PTBP3 Modulates of P53 Expression and Promotes Colorectal Cancer Cell Proliferation by Maintaining UBE4A mRNA Stability

doi:10.21203/rs.3.rs-827706/v1

Background

Colorectal cancer (CRC) is the most common malignancy worldwide and has become the second leading cause of cancer-related death worldwide. The RNA-binding protein polypyrimidine tract-binding protein 3 (PTBP3) was recently reported to play a critical role in multiple cancers, and its molecular mechanisms involve RNA splicing, 3′ end processing and translation. However, the role of PTBP3 in CRC is unclear.

Methods

We analyzed the expression levels of PTPB3 using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets and clinical tissues. The effect of PTBP3 on CRC cell proliferation was measured by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and tumor xenograft assays. A series of experiments were conducted to reveal the mechanisms by which PTBP3 promotes CRC proliferation.

Results

We showed that PTBP3 was upregulated in CRC and associated with a poor prognosis. Knockdown of PTBP3 in CRC cell lines restricted CRC proliferative capacities in vitro and in vivo. Mechanistically, we found that PTBP3 regulated the expression of the E3 ubiquitin ligase ubiquitination factor E4A (UBE4A) by binding the 3' untranslated region (UTR) of its mRNA, thereby preventing its degradation. We also discovered that UBE4A participated in the degradation of P53, and knockdown of PTBP3 in CRC cell lines increased P53 expression. Overexpression of UBE4A rescued PTBP3 knockdown-induced inhibition of CRC cell proliferation and P53 expression.

Conclusions

PTBP3 plays an essential role in CRC cell proliferation by stabilizing UBE4A to regulate P53 expression and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

Clinical Pharmacology

Cancer Biology

Oncology

PTBP3

UBE4A

P53

CRC

mRNA stability

Colorectal cancer (CRC) is the most common malignancy worldwide and has become the second leading cause of cancer-related death worldwide(1, 2). Although survival has improved due to advances in surgical techniques, malignant growth and metastasis are still major causes of cancer-related death(3). Therefore, it is important to increase our understanding of the mechanisms that drive CRC progression.

PTBP3 was first identified as an essential RNA-binding protein in 1999(4) and is a member of the PTB family containing three paralogs, namely, PTBP1, PTBP2 and PTBP3(5). Previous studies showed that PTBP3 was dysregulated and promoted the progression of numerous cancers, including breast cancer(6), hepatocellular cancer(7), gastric cancer(7) and CRC(9). The functions of PTBP3 involved RNA splicing, translational activation and mRNA stability(6–10). However, the specific function of PTBP3 and its potential mechanism in CRC proliferation remain largely unknown.

E3 ubiquitin ligase ubiquitination factor E4A (UBE4A) is a U-box-containing ubiquitylation enzyme(11). Similar to UBE4B, UBE4A belongs to the human homologs of yeast UFD2 ubiquitination factor family, whose members share a conserved domain of approximately 70 amino acids that has been named the U box(12–14). Previous studies implicated UBE4B as a regulator of P53(15–19); the U box catalytic domain is closely related to the RING-finger domain of MDM2 and is responsible for its E3 activity. These two enzymes are considered to be significant regulators of p53 through the ubiquitination process(15–19). However, the function of UBE4A, as a U box catalytic domain-containing E3 ubiquitin ligase, remains largely unknown. In gastrointestinal disease, it has been demonstrated that dysregulation of UBE4A in enteroendocrine cells is associated with Crohn's disease(20), but its function and regulatory mechanism in CRC remain unclear.

In this study, we attempted to identify the function and regulatory mechanism of PTBP3 in CRC. We found that PTBP3 was upregulated in CRC patients with a poor prognosis and led to CRC proliferation, which suggests that PTBP3 may serve as an important factor in CRC development. Mechanistically, we found that PTBP3 regulated the expression of the E3 ubiquitin ligase UBE4A by binding to its 3' untranslated region (UTR) to prevent its mRNA degradation. We also found that UBE4A promoted CRC progression and participated in the degradation of P53. Our results provide new evidence that PTBP3 exerts an oncogenic function and that the PTBP3/UBE4A/P53 axis might serve as a potential therapeutic target for CRC.

Tissue Collection and Ethic Statement

Clinical material were obtained from patients treated at the Third Xiangya Hospital of Central South University (Changsha China) with informed consent and approval of the Medical Ethics Central South University.Tissue specimens were snap frozen and kept in liquid nitrogen until further use.

