Background
Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors and the third most common cause of cancer-related deaths worldwide, often accompanied with invasive fast growing and metastasis [
1,
2]. Although therapeutic strategies including surgery resection, liver transplantation and radiotherapy have greatly improved [
3], there are limited effective therapies for patients with advanced HCC. The five-year post-surgical OS rate remains approximately 16% even after curative resection [
4]. The main causes of poor clinical outcomes in patients with HCC are high recurrence and metastasis rates [
5]. Therefore, the discovery of novel therapeutic target and better understanding of the molecular mechanisms underlying HCC progression are crucial and urgent.
Excision repair cross-complementation group 6-like (ERCC6L), also named as Polo-like kinase 1-interacting checkpoint “helicase” (PICH), which was identified as an embryonic development related proteins, has been shown to play critical roles in regulating the development of embryonic, brain and other tissues [
6‐
8]. Recently several reports revealed that abnormal ERCC6L expression has been detected in several malignant solid tumors consisting of breast cancer [
9], kidney cancer [
10], and neuroblastoma [
11]. In addition, high ERCC6L expression level is related to poor prognosis in breast cancer patients, and may regulate cell proliferation, invasion and metastasis by regulating different single pathways. However, the roles of ERCC6L in HCC progression and its underlying molecular mechanisms remain unclear.
In this study, we explored the effects of ERCC6L on the progression of HCC and confirmed ERCC6L to be a poor prognostic marker. In addition, we assessed the function of ERCC6L in tumor cell proliferation, apoptosis, migration, and molecular mechanisms in vitro and in vivo. These data proposed a potential target for prognosis and therapy in HCC patients.
Methods
Cell culture and transfection
Three human HCC cell line (SMMC7721, HuH-7, Hep3B) and a normal human liver cell line HL-7702 were obtained from the Institutes of Cell Biology at the Chinese Academy of Science (Shanghai, China). The cells were recently authenticated by STR profiling and are free from mycoplasma contamination. Cells were maintained in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY, USA) in a humidified atmosphere with 5% CO2 at 37 °C. Transfection of shRNA (RiboBio Co., Ltd., Guangzhou, China) was performed with the transfection reagent in 2 nM shRNA according to the manuscripts of Lipofectamin 3000 (Invitrogen, Carlsbad, USA). Target sequences as following:
ERCC6L shRNA: 5′-GGACCATATTGATCAAGTA-3′;
Negative control shRNA (NC): 5′-TTCTCCGAACGTGTCACGT-3′.
The ERCC6L cDNA was cloned into a GV219 vector (GenePharma Co. Ltd., Shanghai, China) to overexpress ERCC6L. Cells were transfected for 48 and 72 h, then ERCC6L mRNA and protein expression were verified by quantitative real-time PCR or western blotting analysis respectively [
12]..
Total RNA extraction and quantitative real-time PCR
Total RNA was isolated from cells using TRIzol reagent (Invitrogen) [
13]. 1 μg RNA was reversely transcribed into cDNA using Superscript First-strand Synthesis system (Invitrogen, Carlsbad, USA) according to the manufacturer’s instrucions. Quantitative RT-PCR was performed using SYBR Premix Ex Tag II (Takara Bio Inc.) on a Roche 4800 instrument (Applied Biosystems, USA). The primers were following:
ERCC6L, forward: 5′-AAGGATGAACGGACCAGAAAC-3′,
reverse: 5′-CTGTGAGGAGGAGGCGATTAC-3′;
β-actin, forward: 5′-AGAGCTACGAGCTGCCTGAC-3′,
reverse: 5′- AGCACTGTGTTGGCGTACA-3′.
The experiment was repeated three times, and all data were analyzed by the 2-ΔΔCT method.
