Introduction
Hepatocellular carcinoma (HCC) is the most common primary hepatic cancer worldwide, with an ever-increasing prevalence [
1]. The disease has the characteristics of latent onset, rapid progression, and high malignancy, indicating the dismal prognosis for HCC patients. According to the 2020 Global Cancer Statistics Report, liver cancer ranks the sixth in terms of cancer incidence and the third in mortality [
2]. The treatment of HCC has been continuously improved in recent years. To some extent, targeted medicines and immunotherapy have improved the prognosis and survival time [
3]. Apart from this, the recurrence and metastasis problems of hepatocellular carcinoma patients have not been effectively resolved [
4]. Therefore, searching for applicable biomarkers is critical for the early diagnosis, timely treatment, and prognostic evaluation of hepatocellular carcinoma.
Tumor suppressor candidate 3 (TUSC3) plays an important role in N-glycosylation during the protein folding process by encoding one subunit of the endoplasmic reticulum (ER) Oligosaccharyl transferase (OST) complex, while the unfolded protein response (UPR) is carried on by its loss [
5]. Therefore, TUSC3 gene was originally assumed to be a tumor suppressor candidate. For example, loss of TUSC3 expression in prostate cancer cells results in increased proliferation, migration, and invasion by affecting ER stress via Akt signaling [
6]. In addition, loss of TUSC3 modifies the molecular response to ER stress and causes characteristics of the epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells [
7]. However, the development of cancer is a multi-step process, which involves several molecular events. For example, TUSC3 activates WNT/β-catenin and MAPK signaling to improve the proliferation and migration of colorectal cancer (CRC) cell lines [
8]. Similarly, TUSC3 increases the proliferation of NSCLC cell lines via hedgehog (HH) signaling [
9]. It is assumed that TUSC3 has a possible role beyond N-glycosylation. Moreover, role of TUSC3 in HCC has rarely been reported and deserves to be further investigated.
Lipase C hepatic type (LIPC) was the most related gene to TUSC3 by using TUSC3 microarray analysis. LIPC, a member of the lipase family, plays a key function in lipoprotein metabolism, and its aberrant expression has been linked to metabolic and cardiovascular disorders [
10,
11]. Meanwhile, the aberrant expression of LIPC impacts the onset and progression of cancer, but its role in malignancies has not been concentrated on too much by researchers. In the context of non-small cell lung carcinoma (NSCLC), LIPC expression levels may have both a predictive value and an independent prognostic potential [
12]. LIPC is highly expressed in a cohort of human hepatic metastasis and primary colorectal tumors [
13,
14]. In addition, LIPC promotes the epithelial–mesenchymal transition (EMT) process in Borrmann type 4 gastric cancer [
15] and pancreatic cancer [
16]. It is indicated that LIPC is associated with tumor metastasis. EMT, a reversible cellular program that transforms epithelial cells into quasi-mesenchymal cell states, endows the tumor with several traits including tumor-initiation, which is essential to the malignancy [
17]. Lipid metabolism can accelerate the procedure of EMT in the context of neoplasia, indicating the need for further studies to develop an effective tumor therapy [
18,
19]. However, there are still no relevant reports about TUSC3, LIPC, and EMT in HCC.
In this study, we aimed to demonstrate the expression and roles of TUSC3 in HCC, and to explore the underlying mechanisms of TUSC3 in EMT and the progression of HCC.
Materials and methods
Tissue samples and ethical statement
This study was conducted based on the Declaration of Helsinki and approved by the medical ethics committee of Nanfang Hospital, Southern Medical University, China. Written informed consent was obtained from all patients before the operation. HCC tissues and the adjacent non-tumor tissues collected from 125 HCC patients (109 men and 16 women) between 2017 and 2018 were obtained from Nanfang Hospital, Southern Medical University, Guangdong Province, China. All HCC cases were confirmed by a senior pathologist and staged based on the 2011 Union for International Cancer Control TNM classification of malignant tumors.
Cell culture
The human HCC cell lines, include MHCC97H, Hep3B, HCCLM3, Huh-7, HepG2, Bel-7404, and QGY-7701, LO2 (Normal human liver cell) were obtained from our laboratory. All the cell lines were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) in a humidified atmosphere containing 5% CO2 at 37 °C.
RNA isolation and quantitative real time polymerase chain reaction (qRT-PCR)
Total RNA of cultured cells was extracted using the Ambion Trizol reagent (Thermo Fisher Scientific, Halethorpe, MD) according to the manufactures’ instructions. cDNAs were synthesized using Prime Script TM RT reagent Kit (#RR037A, TaKaRa, Dalian, China) from 500 ng of total RNA. qRT-PCR analysis of mRNA expression was performed as described previously with normalization to α-actin. The gene primers used are listed as following:
Tusc3: forward, 5ʹ-GAGAGCTGATACTTTTGACCTCC-3ʹ.
Reverse, 3ʹ-CCCGAATATGAACATCCGTTCTG-5ʹ.
LIPC: forward, 5ʹ-CCCAGTCCCCCTTCAAAGTT-3ʹ.