Cell lines and cell culture

All colorectal cancer cell lines were purchased from KeyGEN BioTECH (Jiangsu, China). FHC was purchased from American Type Culture Collection(ATCC) (Manassas, Virginia, USA). All cell lines had been authenticated using STR (or SNP) profiling within the last 3 years. All experiments were performed with mycoplasma-free cells.Colorectal cancer cells SW620 (RRID: CVCL_0547) and SW480 (RRID: CVCL_0546) cells were cultured in L15 (KeyGEN BioTECH, Jiangsu, China) medium supplemented with 10% fetal bovine serum (FBS, Biological Industries, Israel) and 1% antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Life Technologies, Inc., Grand Island, NY, USA).HCT116 (RRID: CVCL_0291) and HT29 (RRID: CVCL_0320)cells were cultured in McCoy’s 5 A (KeyGEN BioTECH) medium supplemented with 10% FBS and 1% antibiotics. LoVo(RRID:CVCL_0399) cell was cultured in Dulbecco’s modified Eagle’s medium (DMEM,KeyGEN BioTECH) supplemented with 10% FBS and 1% antibiotics. FHC(RRID:CVCL_3688) cell was cultured in Roswell Park Memorial Institute 1640(RPMI 1640,KeyGEN BioTECH) medium supplemented with 10% fetal bovine serum and 1% antibiotics.All cell lines were grown in a 5% CO₂ cell culture incubator at 37^◦C.

Patients and tissue sampling

All clinical material were obtained from patients who underwent surgical resection for CRC at the Third XiangYa Hospital of Central South University (Changsha, China) after informed consent and approval of the Medical Ethics Central South University.

Quantitative real-time PCR assays

Total RNA from cells and tissues was extracted by TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was generated by ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Quantitative real-time PCR (qRT-PCR) was carried out on a LightCycler 480 Real Time PCR instrument (Roche, Basel, Switzerland).GAPDH was used for normalization of qRT-PCR data. All primer pairs were purchased from Sangon Biotech (Shanghai, China), and sequences are available in Supplementary Table1.

Western blot assays

Whole-cells and tissues were collected and lysed with 1 × RIPA buffer (KeyGEN BioTECH) containing 1% PMSF (KeyGEN BioTECH) to harvest proteins. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and then transferred to polyvinylidene fluoride membranes (PVDF)(Millipore,CA,USA),blocked with 5% skim milk for 2h,then the PVDF membranes were incubated with primary antibodies at 4◦C overnight and secondary antibodies for 1 h, and visualized on an Odyssey CLx Infrared Imaging System (LI-COR Biosciences, NE, USA).The antibodies used for western blot (WB) were provided in Supplementary Table2.

Lentiviral Vector and Transfection

Lentiviruses, including PTBP3 and UBE4A, and their corresponding negtive control,were obtained from Shanghai GenePharma Co., Ltd. To generate stable lentivirus-transduced lines, cells were infected with virus and polybrene following the manufacturer’s recommendations, and stable cell lines were selected with 4µg/ml of puromycin treatment after 72 h of transfection. The efficiency in different cells was determined by the GFP intensity ,qRT-PCR and WB.The shRNA sequences are listed in Supplemental Table 1.

Plasmid Transfection

UBE4A plasmid were obtained from Shanghai GenePharma Co., Ltd.When tranfected the UBE4A plasmid,cells were seeded into 6-well plates and cultured for 24 hours. When the density reached 50–60%, Lipofectamine 3000 reagent (Invitrogen, USA) was used to transfect the UBE4A plasmids according to the instructions.

Cell proliferation and colony formation assays

The CCK8 assay (Dojindo, Kumamoto, Japan) was used to measure cell proliferation in 96- well plates. About 2000 HCT116 cells、2000SW480 cells and 1000 LOVO cells were seeded at per well,with six replicates for each condition. CCK8 was added at 0,24, 48, and 72 h and incubated at 37 °C for 2 h.The absorbance values (A450) were detected using an EnVision microplate reader (PerkinElmer). For colony formation assays , about 100 HCT116 cells、1000 LOVO cells and 1000 SW480 cells were seeded in each well of a 6-well plate in triplicate for each condition and incubated for 7 to 12 days. The colonies were fixed with methanol, stained with crystal violet, and counted. The average colony counts were calculated, and a paired t-test was used to test statistical signifificance. Each experiment was repeated three times.

Flow Cytometric Analysis

Cell cycle analysis was measured by flow cytometry. In general, each group prepared one hundred thousand cells labeled with propidium iodide (PI; Sigma-Aldrich, USA) was analyzed by using FACSCalibur flow cytometer (BD Biosciences). Then the proportions of G0/G1, S and G2/M cells were calculated and compared by using ModFit LT 3.1 software. The results were analyzed by using FACSCalibur flow cytometer (BD Biosciences).

Tumor Xenografts

The nude mouse xenograft tumor growth model experiment was performed according to the guidelines for experimental animal management established by Kagawa University and guidelines for the welfare and use of animals in cancer research(21).In general,the Female BALB/c nude mice (4–5 weeks, 18–20 g) from the Department of Laboratory Animals of Central South University and maintained them under specifific pathogen-free conditions.Then 3 × 10⁶HCT116-shNC(small hairpin carrying negtive control RNAs) cells and HCT116-shPTBP3(small hairpin carrying PTBP3-specifific RNAs) cells and 2× 10⁶LoVo-shNC cells and LoVo-shPTBP3 cells were harvested and injected subcutaneously into the left or right flank of nude mice (n = 3 per group).Recorded the tumors with calipers and an electronic scale to estimate the tumor volume and weight every four days.After 30 days of the injection, the mice were killed by an overdose of pentobarbital (250 mg/kg, intraperitoneal injection), recorded the final results of the tumor volume and weight. The tumor volumes were calculated based on the formula: volume (mm³) = length (mm) × width (mm) × width (mm)/2. Tumors were further embedded in paraffifin for H&E and immunohistochemistry (IHC).

