Proliferation rates were determined at day 1, 2, 3, 4 post-transfection, and quantification was done on a microtiter plate reader (Spectra Rainbow, Tecan) according to the manufacturer’s protocol. Meanwhile, the mimic-transfected cells were trypsinized and replated at 200 cells per well in 6-well plates, cultured for 7 days, then fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min. Western blotting Whole-cell lysate or nuclear extract

was subjected to Western blot analysis as described previously [21]. The following antibodies were used for Western blot: GAPDH (10494-1-AP, Proteintech), PTEN (22034-1-AP, Proteintech). Statistics The statistical HDAC cancer analyses for miR-19a expression in clinical samples, correlation of miR-19a expression with patients’ clinicopathological variables were conducted using the Bonferroni multiple-comparison test. The other statistical analyses were evaluated by independent samples T test (two-tailed). P ≤ 0.05 was considered statistically significant. Results miR-19a is up-regulated in bladder cancer cells To analyze the expression of miR-19a in bladder cancer, q-PCR using Taqman probes

was conducted to measure the levels of miR-19a. We firstly examined the expression of mature miR-19a in immortalized human bladder epithelium (HCV29 and HU609) cells and five human bladder cancer cell lines (J82, HT1376, RT4, T24 and TCCSUP). The expression level of miR-19a in bladder cancer cell lines was significant higher than that in the normal bladder epithelium cells. Expression level of miR-19a in RT4 was a little lower GANT61 mw than that in the four other bladder cancer cell lines (Figure 1A). These data Blebbistatin in vivo demonstrated that the up-regulation of miR-19a might be relevant to the genesis and development of bladder cancer. second Figure 1 miR-19a is significantly up-regulated in bladder

cancer cell lines and in bladder cancer tissues. (A) The expression level of miR-19a in two immortalized human bladder epithelium cells (HCV29 and HU609) and five bladder cancer cell lines (J82, HT1376, RT4, T24 and TCCSUP). Data are shown as mean + s.d. (n = 3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001. (B) The relative expression of miR-19a in 100 pairs of bladder cancer (C) and adjacent non-neoplastic tissues (N). (C) Normalized expression of miR-19a in 100 pairs of bladder cancer and adjacent normal tissues. (D) The correlation of miR-19a expression with tumor grades of bladder cancer tissues. miR-19a is up-regulated in bladder cancer tissues compared with the corresponding adjacent non- neoplastic tissues To further analyze the expression of miR-19a in patients with bladder cancer, we measured the levels of miR-19a in 100 pairs of bladder cancer tissues (C) and the adjacent non-neoplastic tissues (N).