Long Non-Coding RNA TP53TG1 Upregulates SHCBP1 to Promote Retinoblastoma Progression by Sponging miR-33b

70 retinoblastoma and 70 healthy retinal specimens were obtained from surgery, between January 2013 and January 2015, at the First Affiliated Hospital of Xi’an Jiaotong University and The First Hospital of Qingdao University, China. The average age of the tissue donors were 3.12 years (range, 6 months to 12 years). The tumor samples were assessed by two separate pathologists and assigned a grade, according to the 7th edition of the American Joint Committee on Cancer guidelines²⁷. The healthy retinal samples were collected from the ruptured globes of children, averaging 16.3 years of age. Both tumor and healthy specimens were flash frozen shortly after harvest and maintained at −80°C until further analysis. The RB patients were followed up post surgery and the overall survival (OS) rates were recorded.

Both the RB cell lines (SO-RB50, WERI-Rb1, Y79 and RBL-13) and the healthy human retinal epithelial cell line (ARPE-19) were acquired from the American Type Culture Collection (Shanghai, China). They were maintained in RPMI-1640 media (Gibco, Carlsbad, CA) with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) at 37°C in a humidified environment of 95% air and 5% CO2. siRNAs targeting TP53TG1 (si-TP53TG1), negative control siRNA (si-NC), miR-33b mimics, NC mimics (miR-NC), miR-33b inhibitors, and corresponding NC (miR-NC) were acquired from GenePharma (Shanghai, China). Transient transfections were performed in RB cells using lipofectamine 3000 (Invitrogen, Carlsbad, CA), following manufacturers’ protocols.

Both tissue samples and cultured cells were used to extract total RNA using RNAiso (TaKaRa, Japan), following manufacturer’s protocols. Next, 1 µg of the RNA was reverse-transcribed into cDNA with miRNA Reverse Transcription Kit (RiboBio, China) and miR-33b levels were measured using the miRNA kit (RiboBio, China) and normalized using the endogenous U6 control. To verify our findings and to examine alterations in the transcription of relevant genes, 2 µg of total RNA was reverse transcribed using a PrimeScript first-strand cDNA synthesis kit (Takara, Japan) and qPCR was executed using SYBR^® Premix Ex Taq™ II (Takara, Japan) following manufacturer’s guidelines. Supplementary Table 1 lists the primers used in this study. ABI 7900 cycler (Applied Biosystems, Foster City, CA) was employed for the qPCR amplification and GAPDH was employed as the endogenous control. The relative expression of relevant genes were determined with the 2^−ΔΔCt method.

Transfected Y79 cells were trypsinized and re-plated in 96-well plates (5000 cells/ well). Cell growth was measured with a cell counting kit-8 kit (CCK-8; Dojindo Laboratories), based on manufacturer’s guidelines. CP calculations were performed using an enzyme-based immunosorbent assay reader (Thermo Labsystems) at 450 nm.

For colony forming estimation, low concentration cells (1000 cells/plate) were plated and allowed to form into colonies. Media, nourishing the cells, was changed once in 6 days. Upon colony formation, 0.1% crystal violet (Beyotime, China) stain was introduced to the cells and >50 cell colonies were counted and recorded.

To analyze CM capability, the wound healing assay was employed. Briefly, cells were cultured in a 6-well plate and wounded by dragging a pipette tip over the surface. Following this, the cells were PBS-washed to remove debris. Wound images were taken at the time of wounding and at 24 hours after the wounding.

The CM data was further verified with transwell inserts (8-µm pore filter, 24-well cell clusters; Millipore, Corp., Bedford, MA), following manufacturer’s guidelines. Briefly, cells were transfected for 48 h before they were trypsinized and plated (2 × 10⁵ cells/well) on the top chamber with RPMI 1640 media and 2% FBS. The bottom chamber contained only 500 µl complete media and 10% FBS. Next, CM was allowed to occur from the top to the bottom chamber over a period of 24 hours in a humidified environment 37°C in with 5% CO₂. The cells from the top chamber were removed, whereas those on the bottom chamber were fixed with 4% paraformaldehyde at room temperature for 30 min before a 10 min counterstain with 0.1% crystal violet. Lastly, the migrated cells were then counted using 5 random, non-overlapping fields per well using the software ImageJ (Media Cybernetics, Bethesda, MD, USA). CI assays was conducted similar to the transwell assay; only, the top chamber was precoated with Matrigel (BD Biosciences, San Jose, CA), and 1 × 10⁵ cells were introduced to each top chamber.

To conduct the EdU assay, cells were plated in 24-well plates and incorporated with si-TP53TG1 or si-NC. Following this, 40 μM EdU was introduced to the cells for 2 h before fixing, permeabilizing, and EdU staining, following manufacturer’s guidelines. The nuclei staining was done with DAPI (Sigma Aldrich, Louis, MO). And the CP quantification was performed using a fluorosescent microscope (Celenas; Nikon, Japan). The EdU incorporation rate was assessed with ImageJ.

