The online version of this article (https://doi.org/10.1186/s13046-018-0706-6) contains supplementary material, which is available to authorized users.
Wilms’ tumor 1-associating protein (WTAP) plays an important role in physiological processes and the development of tumor such as cell cycle regulation. The regulation of cell cycle is mainly dependent on cyclins and cyclin-dependent protein kinases (CDKs). Recent studies have shown that CDKs are closely related to the tumor diagnosis, progression and response to treatment. However, their specific biological roles and related mechanism in renal cell carcinoma (RCC) remain unknown.
Quantitative real-time PCR, western blotting and immunohistochemistry were used to detect the expression of WTAP and CDK2. The survival analysis was adopted to explore the association between WTAP expression and the prognosis of RCC. Cells were stably transfected with lentivirus approach and cell proliferation and cell cycle, as well as tumorigenesis in nude mice were performed to assess the effect of WTAP in RCC. RNA immunoprecipitation, Luciferase reporter assay and siRNA were employed to identify the direct binding sites of WTAP with CDK2 transcript. Colony formation assay was conducted to confirm the function of CDK2 in WTAP-induced growth promoting.
In RCC cell lines and tissues, WTAP was significantly over-expressed. Compared with patients with low expression of WTAP, patients with high expression of WTAP had lower overall survival rate. Additionally, cell function test indicated that cell proliferation abilities in WTAP over-expressed group were enhanced, while WTAP knockdown showed the opposite results. Subcutaneous xenograft tumor model displayed that knockdown of WTAP could impede tumorigenesis in vivo. Mechanism study exhibited that CDK2 expression was positively associated with the expression of WTAP. Moreover, WTAP stabilized CDK2 transcript to enhance CDK2 expression via binding to 3′-UTR of CDK2 transcript. Additionally, specific inhibitors of CDK2 activity and small interfering RNA (siRNA) of CDK2 expression inhibited WTAP-mediated promotion of proliferation.
These findings suggest that WTAP may have an oncogenic role in RCC through physically binding to CDK2 transcript and enhancing its transcript stability which might provide new insights into RCC therapy.
Additional file 1: Figure S1. WTAP is a prognostic factor in RCC. The KM plot of tumor samples with detailed clinical information which were downloaded from TCGA database (https://cancergenome.nih.gov/). (TIFF 1834 kb)
Additional file 2: Figure S2. The efficiency of WTAP knockdown and overexpression in RCC cell lines. Caki-1 and ACHN cell were infected with WTAP overexpression lentivirus, a negative control, WTAP knockdown lentivirus, and a scramble control. The efficiency of WTAP knockdown (A) and overexpression (B) in Caki-1 and ACHN cell lines was screened by qRT-PCR and western blot. Data represent the mean ± SD from three independent experiments,*P < 0.05. (TIFF 3154 kb)13046_2018_706_MOESM2_ESM.tif
Additional file 3: Figure S5. Overexpression of WTAP could promote cell growth in HK-2 cell line. (A) The efficiency of WTAP overexpression in HK-2 cell lines was screened by western blot and qRT-PCR. (B) Proliferation of HK-2 cell with WTAP over-expressed assessed by CCK8 assays. Data represent the mean ± SD from three independent experiments, *P < 0.05. (TIFF 2677 kb)13046_2018_706_MOESM5_ESM.tif
Additional file 4: Figure S3. WTAP promotes RCC cell migration in vitro. Transwell migration(A,B) and the invasion capability (C,D) indicated that WTAP knockdown significantly decreased the number of cells crossing the membrane (A, C), in contrast, cell migration and invasion were increased after overexpression of WTAP in both Caki-1 and ACHN cell lines (B, D). Data represent the mean ± SD from three independent experiments,*P < 0.05. (TIFF 10013 kb)13046_2018_706_MOESM3_ESM.tif
Additional file 5: Figure S4. The expression of WTAP and CDK2 was positively correlated in RCC tissues. A scatter plot of WTAP and CDK2 relative expression in the tumor samples which were downloaded from TCGA database (https://cancergenome.nih.gov/). (2-tailed Spearman’s correction, R = 0.1604, P = 0.0039) (TIFF 3836 kb)
Additional file 6: Figure S6. WTAP regulated cyclin A2 expression in RCC cells and correlated with cyclin A2 expression in human RCC tissues. (A) Western blot analysis of cyclin A2 expression in Caki-1 cells with WTAP knockdown or overexpression. Cyclin A2 expression was obviously decreased in WTAP-knockdown cells whereas increased in WTAP overexpression cells. (B) The expression of WTAP and cyclin A2 was positively correlated in RCC tissues. A scatter plot of WTAP and cyclin A2 relative expression in the tumor samples which were downloaded from TCGA database (https://cancergenome.nih.gov/) (2-tailed Spearman’s correction, R = 0.25, P = 1.3e-12). (C) WTAP knockdown or overexpression cells were treated with actinomyclin D (Act D). Total RNAs were harvested, and then subjected to quantitative RT-PCR analysis. Knockdown of WTAP could shorten the half-life of cyclin A2 transcript. While, ectopic expression of WTAP could longthen the half-life of cylcin A2 transcript. Data represent the mean ± SD from three independent experiments,*P < 0.05. (TIFF 938 kb)
Additional file 7: Figure S7. WTAP enhanced the stability of the CDK2 transcript. (A) Knockdown of WTAP could shorten the half-life of CDK2 transcript. Cells were treated with 5μg/ml actinomyclin D (Act D) and performed the qRT-PCR. GADPH was used as another stable reference mRNA. The relative quantification was calculated by the 2−ΔΔCt method and normalized based on GADPH. (B) Ectopic expression of WTAP could longthen the half-life of CDK2 transcript. Data represent the mean ± SD from three independent experiments,*P < 0.05. (TIFF 604 kb)13046_2018_706_MOESM7_ESM.tif
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- Wilms’ tumor 1-associating protein promotes renal cell carcinoma proliferation by regulating CDK2 mRNA stability
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