Western blotting
Cell lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in 5% BSA for 1 h, then incubated with primary antibodies overnight at 4 °C. Antibodies were used as following: ERCC6L (Proteintech, 15,688–1-AP, 1:1000), PI3K (Bioss, bs-0128R, 1:1000), p-PI3K (Ser1070; Bioss, bs-6417R, 1:1000),AKT1 (Bioss, bs-0115R, 1:1000), p-AKT1 (Thr34; Bioss, bs-5194R, 1:1000), JAK2 (Bioss, bs-23003R, 1:1000), p-JAK2 (Tyr1007 + Tyr1008; Bioss, 2485R, 1:1000), NF-κB (Bioss, bs-0465R, 1:1000), p-NK-κB (Thr505; Bioss, bs-5663R, 1:1000), and β-actin (Bioss, bs-0061R, 1:5000). The next day membranes were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C. After washed for 3 times in TBST for 5 min, membranes were visualized by chemiluminescence kit and scanned with QuantityOne software (Bio-Rad, Hercules, CA, USA). The bands were analyzed with ImageJ (NIH, Bethesda, MA, USA).
Migration assays
Migration assays were performed using a Transwell assay (8.0 μm, 24well, BD Biosciences). Briefly, 1 × 10
5 cells were added into the upper chambers with serum-free DMEM, and the lower chambers were filled with 600 μL DMEM contained 10% FBS. After incubated for 24 h, the cells that migrated were fixed for 30 min and stained with 0.05% crystal violet. The numbers of migrating cells were counted in five randomly selected visual fields under a microscope at 200× magnification [
14]. The experiments were performed three times.
MTT assays
Cells were inoculated into a 96-well plate at 2 × 103 cells/well and cultured for 24, 48, and 72 h respectively. Then, 20 μL of 5 mg/mL MTT solution (Sigma, USA) was added and incubated for 4 h. 150 μL of dimethyl sulfoxide (DMSO) was added and the optical density (OD) value was measured at 490 nm of the microplate reader. he experiments were performed three times separately.
Cells were seeded into 6-well plates at a density of 500 cells/well for 14 days. Then cells were fixed with 4% paraformaldehyde for 30 min, and stained with 0.05% crystal violet for 20 min. Representative photographs were captured, and colonies with more than 40 cells were counted.
Cell cycle analysis
For cell cycle analysis, cells transfected with shERCC6L or NC for 24 h. Cells were fixed in 70% ethanol, and stained with 50 μg/mL PI for 30 min in the dark at 37 °C. The percentage of cells in the G1, S, and G2 phases were determined with a FACS flow cytometer (Becton Dickinson, San Jose, USA).
Apoptosis assay
Apoptosis was evaluated by flow cytometry using an Annexin V/FITC-PI Apoptosis Detection Kit (Millipore, USA) according to the manufacturer’s instructions. After washing with cold PBS, tumor cells were stained with 10 μL of Annexin V-FITC/PI in the dark for 15 min at room temperature and 400 μL of binding buffer was added. The cells were analyzed by flow cytometry (BD Biosciences, MA, USA). The Q2 and Q3 quadrants were determined as the early and late apoptosis induced cell, respectively. The percentage of total apoptosis (early and late stages) was used for quantitative analysis.
Caspase-3/7 activity
Cell were transfected for with shERCC6L or NC 24 h, then collected and incubated with the working solution provided in the Caspase-Glo® 3/7 Assay Kit (Promega) according to manufacturer’s protocol [
15].
Immunohistochemistry (IHC)
The tissue microarray (HLivH180Su18, Outdo Biotech) was used to validate ERCC6L expression in 90 HCC tissue samples and tumor-adjacent tissues. Briefly, the samples were dewaxed, and antigens were retrieved using a microwave. The paraffin section were dewaxed with xylene (10 min × 3 times), followed by rehydration with gradient ethanol (anhydrous ethanol, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, 5 min each), and then antigens were retrieved using microwave for 15 min. After elimination of endogenous peroxidase activity, sections were blocked with 5% bovine serum albumin and then incubated with primary antibodies against ERCC6L (Bioss, bs-6380R, 1:100) overnight at 4 °C. Then sections were incubated with secondary antibodies (horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (A0208, Beyotime, China) for 1 h. Sections were stained with Diaminobenzidine (DAB) solution and observed under a microscope (Leica DM4000B, Germany) as previously described [
16].
