A novel circRNA, circNUP98, a potential biomarker, acted as an oncogene via the miR-567/ PRDX3 axis in renal cell carcinoma

Abstract

In recent years, plenty of studies found that circular RNAs (circRNAs) were essential players in the initiation and progression of various cancers including the renal cell carcinoma (RCC). However, the knowledge about the circRNAs in carcinogenesis is still limited. Dysregulated expression of circNUP98 in RCC tissues was identified by the circular RNA microarray. RT-PCR was performed to measure the expression of circNUP98 in 78 pairs of RCC tissues and adjacent normal tissues. Survival analysis was conducted to explore the association between the expression of circNUP98 and the prognosis of RCC. The function and underlying mechanisms of circSMC3 in RCC cells were investigated by RNAi, CCK-8, Western blotting, bioinformatic analysis, ChIP assay, circRIP assay and dual luciferase reporter assay. CircNUP98 was up-regulated in both RCC tissues and cell lines, and high expression of circNUP98 was correlated with poor prognosis of RCC patients. Silencing of circSMC3 inhibited the proliferation and promoted the apoptosis in a caspase-dependent manner in RCC cells. Mechanistically, we revealed that silencing of circ NUP98 inhibited RCC progression by down-regulating of PRDX3 via up-regulation of miR-567. Furthermore, STAT3 was identified as an inducer of circ NUP98 in RCC cells. CircNUP98 acts as an oncogene by a novel STAT3/circ NUP98/miR-567/PRDX3 axis, which may provide a potential biomarker and therapeutic target for the treatment of RCC.

Keywords: PRDX3; circNUP98; miR-567; renal cell carcinoma.

Figure 1

Expression of circNUP98 was increased in RCC tissues and correlated with prognosis of RCC patients. A, Heat map showing the circRNA expression profiles of RCC tissues and adjacent normal tissues. Arrow indicates the circNUP98. B, The levels of circNUP98 were measured by RT‐PCR in 78 pairs of RCC tissues and adjacent normal tissues. C, The expression of circSMC3 in human normal renal cells (293K) and RCC cells (A498, 767P, Caki‐1, Caki‐2, 786‐O). D, Overall survival and (E) disease‐free survival of RCC patients with high or low expression of circNUP98. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001

Figure 2

circNUP98 affects cell growth, migration, invasion and apoptosis of RCC cells. A, RCC cells were transfected as indicated, and the expression of circNUP98 was measured by RT‐PCR. B, Cell proliferation analysed by CCK‐8 assay after knockdown of circNUP98 by shRNA for indicated time in RCC cells. C, Colony formation assay was conducted after silencing of circNUP98 in RCC cells. D, Analysis of apoptosis by flow cytometry after silencing of circNUP98 in RCC cells for 48 h. E, Caspase‐3 activity was assayed after silencing of circNUP98 in RCC cells for 24 h. F, Western blot of caspase‐3 after down‐regulation of circNUP98 in RCC cells. G, Wound healing assay was performed after silencing of circNUP98 in RCC cells. H, Tranwell assay was performed after silencing of circNUP98 in RCC cells. I, The indicated proteins were assayed by western blotting after silencing of circNUP98. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001

Figure 3

circNUP98 functions a sponge of miR‐567. A, Subcellular fraction analysis of localization of circNUP96, GAPDH mRNA and U6 mRNA. B, The levels of different miRNAs were measured by RT‐PCR after silencing of circNUP98 in RCC cells. C, The expression of miR‐567 in human normal renal cells (293K) and RCC cell lines (A498, 769‐P, Caki‐2, Caki‐1 and 786‐O) were measured by RT‐PCR. D, The enrichment of circNUP98 and miR‐567 was measured by RIP assay in RCC cells. E, RCC cells were transfected miR‐567 or miR‐NC mimics for 24 h, and then, the levels of miR‐567 were measured by RT‐PCR. F, The predicted binding sites between miR‐567 and circNUP98 (left) and relative luciferase activity in RCC cells co‐transfected with wt or mut luciferase reporters and miR‐567 mimics or corresponding negative control. G, RCC cells were transfected with miR‐567 or miR‐NC inhibitors for 24 h, and then, the expression of miR‐567 was measured by RT‐PCR. H, RCC cells were transfected as indicated for 24 h, and the migration (left) and invasion (right) were measured by wound healing and Matrigel assay, respectively. I, RCC cells were transfected as indicated for 48 h, and cellular apoptosis was analysed. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001