The Beyotime protein extraction kit was employed for the extraction of total protein from cultured cells. Protein measurement was completed with BCA kit (Qiagen, Valencia, CA). 30 µg of total protein was used to perform western blot analysis using 10% sodium dodecyl sulfate-polyacrylamide gel and polyvinylidene difluoride (PVDF) membranes (Millipore, Corp., Bedford, MA). The membrane was then blocked with 5% skim milk at 37°C for 2 h, and incubated with human SHCBP1 primary antibody (1:500, Abcam, Cambridge, MA) and the endogenous control GAPDH (1:1000, CST.) overnight at 4°C. Membranes were then exposed to the corresponding secondary horseradishperoxidase-conjugated antibody (Abcam, Cambridge, MA) for 1 h at 37°C. Finally, protein quantification was performed by analyzing enhanced chemiluminescence signals (Amersham Biosciences, Arlington Heights, IL) using Image Lab 2.0 (Bio-Rad Laboratories, Hercules, CA).

MiRBase (http://www.mirbase.org), TargetScan (http://www.targetscan.org /mamm_31/), and starBase (http://starbase.sysu.edu.cn/index.php) were used to characterize potential targets of miR-33b. The 3’-UTR of SHCBP1 transcript was found to have potential complementary sequence, thereby increasing its likelihood of being a target for miR-33b. Therefore, RB cells were transfected for 48 h with luciferase vectors pGL6-miR (Beyotime, China), a luciferase vector with the wild-type (WT) target gene’s 3’-UTR and one with a mutant-type target gene’s 3’-UTR (MUT) for TP53TG1 or SHCBP1, along with miR-33b mimics or miR-NC, via Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA). Finally, the luciferase luminescence was evaluated with the Dual-Luciferase Reporter Assay System (Promega, WI), using manufacturer’s guidelines, and the firefly luciferase activity was normalized to the control Renilla luciferase activity.

Upon sh-TP53TG1 or sh-NC transfection, Y79 cells (5 × 10⁶) were re-suspended in 150 µl of culture media, in the absence of FBS, and injected into the subcutaneous layer of the right flank of 4-week-old BALB/c nude mice, acquired from the Slac Laboratory Animal (Shanghai, China). The animals were kept in a sterile room under a 12-h light-dark cycle with open access to food and water. Each implantation group consisted of 6 mice. The tumor volume (TV) was determined as follows: TV = (width × width × length)/2, as previous described. The animals were executed with 1% pentobarbital sodium (50 mg/kg, Sigma Aldrich, Louis, MO), decapitated, and the tumors were harvested and weighed 4 weeks post cell implantation. We followed the strict guidelines of the First Affiliated Hospital of Xi’an Jiaotong University for the Care and Use of Animals in all animal studies.

We employed SPSS 19.0 (SPSS) and GraphPad Prism 6 (GraphPad, La Jolla, CA) for all statistical analysis. The chi-square test was employed to determine the correlation between RB patients’ clinical pathological features and the level of TP53TG1 in their cancerous tissues. Statistical significance (P < .05) between groups was analyzed using t-test or one-way analysis of variance (ANOVA). Potential correlations between TP53TG1 and miR-33b or SHCBP1 expressions were evaluated with Spearman’s correlation. Lastly, statistical significance was represented by asterisks.

To determine a possible correlation between TP53TG1 and RB disease progression, the mRNA expression of TP53TG1 was analyzed in RB (n = 70) and normal retina tissues (n = 70). TP53TG1 levels were remarkably high in RB tissues, as opposed to healthy retina (Fig. 1A). To further analyze the relationship between TP53TG1 and RB, TP53TG1 expression was evaluated against the clinical data from RB patients. We found that patients with elevated TP53TG1 levels had shorter OS time, as opposed to patients with low TP53TG1 levels (P < .01, Fig. 1B). Similarly, TP53TG1 levels were also upregulated in other RB cell lines, namely Y79 and RBL-13, relative to healthy retinal cells (Fig. 1C) and were mostly located in the cytoplasm (Fig. 1D). Taken together, these data suggests that TP53TG1 upregulation is closely linked to the progression of RB.

