The human RNA surveillance factor UPF1 regulates tumorigenesis by targeting Smad7 in hepatocellular carcinoma
- Lei Chang†1,
- Cuicui Li†2,
- Tao Guo1,
- Haitao Wang1,
- Weijie Ma1,
- Yufeng Yuan1,
- Quanyan Liu1,
- Qifa Ye2Email author and
- Zhisu Liu1Email author
© Chang et al. 2016
Received: 7 November 2015
Accepted: 7 January 2016
Published: 13 January 2016
In spite of progress in diagnostics and treatment of Hepatocellular Carcinoma (HCC), its prognosis remains poor, and improved treatment strategies for HCC require detailed understanding of the underlying mechanism. In this investigation we studied the role of Up-frameshift 1 (UPF1) in the tumorigenesis of HCC.
We determined the expression level of UPF1 in HCC tissues with quantitative real-time PCR and western blotting and then studied its clinical significance. Sodium bisulfite sequencing was used to investigate the regulation of UPF1. We explored the biological significance of UPF1 with gain-and-loss-of-function analyses both in vitro and in vivo. The relationship between UPF1 and SMAD7 was also investigated by western blotting and immunofluorescence.
A great downregulation of UPF1 due to promoter hypermethylation was observed in tumor tissues compared to their adjacent normal tissues. Meanwhile, patients with low UPF1 expression have significantly poorer prognosis than those with high expression. Functionally, UPF1 regulated HCC tumorigenesis both in vitro and in vivo. Moreover, the decreased UPF1 level in HCC reduces NMD efficiency and leads to up-regulation of Smad7, then affects the TGF-β pathway.
Our findings revealed that UPF1 is a potential tumor suppressive gene and may be a potential therapeutic target for HCC.
KeywordsHepatocellular carcinoma UPF1 Smad7 Hypermethylation
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide . Although recent advances in cancer treatment with respect to surgery, chemotherapy and biologics, majority of HCC remains incurable once it has become metastatic and has a poor prognosis . The details of the molecular mechanisms underlying HCC carcinogenesis remain to be elucidated. Hepatocarcinogenesis has often been described as a multistep process involving a number of genetic alterations eventually leading to the malignant transformation of the hepatocytes .
Tumor suppressor genes protect normal cells from progressing to cancer in general . However, these genes in cancer cells often suffer genetic mutations and aberrant epigenetic modifications [5, 6]. The mutations generate mRNA harboring premature termination codons (PTCs) that are targets of nonsense-mediated mRNA decay (NMD) pathway . Hence NMD regulation and aberrant epigenetic modifications contribute to oncogenesis [8, 9] NMD is an mRNA surveillance pathway that eliminates aberrant mRNA transcript containing PTCs, and prevents the synthesis of potentially toxic truncated proteins . Recent studies have shown that NMD is not only a quality control pathway, but also a regulatory pathway that controls normal gene expression. Gene expression profiling studies have shown that either loss or depletion of NMD factors in species scaling the phylogenetic scale leads to the dysregulation of 3 %–15 % of normal transcripts . The core of the human NMD machinery is composed of seven polypeptides called Up-frameshift 1 (UPF1), UPF2, UPF3, SMG1, SMG5, SMG6, and SMG7 . Human UPF1 also mediates two RNA decay processes, which are independent of canonical NMD as they do not require UPF2: first, it targets those mRNA molecules that are bound to the RNA binding protein Staufen1 for degradation ; second, together with Stem-Loop Binding Protein (SLBP), it promotes degradation of replication-dependent histone mRNAs at the end of S phase and when DNA replication is inhibited .
Recent studies have demonstrated that UPF1 is not only a key player in RNA degradation pathways, but that it is also essential for accomplishing DNA replication during S phase of the cell cycle . UPF1 was required for S Phase progression and genome stability. UPF1 also plays a key role in the process of cell proliferation and differentiation by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGFβ inhibitor, Smad7, and stimulating TGF signaling . UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Liu and his colleagues found that UPF1 was down-regulated in pancreatic adenosquamous carcinoma (ASC) and the UPF1 gene is commonly mutated in pancreatic adenosquamous carcinoma . The discovery of mutations in the UPF1 gene in ASC tumors represents the first known example of genetic alterations in a NMD gene in human tumors. While no studies have assessed UPF1 exact activity in human HCC, which prompted our interest in investigating its biological roles of UPF1 in hepatocarcinogenesis.
In the present study, we demonstrated that UPF1 was significantly down-regulated in HCC tissues as compared with adjacent non-tumor tissues, and this down-regulation of UPF1 was associated with decreased survival of HCC patients. Furthermore, we found that UPF1 was regulated by CpG island methylation of the putative promoter region. Functional analyses indicated UPF1 inhibited both cell growth and tumorigenicity of HCC cells, possible by targeting Smad7 and then effects on TGF-β pathways.
