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@#[Abstract] Objective: To investigate the expression of long non-coding RNA (lncRNA) HULC in bladder cancer tissues and its relationship with the clinicopathological features of patients, as well as the effect of silencing HULC on the proliferation, apoptosis, migration and invasion of bladder cancer 5637 cells. Methods: A total of 102 pairs of cancer tissue and adjacent normal tissue samples from bladder cancer patients who underwent surgical resection in Zhengzhou People’s Hospital from June 2014 to December 2017 were selected, as well as bladder cancer 5637 cell line and human normal bladder epithelial SV-HUC-1 cell line. The expression of HULC in bladder cancer tissues and cells was detected by qPCR, and the correlation between HULC and clinicopathological features of bladder cancer patients was analyzed. The effect of HULC on prognosis was evaluated by Kaplan-Meier survival curve. si-HUL and si-NC plasmids were transfected into 5637 cells by siRNA interference technology, and the effects of silencing HULC on proliferation, apoptosis, migration and invasion of 5637 cells were determined by CCK-8, Flow cytometry, Wound-healing assay and Transwell method, respectively. Results: The expression of HULC in bladder cancer tissues was significantly higher than that in normal tissues (P<0.05), and its expression level was correlated with tumor grade, tumor stage and lymph node metastasis (P<0.05). The OS and PFS of patients with high HULC expression were significantly lower than those with low expression (all P<0.05). The expression level of HULC in 5637 cells was significantly higher than that in SV-HUC-1 cells (P<0.01). After silencing HULC, the proliferation, migration and invasion of 5637 cells were significantly decreased (P<0.01), and the apoptosis rate was significantly increased (P<0.01). Conclusion: lncRNA HULC is highly expressed in bladder cancer tissues and 5637 cells. Silencing HULC expression can inhibit the proliferation, migration and invasion but promote apoptosis of bladder cancer cells.
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@# Objective: To investigate the role and mechanism of Krüppel-like factor 4 (KLF4) in regulating epithelial-mesenchymal transition (EMT) and migration of bladder cancer cells. Methods: Bladder cancer 5637 and T24 cell lines that stably over-expressing KLF4 (LV-KLF4, experiment group) were constructed, and the negative control group (LV-NC) was also established; the mRNA and protein expressions of KLF4 were verified by qPCR and WB, respectively. Transwell chamber assay was used to detect the migration ability of cells in LV-KLF4 and LV-NC groups. WB was performed to detect the expression levels of EMT-related markers (E-cadherin, N-cadherin, Vimentin) and Wnt signaling pathway-related proteins. Immunofluorescence technique was used to detect the distribution of β-catenin in cells after over-expression of KLF4. Results: The 5637 and T24 cell lines over-expressing KLF4 gene were successfully constructed. Compared with the LV-NC group, the mRNA and protein expressions of KLF4 increased in LV-KLF4 groups (all P<0.01); the expression of E-cadherin increased (P<0.01), while the expressions of N-cadherin, vimentin, and the expression levels of total β -catenin, nuclear β -catenin, MMP 9 and c-Myc decreased (all P<0.01); moreover, the migration ability of cells decreased significantly (P<0.01); the fluorescence expression of β-catenin in cells also decreased significantly in LV-KLF4 group as compared to LV-NC group. Conclusion: Over-expression of KLF4 gene in bladder cancer cells may inhibit EMT process by regulating Wnt/β-catenin signaling pathway, and further inhibit the migration of bladder cancer 5637 and T24 cells.
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Objective To analysis the effect of metformin on the expression of heat shock protein and VEGF in 5637 cell line of bladder cancer. Methods 5637 cell line of bladder cancer were selected, divided into blank control group, metformin 2 mM group, 5 mM group, 10 mM group and 20 mM group, 5637 cell line of bladder cancer were treated in each group.The cell proliferation inhibition rate was detected by MTT colorimetric assay, cell clone formation rate was detected by plate clone formation test, flow cytometry was used to detect cell cycle change, the expression levels of HSP-70, HSP-90α, VEGF were detected by Western blot method.Results Compared with control group, the inhibition rate of 5637 cell line of bladder cancer after 24, 48, 72 h were higher in all concentrations of metformin group(P<0.05), the higher the concentration and the longer the action time, the stronger inhibitory effect on cell proliferation.Compared with control group, the clone formation rate of 5637 cell line of bladder cancer in all concentrations of metformin group was lower, the difference was statistically significant(P<0.05).Compared with control group, the proportion of G1 phase cells were higher, the proportion of S phase cells were lower in in all concentrations of metformin group (P<0.05), the proportion of G2 phase cells in metformin 2 mM group, 5 mM group were lower(P<0.05).Compared with control group, the expression levels of HSP-70, HSP-90α, VEGF protein were lower in all concentrations of metformin group, the difference was statistically significant(P <0.05).Conclusion Metformin has obvious inhibitory effects on the expression of HSP-70, HSP-90α, VEGF in 5637 cell line of bladder cancer, can inhibit cell proliferation and cloning, promote the occurrence of G1 block, cause cell apoptosis, the effect was dose dependent.
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Objective To evaluate the effect of rapamycin on proliferation and cell cycle of human bladder cancer cell line 5637. Methods Total?ly 5637 cells were maintained in RPMI 1640 containing 10%fetal bovine serum,100 U/mL penicillin and 100μg/mL streptomycin for adherent cul?ture. Cells were grown at 37℃in a 5%CO2 incubator. The 5637 cells were treated with various concentrations of rapamycin solution(50 nmol/L, 100 nmol/L,200 nmol/L,and 400 nmol/L). The control group was daily treated with single RPMI 1640 without medication. The growth repression rate for 5637 cells was evaluated by MTT. Flow cytometry was used to investigate the effect of rapamycin of different concentrations on cell cycle of 5637 cells. Results Compared to the control group,rapamycin can inhibit the proliferation of 5637 cells in a concentration and time dependent manner. Rapamycin inhibited 5637 cells at G0/G1 thus inhibiting cell proliferation but not inducing apoptosis. Conclusion Rapamycin inhibited growth of 5637 bladder carcinoma cells and arrested cell cycle at G0/G1,indicating that mammalian target of rapamycin may play an important role in proliferation of 5637 cells and act as a new tumor therapeutic target in patients with bladder cancer.