significance of miR-135a and to identify miR-135a-mediated molecular pathways in RCC cells. Restoration of mature miR-135a significantly inhibited cancer cell proliferation and induced G0 /G1 arrest in the RCC cell lines caki2 and A498, suggesting that miR-135a functioned as a potential tumor suppressor. We then examined miR-135a-mediated molecular pathways using genome-wide gene expression analysis and in silico analysis. A total of 570 downregulated genes were identified in miR-135a transfected RCC cell lines. To investigate the biological significance of potential miR-135a-mediated pathways, we classified putative miR-135a-regulated genes according to the Kyoto Encyclopedia of Genes and Genomics pathway database. From our in silico analysis, 25 pathways, including the cell cycle, pathways in cancer, DNA replication, and focal adhesion, were significantly regulated by miR-135a in RCC cells. Moreover, based on the results of this analysis, we investigated whether miR-135a targeted the c-MYC gene in RCC. Gain-of-function and luciferase reporter assays showed that c-MYC was directly regulated by miR-135a in RCC cells. Furthermore, c-MYC expression was significantly upregulated in RCC clinical specimens. Our data suggest that elucidation of tumor-suppressive miR-135a-mediated molecular pathways could reveal potential therapeutic targets in RCC.

Figure 1

Expression of miR‐135a in RCC cell lines and RCC clinical specimens. (a) Expression of miR‐135a in RCC cell lines. MiR‐135a expression was significantly downregulated in RCC cell lines (caki2 and A498) as compared with normal kidney cells. **P < 0.0001. (b) Expression of miR‐135a in clinical RCC specimens. MiR‐135a expression levels are expressed as compared with adjacent non‐cancerous kidney tissues. RNU48 was used as the internal control. (c) Normalized relative expression of miR‐135a is shown in the bar chart. White and black bars indicate expression levels in normal and tumor tissues, respectively.

Figure 2

Effect of mature miR‐135a transfection in the RCC cell lines. (a) Cell proliferation was determined in caki2 and A498 cell lines using XTT assays 72 h after transfection with 10 nM miR‐135a or miR‐control. MiR‐135a transfection significantly inhibited cell proliferation as compared with miR‐control transfection. *P < 0.05. (b) Cell invasion activity was determined by Matrigel invasion assay 48 h after miR‐135a transfection. Relative to miR‐control transfection, the number of invasive cells decreased significantly after miR‐135a transfection. *P < 0.05. (c) Cell cycle analysis of miR‐135a and miR‐control transfectants. G₀/G₁ arrest was induced by miR‐135a transfection. **P < 0.0001 relative to the miR‐control.

Figure 3

Direct regulation of MYC by miR‐135a in the RCC cell lines. (a) MYC mRNA expression in RCC cell lines (caki2 and A498) compared to normal kidney RNA. GUSB was used as an internal control. **P < 0.0001. (b) MYC mRNA expression in RCC cell lines (caki2 and A498) 24 h after transfection with 10 nM miR‐135a. GUSB was used as an internal control. *P < 0.05. (c) MYC protein expression in RCC cell lines (caki2 and A498) 72 h after transfection with 10 nM miR‐135a. GAPDH was used as a loading control. **P < 0.0001, *P < 0.05. (d) MiR‐135a binding site in the 3′UTR of MYC mRNA. Luciferase assays were performed using a vector encoding the full‐length 3′UTR of MYC mRNA. Renilla luciferase values were normalized to firefly luciferase values. *P < 0.05.

Figure 4

Effect of MYC silencing by si‐MYC in A498 cell line. (a) MYC mRNA expression 24 h after transfection with 10 nM si‐MYC in A498 RCC cells. GUSB was used as an internal control. **P < 0.0001. (b) MYC protein expression 72 h after transfection with si‐MYC. GAPDH was used as a loading control. **P < 0.0001. (c) Cell proliferation was determined by XTT assays in A498 cells 72 h after transfection with 10 nM si‐MYC or si‐control. Cell proliferation was significantly inhibited in si‐MYC‐transfected cells in comparison with mock or si‐control‐transfected cells. **P < 0.0001. (d) Cell invasion was measured by Matrigel invasion assay 48 h after transfection of si‐MYC. Cell numbers significantly decreased after si‐MYC transfection. **P < 0.0001 versus mock‐ and si‐control‐transfected cells.

Figure 5

MYC and CCND1 mRNA expressions in RCC specimens and normal kidney tissues. (a) Heat map diagram of 15 genes related to “Cell cycle” pathway based on GEO data sets (53 RCC specimens and 23 adjacent non‐cancerous kidney specimens); the results demonstrate that MYC was upregulated in RCC clinical specimens. (b,c) Expression of MYC and CCND1mRNA in clinical RCC specimens. Relative MYC and CCND1mRNA expression levels are shown in box plots.