Figure 4

PRDX3 is a target of miR‐567. A, RCC cells were transfected with miR‐NC or miR‐567 mimics for 24h, and mRNA levels of PRDX3, GPC5 and TBX4 were measured by RT‐PCR. B, RCC cells were transfected with sh‐NC or sh‐circNUP98 for 24 h, and the expression of circNUP98 was assayed by RT‐PCR. C, RCC cells were transfected with miR‐NC or miR‐567 mimics for 24 h, and the protein levels of PRDX3 were measured by Western blotting. D, The predicted binding sites between miR‐567 and PRDX3 3’UTR region. E, relative luciferase activity in RCC cells co‐transfected with wt or mut luciferase reporters and miR‐567 mimics or corresponding negative control. F, The enrichment of circNUP98, miR‐567 and PRDX3 mRNA was measured by RIP assay in RCC cells. G, RCC cells were transfected with sh‐NC or sh‐PRDX3 for 24 h, and the protein levels of PRDX3 were measured by Western blotting. H, After silencing of PRDX3, the proliferation of RCC cells was measured by CCK‐8 assay at indicated time‐point. I, After silencing of PRDX3 for 24 h, the migration and invasion of RCC cells were measured by wound healing and Matrigel assay, respectively. J, After silencing of PRDX3 for 24 h, the apoptosis of RCC cells was measured by flow cytometry. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001

Figure 5

Overexpression of PRDX3 reversed the effects of silencing of circNUP98. A, RCC cells were transfected with pcDNA3.1 or pcDNA.PRDX3 for 24 h, and the protein levels of PRDX3 were measured by Western blotting. B, RCC cells were transfected as indicated, and the proliferation of cells was measured by CCK‐8 assay at indicated time‐points. C, RCC cells were transfected as indicated, and migration and invasion of cells were measured by wound healing assay and Matrigel assay, respectively. D, RCC cells were transfected as indicated, and cellular apoptosis was measured by flow cytometry. E, RCC cells were transfected as indicated, and cellular lysates were subjected to Western blotting with indicated antibodies. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001

Figure 6

STAT3 induces the expression of circNUP98 in RCC cells. A, RCC cells were transfected with pcDNA3.1 or pcDNA.STAT3 for 24 h, and the mRNA levels of STAT3 were measured by RT‐PCR. B, RCC cells were transfected with pcDNA3.1 or pcDNA.STAT3 for 24 h, and the protein levels of STAT3 were measured by Western blotting. C, RCC cells were transfected with pcDNA3.1 or pcDNA.STAT3 for 24 h, and the expression of circNUP98 was measured by RT‐PCR. D, RCC cells were transfected with sh‐NC or sh‐STAT3 for 24 h, and the mRNA levels of STAT3 were measured by RT‐PCR. E, RCC cells were transfected with p sh‐NC or sh‐STAT3 for 24 h, and the protein levels of STAT3 were measured by Western blotting. F, RCC cells were transfected with sh‐NC or sh‐STAT3 for 24 h, and the expression of circNUP98 was measured by RT‐PCR. G, The predictive promoter region of NUP98, top; the reporter constructs of wild‐type Luc and its mutated derivatives are also shown in bottom. H, The interaction between STAT3 and NUP98 promoter was validated through luciferase reporter assay. I, The binding ability between STAT3 and NUP98 promoter was testified by ChIP assay in RCC cells. Data were presented as mean ± SD. Experiments were performed at least three times. **P < .01; ***P < .001

Figure 7

Silencing of circNUP98 increased chemosensitivity of RCC cells and inhibited the growth of RCC in vivo. A, RCC cells were transfected with sh‐NC or sh‐circNUP98 for 24 h, and then, the cells were exposure to different chemotherapy agents for another 24 h, and cellular apoptosis was analysed by flow cytometry. B, RCC cells stably transfected with sh‐NC or sh‐circNUP98 and inoculated into nude mice, and tumour volumes were measured as different time‐points. C, Mice were killed 30 days after inoculation, and the tumour weight was measured. D, Xenografts were subjected to Western blotting analysis of caspase‐3. E, Xenografts were subjected to caspase‐3 activity assay. Data were presented as mean ± SD. Experiments were performed at least three times. *P < .05; **P < .01; ***P < .001