Clinical specimens and cell lines
HCC specimens and the corresponding adjacent tissues were collected from Zhongnan Hospital of Wuhan University after obtaining informed consent. The diagnosis of HCC was histopathologically confirmed. The protocols used in the study were approved by the Hospital’s Protection of Human Subjects Committee. Overall survival (OS) was defined as the interval between resection and death or the last follow-up visit. Recurrence free survival (RFS) was defined as the interval between treatment and the first diagnosis of metastasis or recurrence. L02, Huh7, Hep3B, HepG2, SMMC-7721 and HCCLM9 included in this study were purchased from the Cell Bank of Type Culture Collection (CBTCC, Chinese Academy of Sciences, Shanghai, China) and cultured in minimum essential medium (Gibco, Carlsbad, CA, USA) with 10 % fetal bovine serum (Gibco).
Quantitative real-time PCR
Primer sequence and target sequence used in this study
Sequence or Target Sequence
Plasmid constructions and transfection assay
UPF1 (Gene-bank: NM_001297549.1) was cloned into pcDNA3.1 vector (Life Technology). UPF1-siRNAs were designed and synthesized by Viewsolid Biotech (Beijing, China). The sequence of siRNAs are presented in Table 1. The expression of UPF1 was confirmed by RT-qPCR and western blotting. Transfection was carried out using FuGene HD transfection reagent (Roche, Indianapolis, IN) according to the manufacturer’s protocol.
Immunohistochemistry (IHC) and Immunofluorescence (IF)
Antibody information used in this study
Cell Signaling Technology
Santa Cruz Biotechnology
Flow cytometry analysis
Cells transiently transfected with siRNA or vector were harvested 48 h after transfection. Following the double staining with Annexin V FITC and Propidium Iodide (PI), the cells were analyzed by flow cytometry (FACScan®; BD Biosciences, San Jose, CA) equipped with CellQuest software (BD Biosciences). For the cell cycle analysis, the cells were stained with PI using the CycleTESTTM PLUS DNA reagent kit (BD Biosciences) according to the manufacturer’s instruction.
Tissular and cellular proteins from each sample were electrophoresed by SDS-polyacrylamide gel electrophoresis (4 % stacking and 10 % separating gels), then transferred onto polyvinylidene fluoride PVDF membranes (Millipore, USA), then incubated with primary antibodies overnight at 4 °C. After the incubation with secondary antibodies, the PVDF membranes were subsequently subjected to immunoblotting analysis using the ECL immunoblotting kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. Antibodies information used in this study was listed in Table 2.
Transwell and wound healing assay
The invasion of cells was assessed using Matrigel-coated chambers with 8 μm pores (BD Biosciences, Franklin Lakes, NY, USA). Briefly, hepatoma cells (1 × 105) were seeded in serum-free medium and were allowed to translocate toward complete media supplemented with 10 % fetal bovine serum after depleted of UPF1. The cells that had invaded through the membrane to the lower surface were fixed, stained and counted after 24 h. HCC cells (1 × 106 cells/well) were treated with the indicated reagents, and wounds were made using a 100 μl plastic pipette tip. The size of the wound was measured after 24 h of wound formation and photographed.
CCK-8 cell proliferation assay
Cell proliferation assay was performed by using Cell Counting Kit-8 (Dojindo, Japan). Briefly, cells were plated in 96-well plates in triplicate at aproximately 3–5 × 104 cells per well and cultured in the growth medium. Cells were then treated with the indicated reagent and the numbers of cells per well were measured by the absorbance (450 nm) at the indicated time points.
Sodium bisulfite sequencing
MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) was used to analyze the CpG islands. Genomic DNA was extracted using DNeasy Tissue Kit (QIAGEN, Valencia, CA). Genomic DNA was treated with sodium bisulfite using the CpGenome DNA Modification Kit (Serologicals Corp, Norcross, GA) according to the manufacturer’s protocol. PCR products were subcloned, and ten constructs representing each region from each sample were randomly selected for sequence analysis. DNA methylation data were analyzed by using BiQ Analyzer (http://biq-analyzer.bioinf.mpi-inf.mpg.de/.).
Tumor formation assay in a nude mouse model
HCCLM9 cells stably transfected with pcDNA3.1-UPF1 or empty vector were suspended at 1 × 107 cells/ml. A total of 100 μl of suspended cells were subcutaneously injected into the male athymic BALB/c nude mice (5 weeks old). Tumor volumes and weights were measured beginning from day 7 after the tumor cell injection.
Tail vein injections into athymic mice
HCCLM9 cells stably transfected with pcDNA3.1-UPF1 or the empty vector were suspended at 2 × 107 cells/ml. Suspended cells (100 μl) were injected into the tail veins of 10 mice (5 weeks old), which were sacrificed 7 weeks after injection. The lungs were removed and visible tumors on the lung surface were counted and used for further analysis.
Data are presented as the means ± standard deviation from at least three independent experiments. Student’s t-test, χ2 test or Fisher’s exact tests were used for comparisons between groups with SPSS 22.0 software (IBM, Chicago, IL, USA). The Kaplan-Meier test was used to estimate OS and RFS. A value of p < 0.05 was considered significant.
