Gambogic Acid Induces Apoptosis Of Human Bladder Cancer Cells Through Long Non-coding RNA GAS5

Backgrounds:Gambogic acid (GA), a natural drug, has been founding powerful to promote apoptosis of various cancers, including bladder cancer in recent years. GA is considered as a potent chemotherapeutic drug. Since, illustrating the effective molecular is crucial. Long Non-coding RNA (LncRNA), which cinstitutes the largest part of non-coding RNAs, are commonly regarded as transcriptional "noise", while recent study shows that they participate in nearly all cellular processes. The dysregulation of LncRNAs is related to various cancers. Among which, GAS5 was found to be downregulated in many cancers, and effective to suppress tumor growth. However, the role and the effect of GAS5 in bladder cancer is still unclear.Purposes:1. To exam the expression of GAS5 in clinical patients, and analysis the relation between GAS5 level and clinical classification and stage.2. Explore the effect of GAS5 on bladder cancer and illustrate the molecular mechanism.3. Complete the mechanisms of the anti-tumor effect of GA.Methods:1. Forty three patients who had undergone radical cystectomy were selected for this study, GAS5 quantities of both tumor and adjacent normal tissues were detected by RT-QPCR.2. GAS5 was knockdown by siRNA or shRNA and full-length GAS5 sequence lacking a poly A tail was synthesized and subcloned into a GV144 vector to overexpress GAS5. SiRNAs, shRNAs and overexpression vectors were transfected into T24 and EJ cells with Lipofectamine 2000.3. MTT assay assessed cell viability, flow cytometric analyzed cell apoptosis rate, and Western blot detected protein expression level.4. Human EZH2 luciferase reporter plasmid was constructed and transfected with GV144-GAS5 together.5. RIP (RNA Binding Protein Immunoprecipitation) was performed to examine the binding between GAS5 and transcription factor E2F4.6. Chromatin Immunoprecipitation (CHIP) was performed to assesses the specific bind between E2F4 and EZH2 promoter.7. EZH2 shRNA and EZH2 overexpression plasmid (PC3.1-EZH2) were transfected into bladder cancer cells, and miR-101 level was assessed by RT-QPCR.8. Human miR-101-2 reporter plasmid and EZH2 shRNA were transfected together to examine the regulation of EZH2 to miR-101.9. Different doses of GA were treated to bladder cancer cells, and RT-QPCR was performed to assess the expression of GAS5.10. After GAS5 was knockdown, cells were then treated with GA, and relative effects of GA were assessed by MTT, flow cytometric, and Western blot.11. EJ cells with GAS5 shRNA or nontarget shRNA stable transfection were injected s.c. into Balb-c nude mice. When tumor volumes reached at an average of approximately 400 mm3, mice were randomly divided into four groups, and GA (dissolved in 0.9% Nacl) or 0.9% Nacl with at 1.8 mg kg-1 was administered i.v. to mice once every other day.14 days after that, the mice were killed, tumors were removed and measured and weighted. RNA was extracted and expression levels of EZH2 mRNA, GAS5 and miR-101 were examined by RT-QPCR. EZH2 protein level was measured by immunohistochemistry and Western.Results:1. GAS5 was downregulated in 33 of the 43 samples (76.74%).59.09% of the facial tumors were GAS5 downregulated, but 95.24% of the invasive tumors were downregulated. Meanwhile, GAS5 was decreased in 78.57% of the specimens without lymph node metastasis and that comes to 75.86% in specimens with lymph node metastasis. Finally,77.5% tissues with distant metastasis presented low expression of GAS5 and 66.67% tissues with distant metastasis presented that.2. With GAS5 overexpression to bladder cancer cells T24 and EJ, cell viabilities were decreased, apoptosis rates increased, and activation of caspase-3 were enhanced. On the contrary, cell viabilities were increased, cell apoptosis rates were decreased, and activation of caspase-3 were declined with GAS5 downregulation.3. Western blot indicated that GAS5 inhibited EZH2 protein expression, RT-QPCR explained that GAS5 suppressed EZH2 mRNA expression, and luciferase reporter gene assay suggested that EZH2 restrained activity of EZH2 promoter.4. By RIP experiment, we found GAS5 could bind to transcription factor E2F4, and by CHIP experiment, we discovered that E2F4 could bind to EZH2 promoter specifically.5. miR-101 was downregulated by GAS5 overexpression and upregulated with GAS5 downregulated. When transfected with GV144-GAS5 together, fireflies fluorescence activity was decreased compared to transfected with control.6. GA upregulated GAS5 expression, and that was positively related to dose. GA promoted bladder cancer cells apoptosis evidently, while that effect was significantly weakened by GAS5 knockdown.7. In Vivo experiments revealed that GA and GAS5 suppressed bladder cancer growth, while the effect of GA could be evidently weakened by GAS5 downregulation.Conclusion:1. GAS5 is downregulated in bladder cancer. The probability of GAS5 low expression is higher in invasive tumors than facial tumors, while that was irrelative to lymph node metastasis or distant metastasis.2. GAS5 inhibits EZH2 expression possibly by binding to transcription factor E2F4 to restrain the activity of EZH2 promoter, and then upregulates miR-101 expression and induces bladder cancer cells apoptosis.3. GA promotes bladder cancer cells apoptosis through blocking EZH2-miR-101 pathway by GAS5.