Objective:To investigate the expression and clinical significance of long noncoding RNA (lncRNA) AP006284.1 in bladder cancer tissues, and to analyze the regulatory effect and downstream mechanism of AP006284.1 knockdown on the proliferation and migration of bladder cancer cells. Methods:The expression of AP006284.1 in bladder cancer tissues, and the relationship between AP006284.1 expression and tumor stage and disease-free survival of bladder cancer patients were analyzed in the gene expression omnibus (GEO) database. AP006284.1 gene expression in bladder cancer cell lines, including MGH-U3, T24, UMUC-3, J82 and 5637, and bladder epithelial immortalized SV-HUC-1 cell were detected by real-time reverse transcription-PCR (RT-qPCR) assay. J82 cells were divided into the control group and the transfection group, and transfected with control plasmid and AP006284.1 knockdown plasmid, respectively. The effect of AP006284.1 knockdown on the proliferation of J82 cells was detected by RT-qPCR and cell counting kit-8 (CCK-8) assay. The effect of AP006284.1 knockdown on the migration of J82 cells was determined by the scratch healing assay. The target gene of AP006284.1 was predicted by LncRNA2Target and LncRNome databases. The target fragment of wild-type AP006284.1 ( AP006284.1-Wt) or mutant AP006284.1 ( AP006284.1-Mut) was constructed into pGL3 plasmid by dual luciferase gene reporter assay. J82 cells were co-transfected with miR-205-3p or miR-negtive control (miR-NC) to validate the targeting relationship between AP006284.1 and miR-205-3p. The correlation between miR-205-3p and AP006284.1 expression in bladder cancer tissues was further analyzed by the GEO database. The effect of AP006284.1 knockdown on the expression of miR-205-3p gene in J82 cells was detected by RT-qPCR assay. The effect of AP006284.1 knockdown on the expression of phosphorylated Ras protein (p-Ras), phosphorylated Raf protein (p-Raf), phosphorylated mitogen-activated protein kinase kinase (p-MEK), and phosphorylated extracellular signal-regulated kinase (p-ERK) of the ERK pathway was detected by Western blotting in J82 cells. Results:GEO database analysis showed that the relative expression of AP006284.1 in bladder cancer tissues ( n=304) was significantly higher than that in normal tissues ( n=28, P<0.01). The relative expression of AP006284.1 was positively correlated with the tumor stage of the bladder cancer patients ( P<0.01). Compared with bladder cancer patients with low expression of AP006284.1, patients with high expression had a lower disease-free survival ( P<0.01). Compared with the SV-HUC-1 cell (1.02±0.34), the expression level of AP006284.1 gene was upregulated in MGH-U3 cell (5.77±0.37), T24 cell (3.02±0.40), UMUC-3 cell (3.62±0.59), J82 cell (7.19±0.24) and 5637 cell (5.59±0.30) (all P<0.01). The expression level of the AP006284.1 gene was the highest in J82 cells, therefore, the J82 cells were selected for the study. The expression level of AP006284.1 gene in the control group (7.20±0.26) was 6.92 times higher than that in the transfection group (1.04±0.28, t=16.16, P<0.01). Compared with the control group (0.74±0.11, 1.35±0.09, 1.63±0.14, 1.74±0.11), the absorbance ( A) values of J82 cells in the transfection group (0.49±0.06, 0.95±0.14, 1.09±0.08, 1.13±0.11) were reduced than those in the control group at the 24, 36, 48 and 60 h after AP006284.1 knockdown (all P<0.05). The migration distance of J82 cells in the control group was significantly longer than that in the transfection group. The migration rate of the control group [(65.03±6.20)%], which was 2.58 times higher than that of the transfection group [(25.22±3.45)%, t=5.61, P<0.01]. The target site of miR-205-3p containing AP006284.1 was