UPF1 was strongly downregulated in HCC and associated with HCC progression
Relationship between UPF1 expression and clinicopathologic parameters of HCC patients
Number of cases
Low(n = 25)
High(n = 25)
≥ 5 cm
< 5 cm
B + C
UPF1 suppressed HCC tumorigenesis in vitro and in vivo
Fluorescence-activated cell sorting (FACS) analysis showed significant increases in apoptotic cells when UPF1 was overexpressed (Fig. 3b). Cell cycle analysis showed that in UPF1-siRNA transfected cells, the percentage of cells in the S phase was more than that of scramble siRNA transfected cells. (Fig. 3c). Similar results were observed in Huh7 cells. Together, these results suggest that UPF1 could suppress HCC cell proliferation by inducing cell cycle arrest.
To further examine the effects of UPF1 on HCC tumorigenesis, the biological consequences of UPF1 in regulating cancer cell invasion and migration were examined using cell biology assays. As shown in Fig. 3d, functionally, knockdown UPF1 increased the cell invasion and migration of HCC cells. While, overexpression of cellular UPF1 significantly decreased the cell invasion, migration and proliferation of HCC cells (Fig. 3e and f).
CpG hypermethylation downregulated UPF1 expression in HCC
Smad7 was strongly up-regulated in HCC
UPF1 suppressed HCC tumorigenesis by targeting Smad7 and affected TGF-β pathway
In this study, we reported the correlation between UPF1 silencing and HCC. Immunohistochemical analysis, western blotting and RT-qPCR experiments showed that the down-regulation of UPF1 was associated with malignant progression of HCC. Epigenetic silencing of UPF1 gene was observed in low UPF1-expressing HCC cell lines. Therefore, we deducted that UPF1 silencing may be required for tumorigenesis and has a potential to be developed as a biomarker for malignant phenotype of HCC, which possesses considerable value in prognosis prediction, progression monitoring and treatment evaluating. For future application, it is critical to design reasonable clinical trial for evaluation of this biomarker. Not only overall survival and recurrence-free survival should be evaluated in randomized, controlled trials, some key factors with regard to HCC, such as HBV infection information, will be considered.
Generally speaking, hypermethylation of tumor suppressor genes and demethylation of oncogenes contribute to tumorigenesis [18, 19]. Cancer-linked hypermethylation and hypomethylation of gene promoter is often associated with cell proliferation and differentiation . In this study, we found UPF1 is a potential tumor suppressor, which negatively regulates proliferation of cancer cells, we also observed that UPF1 is down-regulated in HCC tissues. In HCC tissues and cell lines, the methylation states of the CpGs in UPF1 promoter region were found to be hypermethylated. Moreover, UPF1 gene was also re-expressed following treatment with 5-Ad, a potent demethylating agent, suggesting that status of DNA demethylation was important for the active expression of UPF1.
NMD is an evolutionally conserved mRNA quality-control mechanism that selectively degrades aberrant mRNA containing premature termination codons in order to prevent the accumulation of truncated proteins , which are often non-functional or potentially deleterious in the cells. Three conserved proteins UPF1, UPF2, and UPF3 make up the key NMD machinery with UPF1 as the important member in this protein set [22–24] UPF1 acts in concert with the peptide release factors eRF1/eRF3 to recognize aberrant translation termination events and, together with UPF2 and UPF3, triggers degradation of mRNA in a subsequent step [25, 26]. In addition to its role in NMD, UPF1 also regulates mRNAs in a NMD-independent manner. For example, UPF1 is recruited by the RNA-binding protein Staufen to the downstream of a stop cordon to degrade some mRNAs in a Staufen-mediated decay . Human UPF1 has been shown to regulate the degradation of histone mRNAs in mammalian cells . Moreover, UPF1 is involved in nonsense-mediated altered splicing . Although NMD pathway has been extensively studied, the regulatory mechanism of NMD in cancer is still not well understood. The first report about the relationship between UPF1 and human tumor is pancreatic adenosquamous carcinoma. UPF1 was found down-regulated in pancreatic adenosquamous carcinoma , here, in our study, we found UPF1 was also down-regulated in HCC. In addition, as a key molecule in the TGF-β pathway, Smad7 was an inhibitory SMAD protein. We found UPF1 could suppress the Smad7 level in HCC and then affected the TGF-β pathway.
In conclusion, the current study revealed that UPF1 was down-regulated in HCC cells and tissues. Furthermore, UPF1 suppressed tumorigenesis of HCC. Finally, the reduced UPF1 expression, due to promoter hypermethylation, attenuated NMD, leaded to the dysregulation of Smad7, which indicated aberrant mRNA surveillance mechanism in HCC. All the results indicated that UPF1 played an important role during HCC carcinogenesis and may serve as a putative target for HCC diagnosis and therapy.
This work was supported by the National Natural Science Foundation of China (NSFC; Grant Nos.: 81572450/H1617, 81472268/H1617, 81372552/H1617 and 81272692/H1617).
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