Renal tumors and adjacent tissues were obtained from the Affiliated Hospital of Xuzhou Medical University (Xuzhou, China). This research was approved by the Ethical Review Committee of this hospital. The specimens were collected and stored in liquid nitrogen immediately after the surgery. Total protein was extracted and subjected to WB analysis.

Four human RCC cell lines (ACHN, OSRC2, 786O and Ketr-3) and one normal renal tubular epithelial cell line (HK2) were obtained from the Cell Bank of the Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences. HK2, ACHN and Ketr-3 cells were cultured in DMEM high glucose medium, and the 786O and OSRC2 were cultured in RMPI 1640 medium (Sigma, United States) with 10% fetal bovine serum in a 37°C, 5% CO₂ incubator (Thermo Scientific, United States).

To suppress CPEB4 expression with siRNA, the cells were cultured to 30–50% confluence, and then transfected for 48 h with siRNAs that target CPEB4 or with a nonspecific control (NC). All transfections were performed using siLentFect (Bio-Rad) according to the manufacturer’s instructions. siRNAs specifically targeting CPEB4 or nonspecific control (NC) were synthesized by GenePharm, and their sequences were listed in Supplementary Table S1. The CPEB4 sequence (CPEB4A) was subcloned into the lentiviral vector pCD513B. The virus was packaged in HEK-293T cells and then used to infect 786O and ACHN cells. For stable knockdown of CPEB4, 786O and ACHN cells were separately transduced with each of two CPEB4 shRNAs cloned in the pLKO.1 vector, and selected in the presence of puromycin.

Total RNA was extracted from cultured cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). cDNA was synthesized using a PrimeScript reverse transcription (RT) reagent kit (TaKaRa) in the presence of gDNA Eraser. Quantitative real-time PCR was carried out in a ViiA 7 real-time PCR system (Applied Biosystems) using a SYBR Premix Ex Taq II kit (Takara). The primer sequences used in RT-PCR were listed in Supplementary Table S1.

Cells or tissue specimens were lysed in an NP-40-containing buffer. Equal amounts of protein were separated by SDS-PAGE. After incubation with the indicated antibodies, the immune complexes on the membrane were detected by an ECL kit (Thermo Scientific, #32106). The following antibodies were acquired from commercial sources: rabbit anti-CPEB4 (Proteintech, #25342-1-AP) and rabbit anti-p21 (Santa cruz, #Sc-6246).

Cells grown on cover slips in a 24-well plate were fixed with 4% paraformaldehyde for 20 min and then treated with 0.1% Triton X-100 solution on ice for 4 min. Samples were then blocked with 3% BSA for 1 h followed by antibody incubation at 4°C overnight. Then, the cells were washed 3 × 5 min in PBS and incubated with the fluorescently labeled secondary antibodies for 1 h. DAPI was used to stain the nuclei for 3 min. The slides were mounted with 90% glycerol, and the images were captured by a Zeiss Axio Observer confocal microscope.

Cell proliferation assays were performed using a cell counting kit-8 (CCK-8) from Beyotime Institute of Biotechnology (Nanjing, China). For the colony formation assay, 200 cells were seeded into 6-well plates. After incubation for 14 days, the plates were washed with PBS three times, and the colonies were fixed with 4% paraformaldehyde solution and stained with 0.5% crystal violet. The number of the colonies was counted by ImageJ software. The experiments were independently repeated three times.

To assess the influence of CPEB4 on tumorigenesis in vivo, 6-week-old nude female BALB/c mice were purchased from Beijing HFK Bioscience. The mice were subcutaneously injected with the indicated ACHN and 786O cells (5×10⁶ cells in 100 µl of serum-free medium containing 0.25 v/v Matrigel) in each flank. The tumor volumes were measured every 3–4 days and calculated as length × width² × 0.5. The mice were sacrificed, and the tumors were carefully removed, imaged, and weighed at the end of the experiment.

Approximately 5 × 10⁵ RCC cells were plated in a 6-well plate and maintained in medium containing no serum for 48 h, after which they were released into complete growth medium for 24 h. After release the cells were harvested and fixed using 75% pre-cooled ethanol at 4°C overnight. After being washed three times with phosphate buffered saline (PBS), cells were stained with propidium iodide (400 μg/ml) and RNase A (20 mg/ml) at room temperature for 30min, samples were then analyzed using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA), data on cell cycle distribution were analyzed using ModFit LT 3.0 software.

Cells were collected in lysis buffer (5 mM PIPES. pH 8.0, 85 mM KCl, 0.5% NP40, 1% SDS, 10 mM EDTA, and 50 mM Tris-HCl, pH 8.1) supplemented with a protease inhibitor cocktail and an RNase inhibitor (Thermo Fisher). The cell lysates were precleaned with protein G Sepharose beads and then incubated with the indicated antibodies or IgG control on a rotator at 4°C overnight. The antibody-RNA complexes were collected. The immunoprecipitated RNA was eluted and extracted for real-time PCR analysis.

All data were analyzed with GraphPad Prism 5 and are presented as the mean ± S.D. Student’s t test or one-way ANOVA with Dunnett’s post hoc test was used for statistical analyses of the data when appropriate. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant.

