Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies in humans [
1]. The rate of intrahepatic and extrahepatic metastases is high because of poor prognosis of HCC; moreover, the recurrence rate of HCC is high [
2]. Metastasis usually occurs in patients with advanced HCC, so it is important to develop new therapeutic targets for successful intervention. Metastasis is a complex process; metastatic potential of HCC cells is governed by cell intrinsic identities and external micro-environmental factors [
3]. However, we still need to elucidate the underlying molecular mechanisms that mediate metastatic cascade. By further elucidating the molecular mechanism, we can promote the development of effective metastasis-targeted therapy. This would improve the quality of life and survival of patients with HCC.
Previous studies have shown that metastasis of HCC occurs due to Tiam1 gene, which is a member of Dbl gene family that governs guanine nucleotide-exchange factors (GEFs) [
4]; however, the underlying molecular mechanism is hardly known. We conducted a more in-depth study to further investigate the underlying mechanism of HCC.
In this study, yeast two-hybrid assay was performed to screen the interaction of proteins with Tiam1 gene. We acquired, sequenced, and analyzed 24 positive clones from NCBI database. Finally, we identified six proteins, namely, OSBPLlA, ZNF307, FNDC3B, SRSF5, SYCPl, and SETDB1. After literature mining and bioinformatics analysis, we selected SETDB1 for further verification study.
The protein SETDB1, which is also known as KMT1E, is an H3K9 methyltransferase (HMT). Its multiple functional domains are located on chromosome 1q21. One of the major functions of HMT is covalent histone modification, which involves following processes: acetylation (Ac), methylation (Me), phosphorylation, ubiquitination, and sumoylation. These processes were associated with the development and progression of various tumors [
5]. According to the report that loss of H3K9Me2 is related with poor prognosis of both prostate and kidney cancer [
6,
7]. In addition, H3K9Me3 also serves as a diagnostic marker for the recurrence and distant metastasis of various cancers [
8,
9]. Furthermore, SETDB1 is associated with transcriptional inhibition of euchromatin, while another H3K9 methyltransferase SUV39H1 is mainly responsible for the high expression of structural pericentromeric heterochromatin, which functions as an oncogene in the metastasis of HCC cancer [
10,
11]. A lot of studies have clearly stated that tumorigenesis is promoted by HMTs, including Suv39h1 and G9a (EHMT2) in the past few decades, [
11]. By suppressing the expression of G9a and Suv39h1, cell growth is inhibited and lung epithelial cells are transformed in prostate cancer patients [
12,
13]. These researches support the hypothesis that aberrant histone methylation leads to the activation or repression of certain important genes during tumorigenesis. However, SETDB1, which serves as H3K9 methyltransferase, has been rarely associated with carcinogenesis and migration of liver. Therefore, we need to comprehensively investigate functional and pathological roles of SETDB1 in HCC patients.
Preliminary, Tiam1 was associated with the metastasis of liver cancer. Herein, it was found that SETDB1 is a crucial oncogene in the metastasis of HCC. In addition, we proved that cell invasion and metastasis was enhanced when SETDB1 cooperated with Tiam1. These observations indicate there was a novel pathway to regulate epigenetic modification of genes during HCC metastasis.
Methods
Cell lines and culture conditions
HCC cell lines QGY-7701(Catalogue number:TCHu42), Bel-7402(Catalogue number:TCHu10) and HCCLM3(Catalogue number:TCHu94)were purchased from Shanghai Cell Bank, Chinese Academy of Sciences, MHCC97L cell line(Product number: BNCC337741)was purchased from Bei Na Chuanglian Institute of Biotechnology of Beijing, china. All cell lines were cultured in DMEM or RPMI-1640 medium, which contained 10% FBS (Gibco, USA). As described previously, cells were cultured at 37 °C under 5% CO
2 [
4].
Collection of specimens
Primary HCC specimens and corresponding adjacent non-tumorous (NT) liver specimens were collected from Nanfang Hospital, Southern Medical University in China. A written informed consent letter was obtained from every study participant. The collection of specimens was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, Guangdong Province, China.
RNA isolation, reverse transcription and qRT-PCR
Total RNA was extracted with Trizol Reagent (Invitrogen,USA) according to manufacturer’s protocol. In the PrimeScript RT reagent kit (TaKaRa,Japan), total RNA was used as a template for the production of cDNA. To analyze the expression of mRNA, qRT-PCR was performed with SYBR Green qRT-PCR master mix (TaKaRa,Japan)on a Stratagene Mx3005P qRT-PCR system. The target genes of RT-qPCR were calculated with relative quantification (2−ΔΔCt) method, which was normalized to GAPDH.