Immunohistochemistry

The tissue sections from the nude mouse was embedded by paraffined. Tissue sections were deparaffined and rehydrated. Blocked the tissue sections’ endogenous peroxidase activity with 0.3% hydrogen peroxide for 20 minutes. Then the tissue sections were blocked in 10% BSA for 10 minutes, and incubated with anti-human Ki-67(1:100) antibodies and anti-human PTBP3 antibody (1:100) at 4◦C for 12 h. After that tumor sections were incubated in biotinylated secondary antibodies for 20 minutes at room temperature. Thereafter, the tissue sections were reacted with streptavidin-peroxidase conjugate for10 minutes. Then added 3,3’-diaminobenzidine as the chromogen substrate. Images were captured using an inverted microscope system (Olympus, IX73, Japan).

RIP Assay

The RNA-binding protein immunoprecipitation (RIP) was performed using the EZ-Magna RIP Kit (Merck, KGaA, Darmstadt, Germany, Catalog No. 17-701) according to the manufacturer’s instructions. In general,about 2 × 10⁷ HCT116 and LOVO cells were washed with ice-cold PBS and resuspended with RIP lysis buffer containing protease inhibitor mixture and RNase inhibitor. Then, Magnetic Beads Protein A/G was incubated with 5 µg IgG (negative control) (Merck KGaA) or PTBP3 (SantaCruz) antibody for 30 min at room temperature. Then, the complexes were added cell lysis buffer and immunoprecipitation buffer with EDTA and RNase inhibitor. Thereafter, the complexes were incubated in rotator overnight at 4◦C. The next day, the complex was washed with washing buffer containing with proteinase K and 10%SDS and then heated at 55◦C for 30 min. Finally, RNA was extracted and purified for RT-qPCR analysis. RIP assays were performed in biological triplicates and were detected by RT-qPCR ,the primers are described in Supplemental Table 1.

RNA Antisense Purification Assay

The RNA antisense purification assay was performed using the RNA Antisense Purification(RAP) Kit(Bersin BioTM ,guangzhou,China,CataLog Bes5103-3) according to the manufacturer’s instructions. In general,about 2 × 10⁷ HCT116 cells were washed with ice-cold PBS and resuspended with methanol and 1.375 M Glycine for cross-link cells.Then added the lysis buffer containing protease inhibitor mixture and RNase inhibitor for homogenization.Next added the DNase salt stock、DNase、EDTA、EGTA、DTT for removing DNA.Then added the probes to the processed sample and hybridization at 37℃ for 30 min(the probes are desecribed in Supplemental Table1),incubate degeneration at 50 ℃ for 50 min,hybridization at 37℃ for 180 min one more time.Next added the Streptavidin Beads to the complex incubated for 30 min.After washing with wash buffer, the RAP mix bound to the beads was eluted and then re-suspended in 60μl of 1× loading buffer and boiled for 5 min, and followed with Western blot detection.

Luciferase reporter assays

HCT116sh-NC cells、HCT116sh-PTBP3 cells、Lovosh-nc cells and Lovosh-PTBP3 cells were seeded at a density of 1× 105cells per well in a 24-well plate. Cells were transfected with the pRL-TK (Promega) renilla plasmid by using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). The Renilla luciferase sequence in the pRL-TK vector (Promega, WI, USA) was used as an internal control. The dual luciferase reporter assays were performed according to the protocol using the Dual-Luciferase Reporter Assay System (cat. E1910, Promega). The firefly luciferase activity was normalized to the Renilla luciferase activity. The data are expressed as the percent of luciferase activity in control cells (100%).

Immunoprecipitation Assay

The immunoprecipiton assay was perfermed according to the manufacturer’s instructions using the (Thermo,PierceTM Classic Magnetic,Rockford,USA,LOT:UC283101). In general, HCT116 cell lysates were prepared in immunoprecipitation lysis buffer (20 mM Tris-Cl, pH 8.0, 10 mM NaCl, 1mM EDTA, 0.5% NP-40) containing a protease inhibitor cocktail (Sigma). 2mg cell extracts were pre-cleared with 50μl protein A/G-agarose (Santa Cruz) at 4°C for 2h, and the supernatant was incubated with corresponding antibodies with gently shacking at 4°C overnight, followed by the addition of 50μl of protein A/G-agarose for another 2h. The beads were washed and then re-suspended in 60μl of 1× loading buffer and boiled for 5 min, and followed with Western blot detection.

Bioinformatics analysis

The data of PTBP3 expression analysis was downloaded from the TCGA(https://gdc.cancer.gov/)and GEO( https://www.ncbi.nlm.nih.gov/geo). The survival analysis of PTBP3 was carried out using the Gene Expression Profifiling Interaction Analysis database(http://gepia.cancer-pku.cn/)(22).The correlated gene of PTBP3 was analyzed by the TCGA data and starBase v2.0 (http://starbase.sysu.edu.cn/index.php) (23).The correlated pathway of PTBP3 analyzed by the Gene Set Enrichment Analysis(24).