Reverse, 3ʹ-CAGCTCGCCGATATCCACAT-5ʹ.
Actin: forward, 5ʹ-CTCCCTGGAGAAGAGCTACGAGC-3ʹ.
Reverse, 3ʹ-CCAGGAAGGAAGGCTGGAAGAG-5ʹ.
Protein extraction and western blotting
The protein lysate was obtained by scraping the cells in cold PBS. After centrifugation, PBS was abandoned, and the cells were incubated in lysis buffer (Fdbio Science, China) for 30 min on ice. Then the mixture was centrifuged for another 30 min, and the supernatant was collected as protein lysate. Protein levels were examined by western blotting using the antibodies as follows (Table
1). And protein expression was detected by chemiluminescence (ECL, Pierce). Expression of β-tubulin (Proteintech) was used as a protein loading control.
Table 1
The reagents used in the study
TUSC3 | Proteintech, China | 1:800, #16039 |
LIPC | Proteintech, China | 1:500, #21133 |
β-tubulin | Proteintech, China | 1:2000, #10068 |
pAKT | Cell Signaling Technology, America | 1:1000, #9271 |
AKT | Cell Signaling Technology, America | 1:1000, #9272 |
E-cadherin | Proteintech, China | 1:1000, #20874 |
N-cadherin | Proteintech, China | 1:1000, #22018 |
Vimentin | Proteintech, China | 1:1000, #10366 |
MK2206 inhibitor | Proteintech, China | 5 mg, #S1078 |
Immunohistochemistry assay
The immunohistochemistry (IHC) analysis was performed using the streptavidin-perosidase (SP) method. The sections were deparaffinised and rehydrated, and endogenous peroxidase was inhibited with 0.3% H2O2 methanol (AMRESO) for 15 min. For antigen retrieval, slides were boiled in sodium citrate buffer (0.01 M, pH 6.0) for 5 min in a pressure cooker. After blocking with the 10% normal goat serum, the primary antibodies (rabbit anti-TUSC3, 1:100 dilutions; rabbit anti-LIPC, 1:100 dilutions; Proteintech, China) in blocking buffer were applied and the slides were incubated at 4 °C overnight. After 3× PBS washes, the sections were treated with horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit IgG (1:2000, #7074, Cell Signalling, Danvers, MA). Finally, the visualization signal was developed with 3-3′-diaminobenzidine-hydrogen peroxide (Maixin, Fuzhou, China), and the slides were counterstained in haematoxylin.
The total TUSC3 immunostaining score was calculated as the sum of the percentage positivity of stained tumor cells and the staining intensity. The positive percentage was scored from 0 to 4, with 0 for < 0%, 1 for 1–25%, 2 for 26–50%, 3 for > 51–75% and 4 for > 75%. The staining intensity was scored from 0 to 3, with 0 for no staining, 1 for weakly stained, 2 for moderately stained, and 3 for strongly stained. Then the whole score of TUSC3 expression was calculated with the value of the positive percentage score multiplied by staining intensity score, ranging from 0 to 12. The final expression level of TUSC3 was defined as “negative” (0–3), “low” (4–6), “median” (7–9) and “high” (10–12). As with the TUSC3 immunostaining score, the LIPC immunostaining score was calculated in the same way.
Transfection assays
Overexpression and down-regulation of TUSC3 were performed by lentiviral delivery using the pEZ-Lv105 vector and the psi-LVRH1GP shRNA lentiviral vector (GeneCopoeia, CA) containing TUSC3 shRNA and HEK293T packaging cell line. TUSC3 silenced MHCC97H and Hep3B cell lines, and TUSC3 overexpressed Bel-7404 and QGY-7701 cell lines were constructed. Cells transduced with empty lentiviral vectors were used as negative controls of TUSC3-overexpressed cells. Cells transduced with scrambled shRNA were used as negative controls of TUSC3-silenced cells. Recombinant lentiviruses were produced by transient transfection in 293 T cells using the calcium phosphate method. The transfectants were selected using puromycin reagent (5 μg/ml) for 2 weeks. And the transfective efficiencies were detected through western blotting and qRT-PCR analyses using the protein and mRNA samples.
For the generation of LIPC-overexpressed cells, we purchased the human LIPC expression plasmid from GeneCopoeia (Guangzhou, China). Bel-7404 and QGY-7701 cell lines were transfected with pcDNA3.1-LIPC using Lipofectamine 2000, and transfected cells were selected in the cell culture medium containing 2 μg/ml puromycin. Also, we performed LIPC knockdown in MHCC97H and Hep3B cell lines. The siLIPC was performed with 50 nM of siRNA using Lipofectamine RNAiMAX (Thermmo Fisher Scientific) according to the manufacturer’s instructions. siRNAs were obtained from Ruibo Biotechnology Co. (Guangzhou, China). The siRNA sequences used in the study are: siLIPC-1, si-LIPC-2, siLIPC-3, siControl. All the transfective efficiencies were detected through western blotting and qRT-PCR analyses using the protein and mRNA samples.
