Pumilio homolog 2 (PUM2) is an RNA-binding protein that functions as an oncogene in various types of cancer. However, its role in hepatocellular carcinoma (HCC) has remained to be fully elucidated. In the present study, the role of PUM2 was investigated in HCC and its regulation was assessed by examining its binding to the 3′-untranslated region (UTR) of B-cell translocation gene 3 (BTG3). The expression levels of PUM2 were determined in datasets from the UALCAN and Cancer Cell Line Encyclopedia databases. Furthermore, Gene Expression Profiling Interactive Analysis was used to analyze overall survival in patients with HCC. Reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses were then performed to detect the expression levels of PUM2 and BTG3 in HCC cells. Cell proliferation was assessed using Cell Counting Kit-8 and colony-formation assays. The induction of cell apoptosis was evaluated using TUNEL and western blotting assays. StarBase and RNA-Protein Interaction Prediction were used to determine the possible direct interaction between PUM2 and BTG3. The interaction between PUM2 and BTG3 was then verified by luciferase reporter and RNA-binding protein immunoprecipitation assays. The results indicated that PUM2 expression was upregulated in HCC tissues and cells and that it was associated with the prognosis of patients with HCC. PUM2 silencing inhibited the proliferation and promoted the apoptosis of Huh-7 cells. In addition, PUM2 was confirmed to directly bind to the 3′UTR of BTG3. Downregulation of BTG3 reversed the effects of PUM2 silencing on cell proliferation and apoptosis in Huh-7 cells. Collectively, the results suggested that PUM2 regulated HCC cell proliferation and apoptosis via interacting with BTG3, which may provide a novel therapeutic strategy for the treatment of human HCC.
Hepatocellular carcinoma (HCC) is a common type of malignant tumor of the digestive system, accounting for ~90% of liver cancer cases (
Pumilio homolog 2 (PUM2) is an RNA-binding protein that serves as a translation repressor (
The mRNA levels of PUM2 were analyzed in the UALCAN database (
The HCC cell lines HCC36, HCC-T, HCC-M and HHS-89 were purchased from the American Type Culture Collection. The HCC cell line Huh-7 and human the hepatocyte cell line HHL5 (used as the control cell line) were obtained from the Health Science Research Resources Bank. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Hyclone; Cytiva), 100 U/ml penicillin and 100 µg/ml streptomycin (both Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere with 5% CO2 at 37°C.
The specific short hairpin RNA (shRNA) targeting PUM2 (shRNA-PUM2-1: 5′-GCAATATAGTGTTGTATAA-3′; shRNA-PUM2-2: 5′-CATAGTTGTTGACTGTTAA-3′), the specific shRNA targeting BTG3 (shRNA-BTG3-1: 5′-GATTATGTATGGAGAGAAA-3′; shRNA-BTG3-2: 5′-GATTAATCCTCACATGTTA-3′) and the corresponding control shRNA (shRNA-NC: 5′-CCGGCAACAAGATGAAGAGCACCAACTC-3′) were synthesized with pRNA-U6.1/Hygro vector as the plasmid backbone by Shanghai Integrated Biotech Solutions. These recombinant nucleotides were transfected into Huh-7 cells using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following 48 h of incubation, the cells were collected for subsequent experiments.
Following transfection, Huh-7 cells were placed in 96-well plates (2×104 cells/well) and cultured in DMEM with 10% FBS at 37°C. Following incubation for 24, 48 and 72 h, 10 µl CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and the cells were incubated for 2 h. Finally, the absorbance of each well was detected at 450 nm with a microplate reader (RT-3001; Thermo Fisher Scientific, Inc.).
The cells were seeded in 6-well plates at 500 cells/well and incubated in DMEM with 10% FBS at 37°C. Following incubation for two weeks, the plates were fixed with 4% paraformaldehyde for 15 min at a room temperature and stained with 0.5% crystal violet (Wako Pure Chemical Industries, Ltd.) for 30 min at room temperature. The colonies were imaged and counted by light microscopy (Olympus Corporation). The number of colonies, defined as >50 cells/colony, was counted.
Total RNAs were extracted from Huh-7 cells by TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol (
A TUNEL assay was performed to evaluate Huh-7 cell apoptosis using an apoptosis detection kit (cat. no. 11684795910; Roche Diagnostics) in accordance with the manufacturer's guidelines. The transfected cells were fixed with 4% paraformaldehyde for 10 min at 4°C and incubated with proteinase K (Beijing Solarbio Science & Technology Co., Ltd.) for 15 min at 37°C. Subsequently, the cells were placed in 3% H2O2 for 15 min at room temperature and stained with the reagents from the TUNEL kit, followed by counterstaining with DAPI for 10 min at room temperature. The labeled cells were observed using fluorescence microscopy (Olympus Corporation; magnification, ×200). The number of TUNEL-positive (green) and DAPI-positive cells (blue nuclear stain) was visually counted and at least 10 fields per section were examined. The percentage of apoptotic cells was calculated as (number of TUNEL-positive cells/total number of cells) ×100%.
