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, 9 (2), 238

Long Noncoding RNA GAS5 Promotes Bladder Cancer Cells Apoptosis Through Inhibiting EZH2 Transcription

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Long Noncoding RNA GAS5 Promotes Bladder Cancer Cells Apoptosis Through Inhibiting EZH2 Transcription

Miao Wang et al. Cell Death Dis.

Abstract

Aberrant expression of long noncoding RNA GAS5 in bladder cancer (BC) cells was identified in recent studies. However, the regulatory functions and underlying molecular mechanisms of GAS5 in BC development remain unclear. Here, we confirmed that there was a negative correlation between GAS5 level and bladder tumor clinical stage. Functionally, overexpression of GAS5 reduced cell viability and induced cell apoptosis in T24 and EJ bladder cancer cells. Mechanistically, GAS5 effectively repressed EZH2 transcription by directly interacting with E2F4 and recruiting E2F4 to EZH2 promoter. We previously reported that miR-101 induced the apoptosis of BC cells by inhibiting the expression of EZH2. Interestingly, the present study showed that downregulation of EZH2 by GAS5 resulted in overexpression of miR-101 in T24 and EJ cells. Furthermore, the level of GAS5 was increased under the treatment of Gambogic acid (GA), a promising natural anti-cancer compound, whereas knockdown of GAS5 suppressed the inhibitory effect of GA on cell viability and abolished GA-induced apoptosis in T24 and EJ cells. Taken together, our findings demonstrated a tumor-suppressor role of GAS5 by inhibiting EZH2 on transcriptional level, and additionally provided a novel therapeutic strategy for treating human bladder cancer.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. GAS5 is lowly expressed in human BC tissues, overexpression of GAS5 suppressed viability and promoted apoptosis of BC cells.
a Total RNA was extracted from human normal bladder (N) and the paired tumor (T) tissues of 43 patients, diagnosed with different stages of bladder cancer. GAS5 (GAS5-001), GAS5-005, and GAS5-007 expression levels were then detected by RT-qPCR assay. b GV144-GAS5 or the empty GV144 vector plasmids were transfected into BC cells. Forty eight hour later, overexpression efficient of GAS5 was evaluated by RT-qPCR. c GV144-GAS5 or the empty vector was transfected into BC cells for 24 h, 48 h, 72 h respectively, and cell viabilities were assessed by MTT assay. d Forty eight hour after transfection of GV144-GAS5 or the empty vector, cells were harvested and stained by Annexin V-PE and 7-AAD, and apoptosis rates were analyzed by flow cytometry. e Seventy two hour after transfection of GV144-GAS5 or the empty vector, total protein was extracted, and caspase-3 and cleaved caspase-3 protein levels were then assessed by Western-blot assay, GAPDH was served as the internal control. *P < 0.05 versus control groups
Fig. 2
Fig. 2. GA enhanced GAS5 expression, and knockdown of GAS5 suppressed GA-induced apoptosis of BC cells.
a T24 and EJ cells were treated with different concentrations of GA (0, 1.5, 2, 2.5 μM) for 24 or 48 h, and GAS5 expression levels were assessed by RT-qPCR. b The expression levels of GAS5 in T24 and EJ cells were detected by RT-qPCR assay, with transfection of GAS5 siRNA-1, -2 or the control siRNA for 48 h. c Forty eight hour after transfection of siRNAs, 2.5 μM GA or isopyknic PBS was treated. Twenty four hour later, cells were collected and cell viabilities were evaluated by MTT assay. d Meanwhile, cell apoptosis rates were analyzed by flow cytometry analyzed. Results were presented as the means ± SD of triplicates. *P < 0.05 versus control groups, #a significant difference between GAS5 siRNA and control siRNA groups
Fig. 3
Fig. 3. GAS5 inhibited transcriptional of EZH2, and knockdown of GAS5 suppressed GA-induced inhibition of EZH2 transcription.
a GV144-GAS5 or the empty vector was stably transfected into BC cells, EZH2 protein levels were detected by Western-blot assay. b GAS5 shRNA or the control (nontarget shRNA) was stably transfected into BC cells, and 2.0 μM GA or isopyknic PBS was treated for 48 h, the Western-blot assay indicated that GAS5 shRNA evidently inhibited GA-induced downregulation of EZH2 protein expression and the activation of caspase-3. c BC cells stably transfected with GV144-GAS5 or the empty vector were collected, and EZH2 mRNA levels were assessed by RT-qPCR assay. d BC cells stably transfected with GAS5 shRNA or the control shRNA (nontarget shRNA) were treated with 2.0 μM GA or isopyknic PBS for 48 h, RT-qPCR showed that GAS5 shRNA remarkably reversed the inhibition of EZH2 mRNA expression by GA. e EZH2 transcriptional activities of BC cells stably transfected with GV144-GAS5 or the empty vector were assessed by luciferase reporter assay. f The cells were then treated with 2.0 μM GA or isopyknic PBS. Forty hours later, EZH2 transcriptional activities were analyzed, the activity of firefly luciferase was normalized by renilla luciferase. Results were presented as the means ± SD of triplicates. *P < 0.05 versus controls, #a significant difference between GAS5 shRNA and nontarget shRNA groups