Statistical analysis

Statistical computations was performed using GraphPad Prism 8.0. Data (San Diego, CA) were presented as mean ± s.d. of three independent experiments except where otherwise indicated. To compare the differences between two groups, Student’s t-test was performed.One-way analysis of variance (ANOVA) was used for comparison between the different groups. The relationship between gene expression and clinical pathological indicators is examined through chi-square test,and p < 0.05 was considered statistically signifificant.

PTBP3 is upregulated in CRC, and high PTBP3 expression correlates with a poor prognosis

To identify the role of PTBP3 in CRC development, we first assessed the expression of PTBP3 in CRC tissues. Gene expression data for 471 colorectal cancer samples were downloaded from The Cancer Genome Atlas (TCGA) database. PTBP3 expression was first analyzed in 471 CRC tissues and 41 normal colorectal tissues, and we found that PTBP3 was significantly upregulated in CRC tissues (Fig. 1a). Then, we downloaded and assessed two CRC gene expression datasets, GSE21510 and GSE44076, from the Gene Expression Omnibus (GEO) database, and the results showed the same trend (Fig. 1b). To further confirm this conclusion, we detected the expression of PTBP3 in 30 matched pairs of human CRC tissues and adjacent nontumor tissues by qRT-PCR and 12 matched pairs of tissues by western blotting (WB); the results showed that PTBP3 was significantly upregulated in human CRC tissues (Fig. 1c-d). Next, we summarized the clinicopathological characteristics of the 30 patients in the first cohort, and the data demonstrated that patients with high PTBP3 expression showed larger tumor sizes than patients with low PTBP3 expression (Table 1). Moreover, the expression level of PTBP3 was detected in 5 human CRC cell lines (HT-29, HCT116, SW480, SW620 and LoVo) and normal human colonic epithelial FHC cells, and the results showed that PTBP3 expression was significantly higher in cancer cell lines than in FHC cells (Fig. 1e-f). HCT116 and LoVo cells with relatively high PTBP3 expression were selected for subsequent functional assays. Moreover, we examined the correlation between the PTBP3 expression level and the prognosis of CRC patients through the Gene Expression Profiling Interactive Analysis (GEPIA) database. Kaplan–Meier survival analysis showed that patients with high levels of PTBP3 had shorter overall survival times than those with low levels of PTBP3 (Fig. 1g). Collectively, these results showed that PTBP3 was overexpressed in CRC patients with poor prognosis.

Table 1 Clinic-pathological characteristics of enrolled patients.

Clinicopathological Characteristics	Total (n=30)	PTBP3
Clinicopathological Characteristics	Total (n=30)	High^a (n=15)	low^a (n=15)	P value
Mean age	63.5 ± 1.853	66.07±2.792	60.93 ± 2.343	0.17
Sex				0.6903
Male	21	11	10
Female	9	4	5
Tumor site				0.3940
Left colon	1	0	1
Right colon	12	5	7
Rectum	17	10	7
staging				0.2557
I-II	19	11	8
III-IV	11	4	7
Tumor size				0.0464^b
≥5cm	11	7	2
<5cm	19	8	13
Differentiation				0.9103
CRC,WD	9	5	4
CRC,MD	13	6	7
CRC,PD	8	4	4
Tumor stage				>0.9999
T1-T2	8	4	4
T3-T4	22	11	11
Lymph node				0.0803
N0	19	11	8
N1	7	1	6
N2	4	3	1
Distant metastasis				0.3091
M0	29	15	14
M1	1	0	1

^a Low and high expression groups were determined by the cutoff-point 50% (15 of 30) and 50% (15 of 30) of PTBP3 in 30 tumor tissue specimens. ^b Statistical signifificance (P < 0.05).

PTBP3 is required for the proliferation of CRC cells in vitro and in vivo

To assess the functional significance of PTBP3 in CRC cells, we first silenced PTBP3 expression in HCT116 and LoVo cells with lentiviral vectors carrying PTBP3-specific small hairpin RNAs (shRNAs). Control cells were transfected with lentiviral vectors carrying negative control shRNA. The transfection effect was observed according to green fluorescence protein expression, and the PTBP3 silencing effect was confirmed by qRT-PCR and WB (Fig. S1A-C). Then, we investigated the role of PTBP3 in HCT116 and LoVo cells by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. The results showed that PTBP3 silencing reduced cell proliferation activity (Fig. 2a-b). Next, we assessed the effect of PTBP3 on the cell cycle by using flow cytometry. The results showed that silencing PTBP3 expression increased the proportion of cells arrested in the G0/1 phase and decreased the proportion of cells in S phase for both HCT116 and LoVo cells (Fig. 2c). These findings suggested that PTBP3 plays an important role in CRC proliferation in vitro. To further evaluate the role of PTBP3 in vivo, we injected HCT116 and LoVo with stable PTBP3 knockdown and their corresponding NC cells into nude mice. All mice developed tumors at the injection site (Fig. S2A), but the average size and weight of the tumors generated by PTBP3 knockdown cells were significantly smaller than those generated by NC cells (Fig. 2d-f). Immunohistochemistry (IHC) showed lower Ki67 expression in PTBP3 knockdown tumor tissue group than in the NC group (Fig. S2B), suggesting that PTBP3 knockdown suppressed the proliferation of cancer cells. All tumor tissues were verified by hematoxylin and eosin (H&E) staining (Fig. S2B). Together, these results confirmed the oncogenic activity of PTBP3 in CRC in vivo, which was consistent with what we observed in vitro.