To obtain a 3′-UTR-luciferase reporter plasmid, the 3′UTR of BTG3 was amplified using PCR from genomic DNA of the human HCC cell line Huh-7 (Invitrogen; Thermo Fisher Scientific, Inc.) and cloned into the
The interaction between PUM2 and BTG3 was identified by an RIP assay using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (cat. no. 17-701; MilliporeSigma) according to the manufacturer's instructions. The cells were lysed in complete RIP lysis buffer and the protein extract was then prepared. Anti-PUM2 antibody (cat. no. ab92390; 1:10 dilution; Abcam) and NC normal rabbit IgG (cat. no. NI01; 1/200 dilution; MilliporeSigma) were incubated with the protein extract from the lysed cells at 37°C overnight. The co-precipitated RNAs were detected by RT-qPCR as specified above.
Total protein was extracted from cells using RIPA buffer (Bio-Rad Laboratories, Inc.). Total protein was quantified using a BCA assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. A total of 30 µg protein per lane was loaded on 10% SDS-polyacrylamide gels and after electrophoresis, proteins were transferred to polyvinylidene membranes (MilliporeSigma). The membranes were blocked in 5% non-fat milk (Beyotime Institute of Biotechnology) at room temperature for 2 h and incubated with primary antibodies against PUM2 (1:1,000 dilution; cat. no. ab92390), Bcl-2 (1:1,000 dilution; cat. no. ab32124), Bax (1:1,000 dilution; cat. no. ab32503), cleaved caspase 3 (1:500 dilution; cat. no. ab32042), caspase 3 (1:1,000 dilution; cat. no. ab32351), cleaved poly(ADP-ribose) polymerase (PARP; 1:1,000 dilution; cat. no. ab32561), PARP (1:1,000 dilution; cat. no. ab32138), BTG3 (1:1,000 dilution; cat. no. ab112938) and GAPDH (1:1,000 dilution; cat. no. ab8245; all from Abcam) overnight at 4°C. Finally, the membranes were incubated with horseradish peroxidase-labeled anti-rabbit IgG (cat. no. 7074; 1:1,000 dilution; Cell Signaling Technology, Inc.) or anti-mouse IgG (cat. no. 14709; 1:1,000 dilution; Cell Signaling Technology, Inc.) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham; Cytiva) according to the manufacturer's instructions. The density of each band was quantified by ImageJ software (v.1.8.0; National Institutes of Health).
Statistical analysis was performed using SPSS 22.0 software (IBM Corporation). Values are expressed as the mean ± standard deviation. Significant differences between two groups were analyzed by Student's unpaired t-test, while differences among multiple groups were analyzed using one-way analysis of variance followed by Bonferroni's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
To explore the role of PUM2 in HCC development, the expression levels of PUM2 were initially detected in a public dataset of patients with HCC and in HCC cell lines. According to the data obtained from the UALCAN database, PUM2 was highly expressed in HCC tissues compared with those in the control subjects (
To investigate the effect of PUM2 on HCC cell proliferation and apoptosis, specific shRNA sequences targeting PUM2 were transfected into Huh-7 cells. The transfection efficiency was evaluated by RT-qPCR and western blot analyses (
The mechanisms underlying the regulatory role of PUM2 were further explored in HCC cells. By using the starBase database, PUM2 was predicted to bind to several RNAs. Among these RNAs, PUM2 protein was predicted to bind to the 3′-UTR of BTG3 and the interaction probability was 0.99 according to the RPISeq website. Furthermore, the binding sequence of the PUM2 and BTG3 3′UTR was predicted by Ensembl (
To assess the role of BTG3 in PUM2-mediated HCC progression, BTG3 expression was silenced in Huh-7 cells. RT-qPCR analysis indicated a significant decrease in BTG3 expression following transfection with shRNA-BTG3-1 or −2. shRNA-BTG3-1 exhibited improved knockdown efficiency; therefore, shRNA-BTG3-1 (denoted as shRNA-BTG3 from here onwards) was selected for the subsequent assays (
HCC is a common malignant tumor type of the digestive system with high morbidity and mortality (
The RNA-binding proteins of the Pumilio and FBF (PUF family) have crucial roles in the occurrence and development of multiple diseases (
BTG3 belongs to the B-cell translocation gene/transducer of the Erb-B2 receptor tyrosine kinase 2 protein family (
In conclusion, the results of the present study demonstrated that PUM2 expression was upregulated in HCC and that it was associated with poor prognosis. PUM2 regulated cell proliferation and apoptosis of HCC cells by directly binding to the 3′UTR of BTG3. Rescue experiments indicated that BTG3 silencing reversed the effects of PUM2 expression knockdown on cell proliferation and apoptosis, which implies the potential for this protein to be used as a novel therapeutic target for the treatment of HCC.