To explore the role of TP53TG1 in RB cells, we conducted a TP53TG1 knockdown using TP53TG1 specific siRNA. As shown in Fig. 2A, TP53TG1 levels were markedly reduced after treatment with siRNA. The CCK8 assay, assessing cell viability, revealed low cell survival (Fig. 2B). Similarly, the number of colonies reduced significantly in the TP53TG1 siRNA treated Y79 cells, as compared to those treated with si-NC (Fig. 2C). Moreover, the EdU assay demonstrated markedly reduced EdU positive cells, suggesting vastly reduced CP in the TP53TG1 siRNA treated cells, as opposed to si-NC treated controls (Fig. 2D). Additionally, we examined the TP53TG1 mediated regulation of tumor growth on nude mice xenografts. TP53TG1 was silenced in Y79 cells using recombinant plasmid carrying specific shRNA before implantation into nude mice. Our results demonstrated that TP53TG1-silenced Y79 cells were slow to form tumors and produced smaller sized tumors, as opposed to the tumors originating from sh-NC incorporated cells (Fig. 2E, F). In summary, these results strongly suggest that TP53TG1 silencing suppressed RB cell proliferation.

To characterize the potential regulatory mechanisms whereby TP53TG1 modulated tumor progression in RB, TP53TG1 was knocked down in Y79 cells and the CM and CI properties were assessed. TP53TG1 knockdown was shown to greatly inhibit CM, as evidenced by the wound healing assay (Fig. 3A). This result was further verified using the transwell assay that demonstrated that TP53TG1 knockdown significantly reduced both migratory and invasive behavior of Y79 cells (Fig. 3B). Taken together, this suggests that TP53TG1 knockdown can effectively inhibit both CM and CI in RB cells.

Multiple studies have reported that one of function of lncRNAs is to competitively interact with miRNAs to prevent miRNA-mediated degradation of target transcripts. As such, miRNA candidates that could possibly interact with TP53TG1 and, therefore, be a target of TP53TG1 were searched using miRBase, TargetScan and starBase. The predicted miR-33b binding site within TP53TG1 is shown in Fig. 4A. Comparing between RB tissues and normal retinal tissues, miR-33b showed a marked reduction in RB tissues, but not in normal retina (Fig. 4B). These data were further confirmed by the pearson correlation analysis which demonstrated a strong inverse association between TP53TG1 and miR-33b levels (R = −0.2608, P < .05; Fig. 4C). Moreover, similar to the expression of miR-33b in RB tissues, the levels of miR-33b was remarkably low in all examined RB cell lines, relative to healthy retinal cells (Fig. 4D). To further understand the TP53TG1 and miR-33b interaction, RBL-13 and Y79 cells were first incorporated with either miR-33b mimics or miR-33b inhibitors, respectively. As shown in Fig. 4E, F, cellular miR-33b levels were elevated after miR-33b mimics transfection, and were reduced with miR-33b inhibitors transfection. Next, we constructed luciferase reporter vectors with WT TP53TG1 or TP53TG1 with mutations in the putative binding sites of miR-33b (MUT). Dual-luciferase assays showed a notable reduction in luminescence in RBL-13 cells co-transfected with miR-33b mimics and the WT TP53TG1 expression vector, but not with the MUT (Fig. 4G). Next, we determined whether the TP53TG1-miR-33b interaction modulated miR-33b activity. We introduced TP53TG1 knockdown in RB cells, which resulted in marked increases in miR-33b expression (Fig. 4H). On the contrary, when miR-33b was overexpressed in cells, TP53TG1 levels rose and when miR-33b is downregulated, the TP53TG1 levels fell (Fig. 4I, J). In all, these results confirm that TP53TG1 may serve miR-33b sponge.

TP53TG1 was shown to interact with the 3’ UTR of miR-33b and regulate its expression. We next explored whether TP53TG1 mediated regulation of RB involved miR-33b. CCK-8 assays revealed that cells incorporated with miR-33b inhibitors had higher CP, relative to cells receiving si-TP53TG1 (Fig. 5A). Moreover, using colony formation assay, we demonstrated that miR-33b inhibitor partly reversed the effects of si-TP53TG1 in CP (Fig. 5B). Additionally, cells transfected with miR-33b inhibitor was shown to have higher CM and CI, whereas TP53TG1 knockdown experienced reduced CM and CI (Fig. 5C). Based on these data, miR-33b silencing can significantly diminish the function of TP53TG1 on RB cells, indicating an antagonistic association between TP53TG1 and miR-33b in its modulation of the disease.

Next we investigated which is the downstream moleculars of miR-33b. Bioinformatics analysis predicted that SHCBP1 shared sequence homology with miR-33b at its 3’-UTR (Fig. 6A). Using luciferase reporter assay, we demonstrated a physical interaction between miR-33b and the 3’-UTR of SHCBP1 in Y79 cells (Fig. 6B). Moreover, SHCBP1 mRNA levels declined with si-TP53TG1 transfection of Y79 cells (Fig. 6C). Similarly, SHCBP1 protein expression was elevated in RB cells (Fig. 6D). Alternately, Y79 cells incorporated with miR-33b revealed a large decline in SHCBP1 mRNA and proteins expression (Fig. 6E, F). In all, these data points toward a TP53TG1/miR-33b/SHCBP1 pathway in the regulation of RB progression.