PTBP3 binds to the UBE4A 3' UTR and may stabilize UBE4A mRNA

To study the mechanism related to the oncogenic activity action of PTBP3, we performed gene set enrichment analysis (GSEA) using mRNA expression data from TCGA CRC datasets (Table S3). We found that PTBP3 mRNA expression correlated positively with ubiquitin-mediated proteolysis gene signatures, indicating that this pathway is the most closely related to PTBP3 activity (Fig. 3a). However, in previous studies, PTBP3 was characterized as an RNA-binding protein and was not demonstrated to have a protein domain that participates in ubiquitylation directly(25). Therefore, we speculated that PTBP3 may impact some critical ubiquitylation-related genes to influence the pathway. Consequently, we first analyzed PTBP3-correlated genes in TCGA CRC datasets (Table S4) and downloaded PTBP3 RNA immunoprecipitation sequencing (RIP-seq) data (Table S5)(26). Then, a Venn diagram was drawn, and 136 closely correlated genes were obtained (Fig. 3b). Next, the 136 genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig. 3c), and we found 4 genes (UBE4A, ITCH, CUL4B, HERC4) distributed in the ubiquitin mediated proteolysis pathway (Fig. S3A). To investigate whether PTBP3 impacts the expression of these four genes, we first performed qRT-PCR in HCT116 and LoVo PTBP3 knockdown cells. The results showed that only the mRNA levels of UBE4A were decreased (Fig. 3d). Subsequent WB experiments confirmed this correlation at the protein level (Fig. 3e). It has been demonstrated that PTBP3 can regulate gene expression by 3′ end processing to influence gene expression(6). Therefore, we speculated that PTBP3 may associate with UBE4A to affect its mRNA stability and expression. To verify this hypothesis, we first assessed the RNA decay rate of UBE4A in PTBP3 knockdown CRC cells and the corresponding control cells. UBE4A mRNA expression was initially decreased, and the UBE4A mRNA half-life was consistently markedly shortened upon PTBP3 knockdown (Fig. 3f). Then, we performed RIP assays in HCT116 cells, and primers targeting the UBE4A-3' UTR were significantly more enriched with the anti-PTBP3 antibody than with IgG (Fig. 3g). Additionally, we developed a psiCheck-2 reporter plasmid containing the UBE4A-3' UTR sequence cloned downstream of Renilla luciferase (Rluc) and firefly luciferase driven by the HSV-TK promoter. We observed that Rluc activity in cells with PTBP3 knockdown was significantly higher than that in the control cells (Fig. 3h). These results suggested that PTBP3 binds to the UBE4A-3' UTR directly. Then, we designed 4 probes targeting the UBE4A-3' UTR and performed a biotin-labeled RAP assay(Table S1). The results revealed presence of PTBP3 in the products for probes 1+3 (Fig. 3i). The RNA-induced silencing complex (RISC) is one type of basic eukaryotic cellular machinery that plays a pivotal role in posttranscriptional gene regulation(27). A previous study showed that RNA-binding proteins could maintain mRNA stability by preventing RISC-mediated mRNA degradation(28, 29). Surprisingly, we also observed the presence of AGO2 (a core component of the RISC complex) with the biotin-labeled RNA pull-down assay (Fig. 3I). Therefore, these results suggested that PTBP3 may prevent UBE4A degradation by RISC to stabilize its mRNA. Further immunoprecipitation experiments also revealed interactions between PTBP3 and AGO2 (Fig. 3j). Collectively, our results suggested that PTBP3 stabilizes UBE4A mRNA by binding to its 3' UTR to prevent mRNA degradation mediated by the AGO2-containing RISC complex.

UBE4A promotes CRC cell proliferation in vitro

The above results indicated that PTBP3 regulates oncogenic activity in CRC and regulates UBE4A expression by binding to its 3' UTR. However, we still did not know the function of UBE4A in CRC, and there are no previously published articles about UBE4A in CRC. To assess the function of UBE4A in CRC cells, we silenced UBE4A expression in HCT116 and LoVo cells with lentiviral vectors carrying UBE4A-specific shRNAs. Control cells were transfected with lentiviral vectors carrying negative control shRNA. The transfection effect was observed by detecting green fluorescence (Fig. S4A), and the UBE4A silencing effect was confirmed by qRT-PCR and WB (Fig. S4B-C). Next, we used CCK-8 and colony formation assays to determine the role of UBE4A in mediating the malignant behavior of HCT116 and LoVo cells. The result showed that UBE4A silencing reduced the cell proliferation activity (Fig. 4a-b), which was consistent with the effect of PTBP3 in CRC. Similarly, we also assessed the effect of UBE4A on the cell cycle by using flow cytometry. The results indicated that silencing UBE4A expression increased the proportion of cells arrested in the G0/1 phase and decreased the proportion of cells in S phase for both HCT116 and LoVo cells (Fig. 4c). These findings suggested that UBE4A promotes CRC cell proliferation in vitro, which is consistent with the role of PTBP3 in CRC.