To determine whether CPEB4 is involved in RCC development, we first assessed CPEB4 protein expression in 15 pairs of RCC specimens and adjacent normal tissues, its expression significantly decreased in RCC tissues compared with normal tissues (Figures 1A,B). Additionally, CPEB4 expression in four different RCC cell lines was considerably downregulated compared with normal renal epithelial cell line HK2 (Figures 1C,D). Furthermore, data from the TCGA database proved that CPEB4 expression was decreased in RCC tissues (Figure 1E), and the patients who showed high CPEB4 expression corresponded with longer 5-years overall (p < 0.001) and disease-specific cumulative survival (p = 0.0018) than those with low CPEB4 expression (Figures 1F,G). These results indicated that CPEB4 might be functioned as a tumor suppressor gene in RCC progression.

We next examined the influence of CPEB4 on the biological behaviors of RCC cell lines. CPEB4 stable knockdown (Figures 2A,G) and overexpression (Figures 2D,J) cells were established in ACHN and 786O cell lines. The results of the CCK8 and colony formation assays showed that knockdown of CPEB4 dramatically promoted cell growth and colony formation (Figures 2B,C), and overexpression of CPEB4 suppressed cell growth and colony formation in ACHN cells (Figures 2E,F). The results were confirmed in 786O cells (Figure 2G-l), the phenotypes of the 2 cell lines are identical. According to the basal expression levels of CPEB4 compared in various RCC cell lines (Figure 1D), ACHN cells (CPEB4-high) with CPEB4 stable knockdown and 786O cells (CPEB4-low) with CPEB4 stable overexpression were used to verify the function and mechanism of CPEB4 in the follow-up experiments.

To determine the function of CPEB4 in vivo, we injected CPEB4 stable knockdown and control ACHN cells (Figure 3A) that were mixed with Matrigel into BALB/c nude mice subcutaneously. After 5 weeks, we found that the tumor volume and mean tumor weight were significantly higher in the CPEB4 knockdown group than in the negative control group (Figures 3B–D). In contrast, the overexpression of CPEB4 in 786O cells (Figure 3E) markedly inhibited tumor growth in vivo (Figures 3F–J). Collectively, these findings suggest that CPEB4 inhibits RCC tumorigenesis both in vitro and in vivo.

To determine whether CPEB4 can regulate the duration of the cell cycle, we used serum starvation to synchronize RCC cells and then analysed cell-cycle progression using flow cytometry. Consistent with the CCK-8 and colony formation assay results, we found that CPEB4 knockdown promoted G1/S phase transition (Figures 4A,B). In contrast, the induction of G1 cell cycle arrest was observed when CPEB4 was overexpressed (Figures 4C,D). These results indicated that CPEB4 may inhibit RCC tumor growth by inhibiting G1/S phase transition. Recently, we have reported that CPEB2, as a p53 target, can bind to CPE elements in the p53 3′-UTR and negatively regulate p53 mRNA stability and translation. Therefore, we speculated that CPEB4 may also affect the level of p53. However, we found that CPEB4 overexpression or konckdown cannot change p53 expression, but significantly upregulate p21 expression (Supplementary Figure S1). We then detected major factors involved in cell cycle regulation, and found that only the tumor suppressor p21 mRNA expression was upregulated in 786O cells with stably transfected CPEB4 (Supplementary Figure S2).

Interestingly, the 3′-UTR of p21 containss CPE signal (UUUUUAU) suggesting that p21 is a potential CPEB target. Indeed, knockdown of CPEB4 significantly decreased p21 protein levels, while CPEB4 overexpression substantially enhanced p21 protein levels (Figures 5A,B). Then, we investigated whether CPEB4 regulates p21 by targeting p21 mRNA. Since mRNA is predominantly degraded in the cytoplasm, we examined the localization of ectopically expressed Flag-tagged CPEB4. The majority of CPEB4 localized in the cytoplasm (Figure 5C), which was further confirmed by the nuclear-cytoplasmic separation (Figure 5D). Consistently, CPEB4 knockdown resulted in a decrease in the p21 mRNA levels, whereas CPEB4 overexpression had an opposite effect (Figures 5E,G). Next, we tested whether the changes in the expression of p21 transcripts induced by CPEB4 are due to altered mRNA stability. For this purpose, actinomycin D was used to inhibit de novo mRNA synthesis. Knockdown of CPEB4 accelerated p21 mRNA degradation, while overexpression of CPEB4 stabilized p21 mRNA (Figures 5F,H). In addition, CPEB4 knockdown retarded de novo synthesis of the p21 protein (Figure 5I). Furthermore, the results of RNA immunoprecipitation assays indicated that CPEB4 is associates with the p21 transcript but not with the GAPDH transcript (Figure 5J). Overall, these findings suggest that CPEB4 binds with the p21 transcript and upregulates p21 expression by increasing p21 mRNA stability and translation.

To validate the involvement of p21 in the CPEB4-mediated tumor-inhibiting effect, we introduced stable p21 expression into CPEB4-knockdown cells or control cells with an empty vector (Figure 6A). Forced expression of p21 abrogated the pro-proliferative effect of CPEB4 knockdown, which was demonstrated by accelerated cell proliferation (Figure 6B) and G1/S phase transition (Figures 6E,F). In accordance with this result, silencing of p21 reversed RCC cell proliferation inhibition (Figures 6C,D) and G1 cell cycle arrest (Figures 6G,H) caused by CPEB4 overexpression. Furthermore, the results of EDU staining assays also verified the involvement of p21 in the CPEB4-mediated cell proliferation inhibition (Figures 6I,J). Taken together, these findings clearly demonstrate that CPEB4 modulates RCC cell proliferation by inhibiting cell cycle progression partially through increasing p21 expression.