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All data generated or analyzed during this study are available from the corresponding author on reasonable request.
ZL and CL designed the study, performed the experiments and drafted and revised the manuscript. ZL analyzed the data and performed the literature search. ZL and CL confirmed the authenticity of all the raw data. Both authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
PUM2 is upregulated in HCC tissues and cells. (A) Analysis of PUM2 expression in HCC determined using the UALCAN database. (B) Association between PUM2 expression and overall survival determined using the GEPIA database. (C) mRNA expression and (D) protein level of PUM2 in several HCC cell lines and the human hepatocyte cell line HHL5 (as the control cell line) were detected by reverse transcription-quantitative PCR and western blot assays, respectively. The PUM2 expression in HHL5 was normalized to 1 and the relative PUM2 expression in all other groups was normalized to that in HHL5 cells. GAPDH was used as an internal reference. Values are expressed as the mean ± standard deviation. **P<0.01, ***P<0.001 vs. HHL5. LIHC, liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; HR, hazard ratio; PUM2, Pumilio homolog 2.
PUM2 silencing inhibits cell proliferation of Huh-7 cells. (A) mRNA expression and (B) protein levels of PUM2 in Huh-7 cells after PUM2 was silenced were detected by reverse transcription-quantitative PCR and western blot assays, respectively. (C) A Cell Counting Kit-8 assay and (D) colony-formation assay were used to assess cell proliferation. Values are expressed as the mean ± standard deviation. ***P<0.001 vs. shRNA-NC. PUM2, Pumilio homolog 2; NC, negative control; shRNA, short hairpin RNA; OD, optical density.
PUM2 silencing represses apoptosis in Huh-7 cells. (A) A TUNEL assay was carried out to quantify apoptosis of Huh-7 cells transfected with shRNA-PUM2 (magnification, ×200). (B) Western blot analysis was performed to detect the protein levels of Bcl-2, Bax, caspase3, cleaved caspase3, PARP and cleaved PARP. Values are expressed as the mean ± standard deviation. ***P<0.001 vs. shRNA-NC. PUM2, Pumilio homolog 2; NC, negative control; shRNA, short hairpin RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; PARP, poly(ADP ribose) polymerase.
PUM2 binds to 3′UTR of BTG3. (A) The binding site of PUM2 and 3′UTR of BTG3. (B) mRNA expression and (C) protein levels of BTG3 in Huh-7 cells after PUM2 was silenced were detected by reverse transcription-quantitative PCR and western blot assay, respectively. (D) Luciferase activity of the 3′UTR of BTG3 was measured by a luciferase reporter assay. (E) RIP assay was conducted to verify the binding of PUM2 to the 3′UTR of BTG3. Values are expressed as the mean ± standard deviation. **P<0.01, ***P<0.001 vs. shRNA-NC or lgG. CDS, coding sequence; PUM2, Pumilio homolog 2; NC, negative control; shRNA, short hairpin RNA; RLU, relative luciferase activity; RIP, RNA-binding protein immunoprecipitation; BTG3, B-cell translocation gene 3.
PUM2 silencing inhibits Huh-7 cell proliferation and promotes apoptosis by targeting BTG3. (A) mRNA expression of BTG3 in Huh-7 cells after BTG3 was silenced were detected by reverse transcription-quantitative PCR. (B) A Cell Counting Kit-8 assay and (C) colony-formation assay were used to examine cell proliferation. (D and E) A TUNEL assay was employed to determine apoptosis in Huh-7 cells transfected with shRNA-BTG3. (D) Representative images of TUNEL staining (magnification, ×200) and (E) quantified results. (F and G) Western blot analysis was utilized to evaluate the protein level of Bcl-2, Bax, caspase3, cleaved caspase3, PARP and cleaved PARP. (F) Representative western blots and (G) quantified protein levels. Values are expressed as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001 vs. shRNA-NC in A or vs. shRNA-PUM2 in B-G. PUM2, Pumilio homolog 2; NC, negative control; shRNA, short hairpin RNA; PARP, poly(ADP ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; OD, optical density; BTG3, B-cell translocation gene 3.