Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis

All the RNA duplexes used in present study which possesses 2-nucleotide 3’ overhangs were chemically synthesized by RiboBio Co., Ltd. (Guangzhou, China). A small interfering RNA (siP53) was used to silence p53 expression and a dsControl which lacks significant homology to all known human sequences was used as a negative control [10, 13]. Lenti-dsP53-285 and Lenti-dsControl were purchased from GenePharma (Shanghai, China). The sequences of all the custom dsRNAs are listed in Additional file 1: Table S1.

The human bladder cancer cell lines T24 and EJ (ATCC) were cultured in RPMI 1640 medium (Hyclone, USA) supplemented with 10 % fetal bovine serum (Gibco, USA) in a humidified atmosphere with 5 % CO₂ at 37 °C. The day before transfection, cells were trypsinized and plated to a new 6-well plate with growth medium at a density of 50-60 % without antibiotics. All dsRNAs were transfected at a final concentration of 50 nM by using Lipofectamine RNAiMax (Invitrogen, USA) according to the manufacturer’s instructions. Besides, dsRNA was replaced by MEM in mock transfection.

The day before infection, 3-5 × 10³ cells were seeded in a 96-well plate with 100 μL culture medium, to ensure cells are at a density of 40-60 % in each well when infection. EJ cells were infected with Lenti-dsP53-285 or Lenti-dsControl according to the manufacturer’s protocol with modification. Culture medium was substituted 24 h later. Fluorescence expression was observed at 72-96 h after infection. Then cells were harvested and reseeded into new plates for further experiments.

Total cellular RNA was extracted from bladder cancer cells by using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. After quantified by a Nano Drop ND-1000 spectrophotometer, 500 ng RNA was reversely transcribed into cDNA according to the instructions provided by Takara reverse transcription kit (Takara, China). The resulting cDNA was amplified by SYBR Premix Ex Taq II (Takara, China) conducted on the Mx3000P instrument (Stratagene, USA). All the primers included in this study were provided by Invitrogen (Shanghai, China) and listed in Additional file 1: Table S2. The relative expression of target genes’ mRNA was calculated with the 2^-ΔΔCt method. GAPDH was used as internal control. All experiments were done in triplicate.

Cell proliferation was detected using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay kit (Promega, USA) according to the manufacturer’s protocol. Briefly, cells were transfected with indicated dsRNAs in a 6-well plate. The cells were trypsinized and seeded at 1000 cells/well into a new 96-well plate 24 h later. Cell growth was measured at daily interval from the next day to the fifth day. At each time point, 20 μl of CellTiter 96® AQueous One Solution was added to each well and incubated for 2 h at 37 °C. Absorbance was measured on a microplate reader (Bio-Rad, USA) at 490 nm.

T24 and EJ cells were harvested 24 h after transfection of indicated dsRNAs. 1000 cells were reseeded in each new 6-well plate with complete medium for 10 days. The medium was replaced every 3 days to maintain the cells growth. The colonies were then fixed and stained with 0.5 % crystal violet (Sigma, USA) for 30 min at room temperature. The colony formation rate was calculated using the following equation: colony formation rate = number of colonies/number of seeded cells × 100 %.

Cells were harvested 72 h after transfection and fixed with 70 % ethanol at 4 °C overnight. Then the cells were washed and incubated with RNase A (0.1 mg/mL) for 30 min at 37 °C. Cellular DNA was stained with propidium iodide (PI) (0.05 mg/mL) and analyzed on a FACSort flow cytometer (BD Biosciences, USA). All experiments were repeated 3 times and a total of 10,000 events were analyzed for each sample. The data were processed by CELL Quest software (BD Biosciences, USA).

After 72 h transfection, cells were trypsinized and counted. Approximate 5 × 10⁵ cells were reseeded in each well of a new 6-well plate. With incubation overnight, the confluent cells monolayers were scratched with a 10 μL sterile pipette tip. Then the non-adherent cells were washed off with sterilized PBS and serum-free medium was added into the wells. The gap area caused by the scratch was monitored by the inverted microscope (Olympus, Japan). Three random non-overlapping areas in each well were pictured at 0 h, 12 h and 24 h post-scratch. Scratch width between the two linear regions was quantitated for assessing capacity of cells migration.

The 24-well Boyden chamber with 8 μm pore size polycarbonate membrane (Corning, USA) was used to analyze the cell motility. For invasion assay, the membrane was pre-coated with matrigel (BD Biosciences, USA) to form a matrix barrier. 2 × 10⁴ cells, transfected with dsRNAs for 72 h, were seeded on the upper chamber with serum-free medium. Medium with 10 % serum was added to the lower chamber as a chemoattractant. The membranes were fixed at 24 h and stained with 0.5 % crystal violet (Sigma, USA). After removal of the non-motile cells at the top of the membranes with cotton swabs, 5 visual fields of 200× magnification of each membrane were randomly selected and counted.