PTBP3 regulates P53 expression by facilitating the ubiquitin-mediated degradation of UBE4A

Previous studies have shown that UBE4B can regulate P53 protein stability through the MDM2-P53 pathway(15, 17, 19). UBE4A, as a homolog of UBE4B(15, 17, 19), may also have the same effect on P53. To verify this, we performed qRT-PCR and WB analysis in UBE4A knockdown CRC cell lines and their corresponding negative control cell lines. The results showed that UBE4A knockdown had no effect on the expression of P53 at the mRNA level but significantly increased the expression of P53 at the protein level (Fig. 5a-b), which may suggest that UBE4A affects P53 protein stability. To verify this, we treated HCT116 UBE4A knockdown cell lines and the corresponding negative control cell line with cycloheximide (CHX), a protein synthesis inhibitor. We found that P53 expression was increased after UBE4A knockdown (Fig. 5c left), and the rate of P53 degradation was slow at the indicated time after UBE4A knockdown (Fig. 5c right). The results showed that the stability of P53 was increased by UBE4A knockdown (Fig. 5c). Next, we treated HCT116 UBE4A knockdown cell lines and the corresponding negative control cell line with MG132, a proteasome inhibitor. We found that P53 expression was increased after MG132 treatment. This result suggested that UBE4A affected P53 stability in the context of proteasome-dependent degradation (Fig. 5d). Collectively, these results further confirmed our speculation that UBE4A may affect P53 stability via proteasome-dependent degradation to influence CRC proliferation. It has been demonstrated that PTBP3 can regulate P53 expression in hepatocellular carcinoma, but the specific mechanism remains unclear(7). Here, we performed qRT-PCR and WB analysis of PTBP3 knockdown CRC cell lines, and the results showed that PTBP3 knockdown had no effect on P53 mRNA levels but increased the protein expression of P53 (Fig. 5e-f). Therefore, we speculated that PTBP3 modulates P53 expression by mediating UBE4A expression. Next, we transfected UBE4A plasmids into PTBP3 knockdown HCT116 and LoVo cells (Fig. S4D-E). WB assays showed that overexpression of UBE4A restored P53 expression in PTBP3 knockdown HCT116 and LoVo cells (Fig. 5g). Given that LoVo and HCT116 cells possess the wild-type (WT) TP53 gene, we next used the SW480 cell line with mutant TP53 to examine the effect of PTBP3 and UBE4A on CRC. We first silenced PTBP3 and UBE4A in SW480 cells separately. The transfection effect was observed by detecting green fluorescence (Fig. S5A), and the silencing effect was confirmed by qRT-PCR and WB (Fig. S5B,5E). Then, we assessed cell viability and P53 expression in SW480 cells.The results showed that knockdown of either PTBP3 or UBE4A did not decrease SW480 cell viability or P53 expression (Fig. S5C-E). Collectively, these findings suggest that PTBP3 regulates WT P53 expression by facilitating the ubiquitin-mediated degradation of UBE4A in CRC.

Overexpression of UBE4A restores PTBP3 knockdown-mediated CRC cell proliferation

To investigate the function of UBE4A in PTBP3-mediated promotion of CRC proliferation, we transfected UBE4A plasmids into PTBP3 knockdown HCT116 and LoVo cells and examined the effect of UBE4A overexpression on the proliferation activity and cell cycle distribution. We found that UBE4A overexpression restored the proliferative activity and abnormal cell cycle of PTBP3 knockdown HCT116 and LoVo cells (Fig. 6a-c). These results further proved that PTBP3 promotes CRC cell proliferation by regulating UBE4A.

PTBP3 is a protein-coding gene that plays an essential role in humans(30). Recent studies have shown that PTPB3 is dysregulated in multiple tumors and plays vital roles in carcinogenesis(6–10), but few studies have elucidated the specific mechanisms, especially in CRC. In the present study, we found that PTBP3 is upregulated in CRC patients with a poor prognosis and promotes CRC cell proliferation by meditating UBE4A mRNA stability to regulate P53 expression.