CCK8 assays
Cells were plated in 96-well plates; a final density of 1 × 103 cells was maintained per well. After incubating cells for 24 h, 2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (CCK8) assay was performed by adding 10 μl of reagents (Beyotime, China) to plates. After incubating cells for 1 h, the absorbance of each well was measured with a microplate reader at 570 nm. The absorbance of cell culture was determined continuously for the next six days. All experiments were performed in triplicate.
Clonogenic survival assays
Clonogenic survival assays were carried out as described previously [
4].
Cell migration and invasion assays
Cell migration and invasion assays were performed in Transwell chambers (Corning Costar, USA). Matrigel (BD Bioscience, USA) was added to the chambers in which invasion assay was carried out. Before performing the assay, cells were serum-starved for 24 h. Migration assay was performed as follows: 1× 105 tumor cells were plated into the upper chamber with 0.1% fetal bovine serum (FBS). Then, 10% FBS was used as a chemo-attractant, which was added to the lower chamber. Invasion assay was performed as follows: cells were seeded on filters, which were coated with 20–50 μg/cm2 of reconstituted Matrigel (BD Bioscience, USA) on basement membranes. After incubating cells for 24 h at 37 °C, we used cotton swabs to remove cells that had not migrated or invaded from the top surface of filters. After the cells migrated or invaded into the bottom surface, they were fixed with 100% methanol and stained with 0.5% crystal violet. Permeating cells were observed under a microscope at × 200 magnification; these cells were graphed in six randomly selected fields. The experiment was repeated thrice independently.
Antibodies and western blotting
Western blot analysis was performed according to a procedure described previously [
14].
Yeast two-hybrid
To determine the expression of Tiam1 in yeast cells, a recombinant plasmid pGBKT7-Tiam1/C1199 was fused by recombinant gene technique. The code domain sequence of Tiam1/C1199 was amplified from a commercial Tiam1 cDNA clone, which was then cloned into pGBKT7 vector and confirmed by sequencing. The yeast strain AH109 cells were transiently transformed with pGBKT7-Tiam1/C1199 plasmid. Western blot was performed to confirm whether Tiam1 protein can be expressed normally in Saccharomyces cerevisiae without toxicity or autoactivation.
GST pull-down assay
Inoculate several colonies containing pGEX-4 T-1-Tiam1-PCER, C685, C751, C1199, and control. The recommended proteins were expressed in transformed cells of E.coli. These proteins were then purified. We successfully detected fusion proteins of Tiam1, which were labeled with GST. The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA). The interaction between Tiam1 and SETDB1 was detected and validated in vitro with GST pull-down assay(The detailed procedures could be seen in Additional file
1).
Cross-linking immunoprecipitation
Some different epitope-labeled candidate proteins (Flag and HA) and the recombinant expression vector Tiam1 were constructed by recombinant DNA technology. The recombinant plasmid had different epitope labeling. The recombinant plasmid Tiam1-C1199 was co-transfected into human embryonic kidney cells HEK293T. Cells were fixed at room temperature for 10 min with 10 ml of 1% formaldehyde in phosphate buffered saline (PBS). Then, these cells were sonicated to isolate total cellular protein. Protein, antibody, and protein G were incubated together. Protein samples were analyzed by western blot technique: the presence of proteins was detected with FLAG and HA antibodies in order to determine whether Tiam1 truly interacted with SETDB1 protein.
Establishment of stable cell lines
For the knockdown or overexpression of SETDB1, we purchased lentivirus vector and control vector from Genechem (Shanghai, China). Stably knockdown or overexpression of SETDB1 cells lines were constructed with lentiviruses transfection. Finally, the expression of SETDB1 was quantified by performing western blot analysis.
Xenograft studies
The stably knockdown or overexpression of SETDB1cells were suspended in 25 μL PBS. These cells were injected into the left liver lobe of male BALB/C nude mice aged 6-8 weeks. These cells were purchased from the Medical Experimental Animal Center of Guangdong Province in China. After six weeks, phenobarbital sodium was used to euthanize mice. Then, liver and lungs were removed from the euthanized mice. Xenograft tumor assays were performed as follows: 1 × 106 cells were injected subcutaneously into the flanks of nude mice, which were 4-6 weeks old. When the tumors could be detected, we measured the size of tumors every three days with a slide caliper for 21 days. Animal experiments were performed by strictly adhering to the Regulations for the Administration of Affairs Concerning Experimental Animals. The Institutional Animal Care and Use Committee approved all the procedures performed on animals in this study.