Equivalent amounts EJ cells (about 5 × 10⁶, 200 μL) infected with Lenti-dsP53-285 or Lenti-dsControl were injected subcutaneously into the right back of male BALB/c-nude mice (Hua Fukang Biological Technology Co., Ltd, Beijing, China) at 4 weeks of age, respectively. Tumor length and width were measured using calipers every 4 days for 28 days. Tumor volume was calculated using the formula: V = length × width² × 0.5. Animals were sacrificed 28 days after injection and tumors were weighed.

For in vivo metastasis assay, treated cells (2 × 10⁵) were suspended in 100 μL of PBS and injected intravenously via the tail vein. At 30 days later after injection, the incidence and volume of metastases were estimated by imaging of mice for bioluminescence using the Living Image software (Xenogen, USA). The photon emission level was used to assess the relative tumor burden in the mice lungs. All nude mice were manipulated and cared according to NIH Animal Care and Use Committee guidelines in the Experiment Animal Center of the Tongji medical college of Huazhong University of Science and Technology (Wuhan, China).

Previous study has identified that a specific dsRNA (dsP53-285) can induce p53 expression by targeting sequence position -285 relative to the TSS in the p53 promoter in African green monkey (COS1) and chimpanzee (WES) cells [12]. As human shares almost identical genome sequences with African green monkey and chimpanzee, we speculated that the dsP53-285 may also activate p53 expression in human bladder cancer cells. Hereby, we transfected synthetic dsP53-285 into T24 and EJ cells and analyzed p53 expression 72 h later. Compared with mock and dsControl groups, dsP53-285 caused a significant induction in p53 mRNA (Fig. 1a). This induction was further verified by Immunoblot (Fig. 1b).

It is well known that only wild-type p53 can positively regulate p21 expression [14]. To determine the expression type of p53 in dsP53-285 induced T24 and EJ cells, we further examined the downstream p21 levels. As shown in Fig. 1c, real-time PCR revealed that dsP53-285 profoundly activated p21 levels compared to mock and dsControl treatments. And analysis of p21 induction was further confirmed by western blotting (Fig. 1d). Moreover, time-course experiments revealed that p53 mRNA and protein levels were not induced until 48 h, and continued to increase at 72 h (Fig. 1e and f). Then we silenced wild-type p53 expression by transfecting a small interfering RNA siP53 into both cell lines [13]. The outcomes showed that co-transfection of dsP53-285 and siP53 in the two tested cells markedly abrogated the activating effect of wild-type p53 at both mRNA and protein levels at 72 h (Additional file 2: Figure S3A and S3B). These results suggest that dsP53-285 possesses capacity to induce wild-type p53 expression through RNAa in T24 and EJ cells.

To investigate the effects of wild-type p53 up-regulation mediated by dsP53-285 on bladder cancer cell lines T24 and EJ, we performed CellTiter 96® AQueous One Solution Cell Proliferation Assay. Compared to dsControl group, both tested cells transfected dsP53-285 exhibited progressive retarded growth from the third day following transfection (Fig. 2a). Then we verified whether depletion of wild-type p53 could affect the inhibitory function of dsP53-285 in T24 and EJ cells. As seen from Additional file 3: Figure S1A, knockout of wild-type p53 evidently attenuated the anti-proliferative effect mediated by dsP53-285 in both cells. Then, the EJ cells stably expressing dsP53-285 or dsControl were used to generate the xenograft model in nude mice. As shown in Fig. 2g, Lenti-dsP53-285 significantly reduced xenograft tumor growth. In addition, the average tumor volume and weight in Lenti-dsP53-285 group was markedly smaller and lighter than the control group at day 28 post injection, respectively (Fig. 2f and h).

In order to evaluate the cells growth mode after wild-type p53 activated by dsP53-285, we carried out the colony formation assay and found that dsP53-285 transfected cells formed colonies significantly fewer in number and smaller in size (Fig. 2b). In addition, the colony formation rates of dsP53-285 transfected cells were remarkably lower than dsControl treatment (Fig. 2c). Furthermore, the colony formation ability of the bladder cancer cells was also restored after co-treatment of siP53 (Additional file 3: Figure S1B and S1C).