In this study, we first analyzed the TCGA and GEO online databases. The results showed that PTBP3 mRNA was upregulated in CRC. Then, we performed an assay with clinical tissues to further confirm this conclusion at both the mRNA and protein levels. Next, we analyzed the correlations between the PTBP3 expression level and clinical features, and we found that a high PTBP3 expression level was correlated with clinical features, especially tumor size. Ping et al.’s study also showed that PTBP3 was upregulated and associated with a poorer clinical prognosis(9), which supported our findings. Additionally, PTBP3 overexpression was also found in hepatocellular cancer(7), gastric cancer(8), and breast cancer(10) and was correlated with a poorer clinical prognosis, which suggested that PTBP3 may serve as an oncogene in multiple cancers. However, all of these studies, including ours, lacked a sufficient amount of data to draw reliable conclusions; thus, further multicenter clinical trials are needed. To further investigate the oncogenic role of PTBP3 in CRC, we silenced PTBP3 in HCT116 and LoVo cells. After knockdown of PTBP3, we found that CRC cell proliferation was significantly inhibited in HCT116 and LoVo cell lines in vivo and in vitro. This was partly consistent with the results of the study of Ping et al(9), who used HCT116 and SW480 cells for research but only conducted a CRC cell proliferation assay in HCT116 cells and not in SW480 cells. Considering that PTBP3 was found to regulate P53 expression in our study, we also silenced PTBP3 in the SW480 cell line, a TP53-mutant gene cell line. However, we found that silencing PTBP3 in SW480 cells had no effect on CRC cell proliferation. Similarly, it has been reported that PTBP3 has no effect on gastric cancer proliferation but promotes breast cancer and hepatocellular cancer growth(6–8). We speculated that PTBP3-mediated regulation of P53 expression might be one of the reasons. Mechanistically, we found that PTBP3 was positively correlated with the ubiquitin-mediated proteolysis signaling pathway according to GSEA. However, PTBP3 does not have a protein domain that participates in ubiquitination(25). Therefore, we investigated four candidate genes related to the ubiquitin-mediated proteolysis signaling pathway by PTBP3 RIP-seq and identified PTBP3-correlated genes through TCGA database analysis. We found that knockdown of PTBP3 decreased UBE4A expression at both the mRNA and protein levels. We hypothesized that PTBP3 may influence UBE4A mRNA stability. We examined the decay rate of UBE4A mRNA, and the results proved our hypothesis. Then, we performed the RIP assay and luciferase reporter assay to prove PTBP3 binding to the UBE4A 3' UTR, and the RAP assay further proved this. The presence of AGO2, a core component of RISC, in the RAP assay suggested that PTBP3 may stabilize UBE4A mRNA by preventing RISC-mediated degradation of UBE4A mRNA. Further immunoprecipitation assays showed that PTBP3 and AGO2 could bind to each other. Collectively, our results first proved that PTBP3 has a great impact on the ubiquitin-mediated proteolysis signaling pathway by stabilizing UBE4A mRNA in CRC.

It has been demonstrated that the ubiquitin-mediated proteolysis signaling pathway plays a role in every cellular function, including processes of paramount importance for carcinogenesis, such as proliferation, apoptosis, and angiogenesis(31, 32), which commonly occur in CRC(33, 34). UBE4A, an E3 ubiquitin ligase, has seldom been reported in the context tumorigenesis. UBE4A represses ILEI protein expression to inhibit prostate cancer progression(35). However, in thyroid carcinoma, UBE4A was reported as a ubiquitin ligase that is inversely correlated with PCBP1 protein expression and promotes cancer progression(36). Therefore, it is necessary to discuss the specific function and mechanism of CRC. In the present study, we first demonstrated that knockdown of UBE4A could promote CRC proliferation and increase P53 expression at the protein level in CRC cell lines expressing the WT TP53 gene but not in CRC cell lines expressing the mutant TP53 gene. Further experiments indicated that UBE4A might influence P53 stability. However, we did not elucidate the specific mechanism by which UBE4A regulates P53 stability in this study, which needs further research, especially concerning the relationship between UBE4A and mutant P53 expression. In addition, we found that PTBP3 knockdown could increase WT P53 expression, and a similar finding was reported in hepatocellular carcinoma(36). On the other hand, we found that knockdown of PTBP3 could decrease UBE4A expression in CRC cells lines with both WT and mutant TP53, which led us to believe that PTBP3-regulated UBE4A stability is a common phenomenon in CRC. Finally, we overexpressed UBE4A in PTBP3 knockdown CRC cells, and the results showed that UBE4A restored P53 expression and inhibited CRC cell proliferation. Collectively, these results suggested that PTBP3 mediates UBE4A mRNA stability to regulate WT P53 expression to promote CRC proliferation.

In summary, our results revealed that PTBP3 is overexpressed and correlated with a poor prognosis and plays an oncogenic role, contributing to CRC proliferation. Additionally, we identified that PTBP3 was closely related to the ubiquitin-mediated proteolysis signaling pathway-related gene UBE4A and may mediate its mRNA stability to regulate its expression. On the other hand, we demonstrated for the first time that knockdown of UBE4A could inhibit CRC proliferation, which may be connected to P53 stability. In conclusion, these results indicate that the PTBP3/UBE4A/P53 axis might be a potential prognostic marker and therapeutic target in CRC, potentially providing new insight into CRC progression and treatment.