Two pathologists who were separately blinded to the clinicopathological data evaluated the stained results independently. Staining intensity was scored as follows [
17,
18]: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive-stained cells was divided according to density: 0–10%; 11–25%; 26–50%; 51–75%; ≥ 76%, and score was 0, 1, 2, 3, 4 respectively. The total scores was calculated by multiplying the intensity scores by the percentage scores; total scores of 0–3 or 4–12 were defined as low or high expression.
Xenografts tumor growth
BALB/c nude mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (male, 6 weeks old, 18-20 g). Animal experiments were approved by the Institute of Basic Medicine, Shandong First Medical University & Shandong Academy of Medical Sciences and were performed according to the established guidelines. Mice were fed under the condition of specific pathogen-free. shERCC6L and NC were used to construct lentiviral particles (GenePharma), and the lentivirus were infection to establish stable transfected cells. Mice were randomly grouped two groups (with 7 mice each group): negative control group (NC group, injected with SMMC7721 transfected with NC) and shERCC6L group (injected with SMMC7721 transfected with shERCC6L). A total of 1 × 107 cells were suspended in 100 μL serum-free DMEM and injected subcutaneously into the right back of mice. After injection, the long diameter (L), width diameter (W) of tumors were measured every 5 days, and the volumes of tumor were calculated as following Volume (V) = (W2 × L)/2. Mice were anaesthetized by intraperioneal injection of pentobarbital sodium overdose (100 mg/kg) on the 30th day. Euthanasia was considered to be successful if there was cardiac arrest and no spontaneous breath for 3 min, and then tumors were taken out and weighed.
Statistical analysis
All statistical analyses were performed using SPSS 22.0 Software (NY, USA). All measured data were presented as the mean ± SD, and Student’s t-tests were used for comparing the significance of differences between two groups. OS were calculated using the Kaplan–Meier method, and survival curve comparisons were performed using the log-rank test. Univariate and multivariate Cox regression was used for analysis factors predicting OS. p < 0.05 was considered the statistical significance.
Discussion
HCC is an aggressive malignancy, which metastasis, invasion, and recurrence are the main causes of death in HCC patients [
23]. Rapid HCC progression and difficulty detecting early disease are major obstacles to curative treatment [
24]. It is urgently required to screen novel diagnostic or prognostic biomarkers for targeted therapy. In this study, we found that higher ERCC6L expression levels are detected in tumor tissues than in adjacent normal tissues, suggesting that ERCC6L may play a crucial role in the progression of HCC. Moreover, high ERCC6L expression is a prognostic factor for reduced OS in HCC patients. Knockdown ERCC6L could inhibit HCC cell proliferation, invasion in vitro and vivo. In the aspect of molecular mechanism, we confirmed that PI3K/AKT and NF-κB pathway were involved in the regulation of ERCC6L. Taken together, our findings reveal that ERCC6L overexpression correlates with the development of the malignant tumor and may be an effectively prognostic factor and potential therapeutic target for HCC patients.
Increasing evidences have demonstrated that ERCC6L is an important oncogene in tumor progression. Pu et al. [
10] observed that knockdown ERCC6L expression inhibited the proliferation of breast and kidney cancer cell in vitro
and vivo. Upregulated ERCC6L mRNA was notably correlated with the progress of tumor and associated with poorer outcomes in breast and kidney cancer patients. They also found that RAB31 may be as ERCC6L downstream protein involved in the progression of cancer via phosphorylated MAPK and CDK. Zhang et al. [
25] analyzed a tissue microarray containing 150 renal cell carcinoma (RCC) samples showed that compared with adjacent tissue, ERCC6L was overexpressed in the tumor tissues, and abnormal expression was positively correlated with the cancers progression. Meanwhile, knockdown ERCC6L expression inhibited RCC cells viability and induced apoptosis accordingly. In terms of mechanism, they confirmed that MAPK signaling pathway was involved in the regulation of ERCC6L on cellular process of RCC. Zhong et al. [
11] showed that four genes (ERCC6L, AHCY, STK33, and NCAN) have been identified to increase in the neuroblastoma and predicted poor prognosis of neuroblastoma patients. Of these, ERCC6L was an independent prognostic factor of overall survival and event-free survival. Furthermore, they identified that some special genes such as MAD2L, CCNB1 and BIRC5, which had a close relationship with ERCC6L were involved in the cell cycle pathway, thus ERCC6L may play an important role in neuroblastoma. Recently, Xie et al. [
26] found that ERCC6L was abnormal overexpressed in colorectal cancer (CRC) tissues and cell lines, and reducing ERCC6L expression in CRC cells significantly inhibited the proliferation, cell cycle progression, and arrested cell cycle at G0/G1 phase. These findings demonstrated that ERCC6L may exert an essential role in tumor growth and may be an efficient target for tumor detection, diagnosis and therapy.