Nest we performed flow cytometry to measure cell cycle distribution. In both bladder cancer cell lines, transfection with dsP53-285 led to a significant increase in the G0/G1 population and a corresponding decrease in the S phase population compared with dsControl group (Fig. 2d and e). Additionally, p53 silencing remarkably alleviated G0/G1 cycle arrest mediated by dsP53-285 in both cell lines (Additional file 3: Figure S1D and S1E). These data together implied that dsP53-285 could inhibit bladder cancer cells proliferation, suppress colony formation and induce cell cycle G0/G1 arrest mainly via manipulating wild-type p53 expression.

As we know, wild-type p53 suppresses epithelial-to-mesenchymal transition (EMT) which is a process that plays crucial roles in the early stage of metastases, invasiveness and wound healing of bladder cancer [15]. Hereby, we examined the potential roles of dsP53-285 on migration and invasion capacities of bladder cancer cells. At 72 h after transfection, cells were reseeded and then scratches were made 24 h later. Transfection of dsP53-285 led to retarded wound closing compared with dsControl group from 12 h in both T24 and EJ cells (Fig. 3a). Then the wound widths of each group were measured and normalized to corresponding 0 h of dsControl. The relative distances between wound edges of dsP53-285 groups were markedly wider than those of dsControl group in both cell lines from 12 h post-scratch (Fig. 3b). Furthermore, compared to dsP53-285 treatment alone, siP53 co-transfected T24 and EJ cells recovered to close the wound faster within 24 h (Additional file 4: Figure S2A). Further quantitative analysis of wound width indicated relative scratch widths in co-treatment group were significantly less than dsP53-285 transfection groups from 12 h (Additional file 4: Figure S2B).

Subsequently, the transwell assay was conducted to further assess cells migration and invasion capability in vitro. T24 and EJ cells transfected with dsP53-285 or dsControl were allowed to migrate through a transwell membrane into complete media. Compared with control, dsP53-285 exerted a potent inhibiting effect on migration of the both cell lines (Fig. 3c and d). Likewise, invasion capability of bladder cancer cells transfected dsP53-285 was detected by Matrigel invasion chamber assay. As expected, dsP53-285 can notably repress their invasion ability compared with dsControl treatment (Fig. 3c and d). In intravenous injection assay, bioluminescence imaging revealed that fluorescence signal in Lenti-dsP53-285 group was significantly weaker than lenti-dsControl group, which mean that less metastasis is formed in lung after dsP53-285 overexpression (Fig. 3e and f). Moreover, depletion of wild-type p53 dramatically restored cells migration in response to dsP53-285 transfection (Additional file 4: Figure S2C and S2D). Consistent with this, cells migrated the membranes pre-coated with matrigel were more in co-transfection group than dsP53-285 group (Additional file 4: Figure S2C and S2D). These results demonstrate that dsP53-285 can suppress the migration and invasion of T24 and EJ cells mainly by regulating wild-type p53 expression.

We further determined the effects of dsP53-285 transfection on the expression of down-stream genes associated with cell cycle and mitotic checkpoint in bladder cancer cells. As shown in Fig. 4a, transfection of dsP53-285 caused a significant decrease of mRNA levels of Cyclin D1 and CDK4/6 in both tested cell lines. The suppressing effects on protein levels of these genes were further identified by western blotting analysis (Fig. 4b). Moreover, some essential genes related to the EMT process were also detected post dsP53-285 transfection. As seen from Fig. 4c, compared to dsControl group, dsP53-285 significantly up-regulated the mRNA expression of epithelial markers, E-cadherin and β-catenin, and down-regulated mesenchymal markers, ZEB1 and Vimentin in both cell lines, respectively. Immunoblot analysis also revealed a robust increase in E-cadherin and β-catenin, and decrease in ZEB1 and Vimentin protein levels of the two cell lines after dsP53-285 transfection (Fig. 4d).

Next, we blocked wild-type p53 expression and examined the key genes associated with cell cycle. As shown in Figure Additional file 2: Figure S3C, dsP53-285 failed to down-regulated Cyclin D1 and CDK4/6 mRNA levels after siP53 co-transfection. The protein analysis of immunoblot further proved that (Additional file 2: Figure S3D). We next assessed the expression levels of the specific genes mediated EMT process after siP53 transfection. Compared to dsP53-285 group, the mRNA of epithelial markers (E-cadherin and β-catenin) was downregulated, whereas mesenchymal markers (ZEB1 and Vimentin) was upregulated after co-treatment of siP53 for 72 h in both T24 and EJ cells (Additional file 2: Figure S3E). Additionally, immunoblot analysis further confirmed that (Additional file 2: Figure S3F). Taken together, our data strongly imply that dsP53-285 modulates bladder cancer cells cell cycle and EMT associated genes largely depended on modulating wild-type p53 expression.