predicted by LncRNA2Target and LncRNome databases. Compared with miR-NC group (1.00±0.11), the relative activity of dual luciferase of AP006284.1-Wt gene was significantly downregulated in the miR-205-3p group (0.31±0.07, t=5.47, P<0.01). Compared with the miR-NC group (0.97±0.14), the relative activity of dual luciferase of AP006284.1-Mut vector (0.98±0.07) was not significantly change ( t=0.09, P>0.05). The GEO database analysis showed that the expression of AP006284.1 in bladder cancer tissues was negatively correlated with the expression of miR-205-3p ( P<0.01). The expression level of miR-205-3p gene in the transfection group (5.42±0.24) was 5.21 times higher than that in the control group (1.04±0.40, t=9.40, P<0.01). Compared with the control group (3.22±0.17, 5.56±0.19, 4.38±0.17, 5.74±0.36), the expressions of p-Ras (2.33±0.12), p-Raf (1.61±0.20), p-MEK (1.57±0.25), and p-ERK (2.40±0.28) of the ERK pathway were decreased in the transfected J82 cells (all P<0.01). Conclusions:AP006284.1 is highly expressed in bladder cancer tissues. Knockdown of AP006284.1 can inhibit the proliferation and migration of bladder cancer cells by regulating the miR-205-3p and ERK pathway proteins.

Objective : To analyze the expression and clinical significance of circ⁃RACGAP1 in bladder cancer tissues , and to explore the influence and mechanism of circ⁃RACGAP1 on the malignant biological behavior of bladder cancer cells. Methods : The expression of circ⁃RACGAP1 in bladder cancer tissues was explored through the TCGA database , and the relationship between the expression of circ⁃RACGAP1 and the clinicopathological features of bladder cancer patients was analyzed. The expression of circ⁃RACGAP1 in cell lines 5637 , T24 , J82 , RT⁃4 and UM⁃UC⁃3 was analyzed by quantitative real⁃time PCR (qPCR) . The circ⁃RACGAP1 knockdown plasmid was transfected into T24 cells by lipofection technology. Colony formation assay , scratch assay and Transwell assay were used to analyze the effects of knocking down circ⁃RACGAP1 on the proliferation , migration and invasion of T24 cells , respectively. The targeted binding between circ⁃RACGAP1 and miR⁃4324 was verified using deepBase , Circbank , CircInteractome , circRNABase databases and a fluorescent reporter system. The effect of knocking down circ⁃RACGAP1 on the expression of miR⁃4324 in T24 cells was detected by qPCR. Western blot was used to detect the effect of knocking down circ⁃RACGAP1 on the expression of recombinant rac⁃GTPase activating protein 1 (RACGAP1) protein and phosphatidylinositol⁃3 ⁃kinase/protein kinase B (PI3K/AKT) signaling pathway proteins in T24 cells. Results circ⁃RACGAP1 was highly expressed in bladder cancer tissues (P < 0. 01) , and its expression increased with the clinical stage of the patients ( P < 0. 01) . The expression of circ⁃RACGAP1 in bladder cancer cell lines was significantly higher than that in normal human bladder epithelial cells ( all P < 0. 01) . Compared with the control group , the proliferation , migration and invasion abilities of T24 cells in the sh⁃circ⁃RAC⁃GAP1 group significantly decreased (all P < 0. 01) . circ⁃RACGAP1 could target and inhibit the expression of miR⁃4324 (P < 0. 01) . Compared with the control group , the expression level of RACGAP1 protein in T24 cells in the sh⁃circ⁃RACGAP1 group decreased (P < 0. 01) , and the expression levels of PI3K/AKT signaling pathway proteins phosphatidylinositol⁃3 ⁃kinase (p⁃PI3K) , phosphorylated protein kinase B (p⁃AKT) , nuclear factor kappa B (NF-κB) decreased (all P < 0. 01) . Conclusion circ⁃RACGAP1 is highly expressed in bladder cancer tissues and cell GAP1 plays a role by inhibiting the expression of miR⁃4324 and activating the PI3K/AKT signaling pathway.