In the current study, we evaluated the expression of ERCC6L in HCC patients and demonstrated that ERCC6L levels were overexpressed in the tumor tissues than in paired tumor-adjacent tissues. Meanwhile, ERCC6L expression levels were positively associated with clinicopathological characteristics, including Edmondson stage, tumor encapsulation, and female gender. Furthermore, abnormal expression of ERCC6L was associated with shorter OS compared with low ERCC6L expression in patients, indicating that ERCC6L expression is markedly significant for the prognosis of HCC patients. Epidemiological studies of HCC have showed that it was notably more prevalent in male than in female, with a male-to- female ratio ranging from 2:1 to 8:1 [
27]. We also found that ERCC6L expression was higher in man than that of women. Although in line with this trend, however the proportion of female patients in our sample was lower, than it may need to further expand the sample size for further research. High ERCC6L expression was also found in the patients with non-tumor encapsulation or III-IV pathological stage, which often indicating highly invasive, metastatic, and poor prognosis.
Similarly, the level of ERCC6L mRNA and proteins expression was also elevated in HCC cell lines than in normal human liver cells. Then, we revealed that interfering with ERCC6L expression with shRNA in SMMC-7721 and HuH-7 cells significantly inhibited their proliferation in vitro and in vivo. Apoptosis is a cellular mechanism characterized with programmed cell death and is result of chain of reaction modulated by many effectors for regulating coordinates cell proliferation and cell death [
28]. Previous studies have observed that caspase3 is considered as a crucial executioner of apoptosis and activated caspase3/7 may cleave the majority of polypeptides which undergo proteolysis in cells, and was an independent prognostic factor for tumor [
29] . Knockdown ERCC6L promoted the G1 phase cell arrest and increased the proportion of apoptosis cells by caspase3/7 dependent manner. Collectively, these data indicated that ERCC6L may function as an oncogene in the occurrence and development of the HCC.
Numerous studies have shown that the PI3K/AKT signaling pathway plays a crucial role in malignant transformation and the subsequent growth, proliferation, and metastasis of human tumors [
30,
31]. The downstream effectors of abnormal activated PI3K/AKT pathway may contribute to its role in progression of tumor growth, apoptosis, altered endothelial cell function, and angiogenesis, such as NF-κB and mTOR [
32‐
35]. In a clinical study, PI3K/AKT pathway activation was associated with tumor progression and the reduced survival of HCC patients [
36]. Caspases-7 and caspases-3 are involved in the initiation and execution of apoptosis, respectively. Activated AKT can phosphorylate caspases-7 and caspases-3 to prevent caspase-9 and caspase-3 activation [
37]. We investigated the mechanism by which ERCC6L shRNA inhibited HCC progress. Results revealed that knockdown-ERCC6L expression reduced the level of phosphodiesters related to the PI3K/AKT and NF-κB. Furthermore, the rescue experiments clarified that AKT inhibitor attenuated the enhancement of upregulating mediated p-PI3K/p-AKT and p-NF-κB protein expression. These data suggested that ERCC6L may regulate HCC via PI3K/AKT pathway (Fig.
5d). This may provide a new direction for studying ERCC6L function.
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