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. 2019 Nov 15;11(11):6838-6849.
eCollection 2019.

Hypoxia-induced circular RNA has_circRNA_403658 promotes bladder cancer cell growth through activation of LDHA

Yinsheng Wei  1 , Yudong Zhang  2 , Qingjun Meng  1 , Lingang Cui  1 , Chang Xu  3
Affiliations

Hypoxia-induced circular RNA has_circRNA_403658 promotes bladder cancer cell growth through activation of LDHA

Yinsheng Wei et al. Am J Transl Res. .

V体育官网入口 - Abstract

Bladder cancer (BC) is one of the most common cancers in male patients, and the leading cause of cancer-related death in men. Hypoxia plays a critical role in carcinoma biology, including in bladder cancer. However, whether circular RNAs are associated with hypoxia-mediated progression of bladder cancer remain unknown. In this study, our aim was to investigate the role of circular RNA on the hypoxic adaptive response in bladder cancer. Here, we identified a hypoxia-inducible circular RNA, has-circRNA-403658 that contributes to bladder cancer progression. Has-circRNA-403658 is spliced from its host gene, ZNF292, through back-splicing between the 1st and 4th exon. We demonstrated that has-circRNA-403658 was an important circRNA that upregulated in bladder cancer cells under hypoxia, and higher has-circRNA-403658 levels were associated with poorer survival outcome. Silencing has-circRNA-403658 in bladder cancer cells inhibited cell growth and induced cell apoptosis. In addition, has-circRNA-403658 was induced by HIF1α and silencing has-circRNA-403658 inhibited LDHA-mediated aerobic glycolysis, inhibiting bladder cancer cell growth. Thus, our results suggest that has-circRNA-403658 may function as a novel therapeutic target in human bladder cancer VSports手机版. .

Keywords: Bladder cancer; HIF1α; LDHA; aerobic glycolysis; circular RNA. V体育安卓版.

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"V体育平台登录" Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Expression of has_circRNA_403658 in bladder cancer. A. Heatmap of cluster analysis in bladder cancer cells (ccc-HB2, 5637, J82, T24, SW780 and RT4) under hypoxic and normal condition. B. qPCR was performed to confirm the differentially expressed circRNAs in 5637 cells under hypoxic and normal condition. C. Expression of has_circRNA_403658 in bladder cancer tissues and the matched adjacent tissues. D. Kaplan-Meier curves of overall survival in patients with bladder cancer with low or high has_circRNA_403658 expression. *P<0.05.
Figure 2
Figure 2
Silencing has_circRNA_403658 inhibited bladder cell growth and invasion. (A) qRT-PCR analysis for has_circRNA_403658 in bladder cancer cell lines, including ccc-HB2, 5637, J82, T24, SW780 and RT4. (B) qRT-PCR analysis for has_circRNA_403658 in 5637 and T24 cells after has_circRNA_403658 siRNA 1# and siRNA 2# transfection. (C, D) Brdu assay was performed to measure the proliferation rate of 5637 (C) and T24 (D) cells after has_circRNA_403658 siRNA transfection. (E, F) Apoptosis analysis for 5637 (E) and T24 (F) cells after has_circRNA_403658 siRNA transfection. (G, H) Transwell assay for 5637 (G) and T24 (H) cells after has_circRNA_403658 siRNA transfection. (I) T24 cells transfected with has_circRNA_403658 siRNA were injected into the back of BALB/c nude mice. The tumor volumes were measured weekly. (J) Immunohistochemistry analysis for Ki67 and c-PARP in xenografted tumor tissues (left), and quantification by imageJ (right). scan bar, 200 μm. *P<0.05 vs control.
Figure 3
Figure 3
Effects of circRNA_403658 knockdown on glycometabolism relevant gene expression. A, B. 5637 and T24 cells were transfected with circRNA_403658 siRNA 2# for 48 h, and then qPCR was performed to determine the expression of glycometabolism relevant genes. circRNA_403658 knockdown significantly decreased the expression of LDHA. *P<0.05.
Figure 4
Figure 4
Silencing has_circRNA_403658 inhibits lactate production and LDH activity in bladder cancer cells. A. Lactate concentrations were determined in 5637 and T24 cells after 48 h transfection with has_circRNA_403658 siRNA 2#. B. LDH activity was determined in 5637 and T24 cells after 48 h transfection with has_circRNA_403658 siRNA 2#. C. ATP concentration was determined in 5637 and T24 cells after 48 h transfection with has_circRNA_403658 siRNA 2#. D. Glucose uptake was determined by Glucose Uptake Colorimetric Assay kit in 5637 and T24 cells after 48 h transfection with has_circRNA_403658 siRNA 2#. E. 5637 and T24 cells were transfected with has_circRNA_403658 siRNA and then treated with oxamate sodium (Oxamate, 80 mM) for 48 h. The cell viability was measured using a Brdu assay. F. 5637 and T24 cells were transfected with has_circRNA_403658 siRNA and then were treated with 6 mM 2-DG for 48 h. Cell viability was measured using a Brdu assay. *P<0.05.
Figure 5
Figure 5
HIF1α induces has_circRNA_403658 expression. (A) Western blot analysis of the expression of has_circRNA_403658 in 5637 and T24 cells after HIF1α expressed plasmid transfection. (B) The expression of has_circRNA_403658 was determined in 5637 and T24 cells after HIF1α expressed plasmid transfection. (C, D) Brdu assay was performed to measure the proliferation rate of 5637 (C) and T24 (D) cells after HIF1α expressed plasmid or plus has_circRNA_403658 siRNA transfection. (E, F) Transwell assay for 5637 (E) and T24 (F) cells after HIF1α expressed plasmids or plus has_circRNA_403658 siRNA transfection. *P<0.05.
Figure 6
Figure 6
LDHA blocked has_circRNA_403658 siRNA-mediated tumor-suppressive effects in bladder cancer cells. A. Western blot analysis of LDHA, EGFR, VEGFR in 5637 and T24 cells after has_circRNA_403658 siRNA transfection. B. RNA pull-down assay for the amount of has_circRNA_403658 and LDHA with either has_circRNA_403658 or control probe in 5637 cells. C. Luciferase reporter assay for the luciferase activity of pGL3-LDHA in 5637 cells co-transfected with has_circRNA_403658. D. Brdu assay was performed to measure the proliferation rate of 5637 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. E. Transwell assay for 5637 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. *P<0.05.
Figure 7
Figure 7
LDHA reverses has_circRNA_403658 siRNA-inhibited aerobic glycolysis in bladder cancer cells. A. Lactate concentrations were determined in 5637 and T24 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. B. LDH activity was determined in 5637 and T24 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. C. ATP concentration was determined in 5637 and T24 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. D. Glucose uptake was determined by Glucose Uptake Colorimetric Assay kit in 5637 and T24 cells after LDHA expressed plasmid or plus has_circRNA_403658 siRNA transfection. *P<0.05.
Figure 8
Figure 8
Correlation between LDHA and circRNA_403658 in bladder cancer tissues. A. Representative IHC images for LDHA in bladder tissues (upper), and quantification by ImageJ (lower). Bar=200 μm. B. Pearson correlation analysis for LDHA and circRNA_403658 in bladder cancer tissues.
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