Fig. 4
Fig. 4. GAS5 bind and recruited transcription factor E2F4 to EZH2 promoter to inhibited EZH2 transcription.
a E2Fs binding sites in EZH2 promoter: a, b represents putative binding sites, +1 represents the most 5′ nucleotide in the longest identified cDNA published in the NCBI database. b GV144-GAS5 or the empty vector was transfected into BC cells, and chrome immunoprecipitations were performed by using specific anti-E2F1 or anti-E2F4 antibodies. c E2F4 siRNAs (E2F4-1, 2) or the control siRNA were transfected into BC cells for 48 h, E2F4 and EZH2 mRNA levels were then assessed by RT-qPCR. d BC cells transfected with E2F4 siRNAs or the control siRNA for 72 h were collected, and EZH2 and E2F4 protein levels were detected by Western-blot assay. e RNA immunoprecipitations were performed in BC cells, and the relative quantities of GAS5 were detected by RT-qPCR assay, normalized to the input groups. IgG and E2F4 represented for the groups coprecipitation with IgG protein and anti-E2F4 antibody respectively. f Total proteins were extracted from T24 and EJ cells, and then lncRNA GAS5 pull-down assay was performed. The E2F4 protein levels were evaluated by Western-blot. GAS5 probe represented the biotin-labeled GAS5 probe group and control stood for the oligo probe group. g GST and GST-E2F4 fusion proteins were applied for lncRNA GAS5 pull-down assay, and the GST protein levels were analyzed by Western-blot. h, i BC cells with GV144-GAS5 or the empty vector was harvested, and expression levels of E2F4 protein (h) and E2F4 mRNA (i) were detected. *P < 0.05 versus the control groups, NS no significant difference between the groups, #a significant difference between GV144-GAS5 and empty vector groups
Fig. 5
Fig. 5. Knockdown of EZH2 improved miR-101 transcription, and overexpression of EZH2 suppressed GA-stimulated upregulation of miR-101.
ac EZH2 shRNA was stably transfected into BC cells, RT-qPCR was performed to assess EZH2 mRNA (a) and miR-101 (b) expression, luciferase reporter assay was performed to detect miR-101 transcriptional activity (c). d BC cells stably transfected with PC3.1-EZH2 or the empty PC3.1 vector was harvested and the transfection efficiency was evaluated. e, f Then, 2.0 μM GA or isopyknic PBS was added. Forty eight hour later, miR-101 expression levels were assessed (e), and 40 h later, miR-101 transcriptional activities were detected (f). *P < 0.05 versus controls, #a significant difference between PC3.1-EZH2 and empty vector groups
Fig. 6
Fig. 6. Knockdown of GAS5 inhibited GA-induced apoptosis of BC cells in vivo.
Four-week-old Balb-c nude mice were randomly divided into two groups, and EJ cells (1 × 106) with GAS5 shRNA or the nontarget shRNA stable transfection were injected s.c. respectively. Two weeks later, GA (1.8 mg kg−1) or the vehicle was administered i.v. once every other day. Sixteen days after that, the mice were sacrificed and the tumors were removed, weighed (a), and photographed (b). c Then, total RNA was extracted from the tumor tissues, and expressions of GAS5, EZH2 mRNA and miR-101 were detected by RT-qPCR (c). d Immunohistochemistry analysis of EZH2 was obtained from tumors (100×, 400×). e Total protein was extracted and expression of EZH2 protein was assessed by Western-blot assay. *P < 0.05 versus controls, #a significant difference between GAS5 shRNA and nontarget shRNA groups
Fig. 7
Fig. 7. A hypothetical working model of the role of GAS5 in bladder cancer apoptosis.
Overexpression of GAS5 effectively increases the binding of E2F4 to EZH2 mRNA promoter, resulting in repression of EZH2 transcription that promotes tumor cell apoptosis. Meanwhile, this study shows that EZH2 negatively regulated miR-101 transcription in BC, and as we known, miR-101 is a repressor of EZH2. Thus, upregulation of GAS5 results in a positive feedback loop between EZH2 and miR-101. Furthermore, Gambogic acid (GA), a promising natural anticancer compound, could induce bladder cancer cell apoptosis though regulating the expression of both GAS5 and miR-101

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