Mir-135a enhances cellular proliferation through post-transcriptionally regulating PHLPP2 and FOXO1 in human bladder cancer

Abstract

Background: Bladder cancer is the most common malignancy in urinary system and the ninth most common malignancy in the world. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation and play essential roles during cancer development. We investigated the expression of miR-135a in bladder cancer and explored its bio-function during bladder cancer progression.

Methods: The expression of miR-135a in bladder cancer cells and tissues are performed by using Real-time PCR assay. Cell viability assay (MTT assay), colony formation assay, anchorage-independent growth ability assay and Bromodeoxyuridine labeling and immunofluorescence (BrdUrd) assay are used to examine cell proliferative capacity and tumorigenicity. Flow cytometry analysis is used to determine cell cycle progression. The expressions of p21, p27, CyclinD1, Ki67, PHLPP2 and FOXO1 are measured by Western blotting assay. Luciferase assay is used to confirm whether FOXO1 is the direct target of miR-135a.

Results: miR-135a is upregulated in bladder cancer cells and tissues. Enforced expression of miR-135a promotes bladder cancer cells proliferation, whereas inhibition of miR-135a reverses the function. Furthermore, for the first time we demonstrated PHLPP2 and FOXO1 are direct targets of miR-135a and transcriptionally down-regulated by miR-135a. Suppression of PHLPP2 or FOXO1 by miR-135a, consisted with dysregulation of p21, p27, Cyclin D1 and Ki67, play important roles in bladder cancer progression.

Conclusion: Our study demonstrates that miR-135a promotes cell proliferation in bladder cancer by targeting PHLPP2 and FOXO1, and is performed as an onco-miR.

Figure 1

Expression of miR-135a is elevated in bladder cancer cells. A. Real-time PCR analysis of miR-135a expression in bladder cancer cells, including primary culture cells of bladder cancer tissues (shown as T#1, T#2, T#3) and bladder cancer cell lines, EJ, T24, BIU87, SCaBER, and 5637, compared to normal bladder cells as controls. The normal control for bladder cancer cell lines are primary cultures of normal bladder epithelial cells established from fresh specimens of normal bladder tissues (shown as NC #1, #2, #3). B. The expression of miR-135a was examined in seven pairs of cancerous tissues (T) and their adjacent non-cancerous bladder tissues (ANT). The average miR-135a expression was normalized using U6 expression. Bars represent the mean ± SD of three independent experiments. **P <0.01, *P <0.05.

Figure 2

MiR-135a induces proliferation of bladder cancer cells. A. Effects of miR-135a on proliferation of the indicated cells, as analyzed by MTT assays. B. Representative micrographs (left) and quantifications (right) of crystal violet stained colonies formed by the indicated cells. C. Effects of ectopic miR-135a expression on the tumorigenicity of the indicated cells, as determined by anchorage-independent growth ability assays. D. Effects of ectopic miR-135a expression on cell cycle progression of the indicated cells, as analyzed by flow cytometry. E. Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in the indicated cells. All these experiments were done with T24 and 5637 cells stably overexpressing miR-135a or pMSCV-vector. Bars represent the mean ± SD of three independent experiments. *P <0.05.

Figure 3

Inhibition of miR-135a suppresses the growth of bladder cancer cells. A. Effects of miR-135a inhibitor (shown as miR-135a-in) or negative control (shown as NC) on the proliferation of bladder cancer cells, as analyzed by MTT assays. B. Representative micrographs (left) and quantifications (right) of crystal violet stained colonies formed by the indicated cells. C. Effects of miR-135a inhibitor or negative control on the tumorigenicity of the indicated cells, as determined by anchorage-independent growth ability assays. D. Effects of miR-135a inhibitor or negative control on cell cycle progression of the indicated cells, as analyzed by flow cytometry. E. Representative micrographs (left) and quantification (right) of the BrdUrd incorporation assay in the indicated cells. Bars represent the mean ± SD of three independent experiments. *P <0.05.

Figure 4

MiR-135a modulates expression of cell cycle regulators, including p21 ^Cip1 , p27 ^Kip1 , Cyclin D1 and Ki67. A. Western blotting analysis of p21^Cip1, p27^Kip1 , Cyclin D1 and Ki67 in indicated bladder cancer cells. α-Tubulin was used as a loading control. B. Real-time PCR analysis of p21 ^Cip1, p27 ^Kip1, Cyclin D1 and Ki67 mRNA in bladder cancer cells transfected with miR-135a or NC. C. Real-time PCR analysis of p21 ^Cip1 , p27 ^Kip1, Cyclin D1 and Ki67 mRNA in bladder cancer cells transfected with the miR-135a inhibitor or NC. GAPDH was used as a loading control. Bars represent the mean ± SD values of three independent experiments; *P < 0.05.

Figure 5

PHLPP2 and FOXO1 are potential targets of miR-135a. A. Sequence of PHLPP2- or FOXO1-3′UTR miR-135a binding seed region, miR-135 and mutation of miR-135a (shown as miR-135a-mut). B. Upper: Western blotting analysis of PHLPP2 or FOXO1 in bladder cancer tissues, compared with normal bladder tissue. α-Tubulin was used as a loading control. Lower: Real-time PCR analysis of miR-135a in bladder cancer tissues, and the correlation between miR-135a and PHLPP2 or FOXO1 expression in bladder cancer tissues. C. The expression level of PHLPP2 or FOXO1 protein in bladder cancer cells transfected with miR-135a or miR-135a inhibitor, respectively, compared with control cells (shown as NC), by Western blotting analysis. α-Tubulin was used as a loading control. D. PHLPP2 or FOXO1 luciferase reporter activity in the indicated cells, measured by luciferase assay. pRL-TK Renilla luciferase was used as the normalization control; Bars represents the mean ± SD of three independent experiments. *P <0.05.

Figure 6

MiR-135a promotes proliferation of bladder cancer cells by inhibiting PHLPP2 and FOXO1. A. The expression level of PHLPP2 or FOXO1 in miR-135a-inhibitor transfected bladder cells that were transfected with PHLPP2- or FOXO1-siRNA, measured by Western blotting assays; α-Tubulin served as the loading control. B. The growth rates in PHLPP2- or FOXO-silenced cells, measured by MTT assays. C. Representative micrographs (left) and quantifications (right) of crystal violet stained colonies formed by the indicated cells. D. Representative images (left) and quantifications (right) of colonies formed by the indicated cells determined by anchorage-independent growth assay. Bars represent the mean ± SD from three independent experiments. *P < 0.05.

Figure 7

The model of miR-135a-mediated Akt signaling activation through down-regulation of PHLPP2 and FOXO1 that results in the promotion of bladder cancer cell proliferation and tumorigenesis.