Statistical analysis
Data were expressed as mean ± standard deviation (SD), and a P value of < 0.05 was considered to be statistically significant in all the experiments. Results were analyzed by performing ANOVA or a two-tailed Student’s t-test. Statistical analysis was performed with SPSS 13.0 software (SPSS; North Chicago, IL, USA). Western blot results were quantified with Bio-Rad lab image software. In this study, all the experiments were performed in triplicates.
Discussion
The gene Tiam1 is is a member of the guanine nucleotide exchange factor (GNEF) family which is located on the human chromosome 21. Previous studies showed that the expression of Tiam1 gene is up-regulated in following types of carcinomas: T lymphoma, B lymphoma, pancreatic carcinoma, gastric cancer, breast cancer, and lung cancer [
15‐
18]. The expression of Tiam1 was regulated by epigenetic mechanisms [
19‐
21]. Previous studies have reported that Tiam1 gene interacts with many proteins, such as IB2/JIP2, nm23H1, Arp2/3, CD44, PAR, c-myc, EphB2, and Par-3. Therefore, Tiam1 gene is involved in many biological processes [
22‐
25]. However, researchers have not been successful in elucidating the inherent molecular mechanisms associated with HCC cells; these mechanisms are apparently mediated by Tiam1.
In this study, yeast two-hybrid method was used to discover and validate the interaction between candidate proteins and Tiam1. The results indicate that OSBPLlA, ZNF307, FNDC3B, SRSF5, SYCPl, and SETDB1 were candidate proteins. The interaction between Tiam1 and candidate proteins was confirmed by cross-linking IP.
The candidate protein SETDB1 is a major histone methyltransferase (HMT), which is associated with transcription repression through H3K9me3 [
10].
The candidate protein SETDB1 plays an significant role on transcriptional repression of euchromatin, the activity of which is important for the maintenance of embryonic stem cell state by repressing lineage-specific gene expression [
10,
26,
27]. Previous studies have reported the SETDB1 overexpression in patients was detected in certain tumors such as melanoma and lung cancer [
28,
29]. Previous research studies have reported SETDB1 accelerates tumorigenesis by regulating WNT signaling pathway [
29]. Recently, SETDB1 was reported as a novel oncogene in zebrafish melanoma model [
30]. However, we have not completely explored tumorigenesis mechanism of SETDB1, which is associated with the growth and metastasis of HCC tumor.
The interaction between Tiam1 and SETDB1 was determined by performing crosslinking IP and GST-pull down assays. It is important to note that SETDB1 is a major histone methyltransferase (HMT). During tumorgenesis, critical genes are activated or repressed by aberrant histone methylation. Moreover, H3K9Me3 serves as a significant diagnostic marker of recurrence and distant metastasis in cancer patients [
8,
9].
We investigated whether SETDB1 acts as an oncogene in patients with HCC. The proliferation, migration, and invasion of HCC cells were promoted with the overexpression of SETDB1. Compared to adjacent liver tissues, the expression of both SETDB1 and Tiam1 was unregulated in HCC samples; moreover, the expression of SETDB1 and Tiam1 was positively correlated with HCC samples. The effect of SETDB1 was restored on cells following the knockdown of Tiam1. In this study, a complex was formed following the interaction of SETDB1 with Tiam1. This indicates that SETDB1 functions as an oncogene in HCC cells.
Conclusions
In our work we confirm that proliferation, migration, and invasion of HCC cells could be promoted with the overexpression of SETDB1. Compared to adjacent liver tissues, the expression of both SETDB1 and Tiam1 was up-regulated in HCC samples; moreover, there was positive correlation between the expression of SETDB1 and Tiam1. In our study, SETDB1 interacted with Tiam1 to form a complex. This establishes that SETDB1 functions as an oncogene in HCC samples. These results support a novel link between SETDB1 and Tiam1; moreover, the complex SETDB1-Tiam1 is associated with the growth and metastasis of HCC tumor. They also suggest that SETDB1-Tiam1 complex is involved in a novel pathway, which regulates epigenetic modification of gene expression in HCC.
Acknowledgements
Thank you for the help of Dr. Zheng Lin in the course of the experiment.