CRC

Colorectal cancer; PTBP3:Polypyrimidine tract-binding protein 3; TCGA:The Cancer Genome Atlas; GEO:Gene Expression Omnibus; CCK8:Cell counting kit 8; UBE4A:Ubiquitination factor E4A; ATCC:American Type Culture Collection; qRT-PCR:Quantitative real-time PCR; WB:Western Blot; shNC:Small hairpin carrying negtive control RNAs; shPTBP3:Small hairpin carrying PTBP3-specifific RNAs; shUBE4A:Small hairpin carrying UBE4A-specifific RNAs; IHC:Immunohistochemical; RIP:RNA-binding protein immunoprecipitation; RAP:RNA Antisense Purification; GEPIA:Gene Expression Profifiling Interaction Analysis ; GSEA:Gene set enrichment analysis; KEGG:Kyoto Encyclopedia of Genes and Genomes; RISC:RNA-induced silencing complex; CHX:Cycloheximide; WT:Wilde Type; UTR:Untranslated region; H&E:hematoxylin and eosin.

Ethics approval and consent to participate

The study was approved by the ethics committee of the Third XiangYa Hospital of Central South University (No.2019-S082).

Consent for publication

Thirty pairs of CRC tissues and adjacent normal tissues used were obtained from the 30 patients who underwent surgical resection of CRC at the Third XiangYa Hospital of Central South University (Changsha, China) after informed consent was obtained.

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing Interests

The authors declare that they have no conﬂict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (No.81602568, No.81773130, No.82172833), China Postdoctoral Science Foundation (No.2018M643009), the National Natural Science Foundation of Hunan Province (No.2019JJ50906, No.2019JJ80030, No.2018JJ2599, No.2020JJ4863) and Hunan Cancer Hospital Climb Plan (No.2020NSFC-A004).

Authors' contributions

CBX, CWL and GH provided study concept and design.CBX, LL, MM, FL, RLW,YZ,LHH,JC, and NG collected and analyzed the data. CBX, LL, MM, FL, RLW,YZ,JC,NG and LHH interpreted the data. CBX, LL, MM and FL performed the experiments.CBX and LL collected the patients’ samples. CBX,CWL and GH wrote the manuscript. All authors approved the final version of manuscript.All authors read and approved the final manuscript.

Acknowledgements

We thank all individuals who participated in this work.

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Fig.S1.jpg
Stably knock down PTBP3 in HCT116 and LoVo cell lines.(A)Representative images of HCT116 and LoVo cell lines after transfected with UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(B)Relative mRNA expression level of UBE4A in HCT116 and LoVo cells transfected with lentivirals carrying PTBP3-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(measured by qRT-PCR; GAPDH was used as an internal control).(C)Relative protein level of PTBP3 in HCT116 and LoVo cells transfected with lentivirals carrying PTBP3-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(measured by Western Blot).The results are presented as the mean±s.d. and are representative of at least three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.
Fig.S2.jpg
Xenograft tumors of PTBP3 knockdown in HCT116 and LoVo cell lines.(A)Image of the injected mouse xenograft tumors.(B)Representative images of H&E staining of mouse xenograft tumors,PTBP3 and Ki67 IHC of mouse xenograft tumors(scale bar, 20µm;).
Fig.S3.jpg
PTBP3 correlated gene in ubiquitin mediated proteolysis pathway.(A)The correlation of PTBP3 with four ubiquitin mediated proteolysis gene.
Fig.S4.jpg
Stably knock down UBE4A and transient transfenct plasmid UBE4A in HCT116 and LoVo cell lines.(A)Representative images of HCT116 and LoVo cell lines after transfected with UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(B)Relative mRNA expression level of UBE4A in HCT116 and LoVo cells transfected with lentivirals carrying UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(measured by qRT-PCR; GAPDH was used as an internal control).(C)Relative protein level of UBE4A in HCT116 and LoVo cells transfected with lentivirals carrying UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC).(measured by Western Blot).(D)Relative mRNA expression level of UBE4A in HCT116 and LoVo cells transfected with UBE4A and Vector plasmids.(measured by qRT-PCR; GAPDH was used as an internal control).(E)Relative protein level of UBE4A in HCT116 and LoVo cells transfected with UBE4A and Vector plasmids.(measured by Western Blot).The results are presented as the mean±s.d. and are representative of at least three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.
Fig.S5.jpg
Knock down PTBP3 and UBE4A separately in SW480 cell line have no effect on its proliferation and P53expression.(A)Representative images of SW480 cell line after transfected with PTBP3-specific small hairpin RNAs(shPTBP3)、UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC) separately.（B）Relative mRNA expression level of PTBP3 and UBE4A in SW480 cell line after transfected with PTBP3-specific small hairpin RNAs(shPTBP3)、UBE4A-specifific small hairpin RNAs (shUBE4A) and their corresponding lentivirals carrying negative control shRNA (shNC) separately.(C)The colony formation ability of shPTBP3 SW480 and shUBE4A SW480 cells compared with that of the control (NC) cells by a colony formation assay. The bar graph indicates the number of colonies.(D)The proliferation ability of shPTBP3 SW480 and shUBE4A SW480 cells compared with that of the control (NC) cells by a CCK8 assay.(E)Relative protein level of UBE4A,PTBP3,P53 in shPTBP3 SW480 and shUBE4A SW480 cells compared with that of the control (NC) cells.The results are presented as the mean±s.d. and are representative of at least three independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001, ns p > 0.05.
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PTBP3 Modulates of P53 Expression and Promotes Colorectal Cancer Cell Proliferation by Maintaining UBE4A mRNA Stability

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