Cells were seeded in 6-well plates at a density of 1 × 103 per well. After 2 weeks, the cells were fixed in formalin and stained with crystal violet. The number of colonies containing ≥ 50 cells was counted under a microscope. The experiment was performed with three replicates for each cell line.
Cell counting kit-8 assay (CCK8)
1 × 103 cells/well of HCC cell lines or the control cells were seeded in 96-well plate. At 0, 1, 2, 3, 4, 5 days, 10 μl CCK8 solution (Dojindo, Tokyo, Japan) with 90 μl culture medium was added into each well and mixed for 2 h. The Microplate Autoreader (BioTek, Winooski, VT, USA) was used to measure optical density at 450 nm.
Cell wound healing assay
Cells were seeded on six-well culture plates and incubated for 24 h (80–90% confluence). After two washes with PBS, scratch wounds were produced in each well using a 10 μl pipette tip. Migration was monitored for up to 48 h and wound margins were photographed. Images were captured using an image-analyzing frame-grabber card (LG-3 Scientific Frame Grabber; Scion, Frederick, MD, USA) and analyzed with image analysis software (Image J). Cell motility was quantified by measuring the distance between the advancing margins of cells in three randomly selected microscopic fields (× 200) at each time point.
Transwell assay
About 1 × 105 cells mixed in 200 μl serum-free media were placed in the upper compartment of 8-μm-pore transwells (Costar, Corning, Cambridge, MA, USA) and 400 μl of 10% FBS in free medium (Gibco, Invitrogen, Carlsbad, CA, USA) was added to the lower compartment. And we have checked the migration every 12 h. The cells were allowed to migrate within 72 h. For quantification, the cells in the lower compartment were stained with crystal violet and counted in five randomly chosen fields (× 200) under a light microscope. The experiment was conducted with three replicates.
In vivo tumor growth assay
Nude mice (aged 3–4 weeks) were purchased from the Experimental Animal Center of the Southern Medical University, Guangzhou, and housed in a pathogen-free facility. To figure out the impact of the aberrant TUSC3 level on the tumor genesis, 1 × 106 cells from the transfected HCC cell lines together with their control groups were injected subcutaneously into the flanks of nude mice. The length and width of tumors were measured every week with a caliper to calculate the tumor volume using the formula: V = L × W2/6(V, volume; L, length; W, width)). At the endpoint, the xenograft tumors were isolated and processed for HE assays. All experimental procedures were performed in accordance with protocols admitted by the Institutional Animal Care and Research Advisory Committee of Southern Medical University.
Immunofluorescence assays
Cells were seeded evenly on the confocal dish at a density of 5 × 104 per well for 48 h and then probed with primary antibodies against TUSC3 (#SAB4503183, Proteintech, China). Next, the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated goat antibodies against rabbit IgG (anti-rabbit IgG, #ab6940, Abcam, MA). From then on, the dishes were protected from light. After washing with PBS in a dark place, the dishes were incubated with primary antibody against LIPC (#3538, Proteintech, China), and then incubated with rhodamine-conjugated goat antibodies against rabbit IgG (anti-rabbit IgG, #ab6940, Abcam, MA). Following counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, MO), images were captured using an Olympus FV1000 confocal laser-scanning microscope (Olympus America Inc., NY).
Co-immunoprecipitation
For co-immunoprecipitation, cell lysates were prepared as described above from the HCC cell line MHCC97H. The cell lysates were pre-cleared by incubating with pre-blocked Protein A Sepharose beads (Zymed, San Francisco, CA, USA). Then individual antibodies (TUSC3, 1:250, #SAB4503183, Proteintech, China; LIPC, 1:500, #C2206, Proteintech, China; normal rabbit IgG, (AB_2771930, Abclonal, China) were added and incubated overnight in the shaking bed at 4 °C before harvesting of complexes with protein A Sepharose (GE Healthcare, Piscataway, NJ, USA) and brief centrifugation. Binding proteins were separated with SDS/PAGE, followed by visualization using western blotting.
Statistical analysis
All statistical analyses were performed using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and the data were expressed as the mean ± s.d. P < 0.05 was considered statistically significant. The relative quantification of gene expression detected by qRT-PCR was log 2 transformed and analyzed by Student’s t-test. Linear or rank correlation analysis was performed to determine the correlation between the gene expression levels. Pearson’s Χ2-test was used to analyze the associations of TUSC3 or LIPC with clinical pathologic features. For cell line experiments and animal assays, data was subjected to a two-tailed Student t-test or one-way ANOVA (T-test for two-group comparisons, otherwise one-way ANOVA).
Discussion
In this study, the expression of TUSC3 was downregulated in HCC tissues, and the expression of TUSC3 in HCC tissues were inversely related to tumor size, degree of differentiation, and BCLC stage. These outcomes basically agreed with Sheng’s conclusion [
20]. However, we did not have enough prognosis data used for survival analysis. In addition, gain-of-function and loss-of-function assays showed that TUSC3 inhibited the proliferation and migration of HCC cells. Additionally, TUSC3 may alter the activity of the LIPC/AKT axis to encourage the progression of HCC. All the above results indicated that the decline of TUSC3 expression was associated with the malignant process of HCC and that TUSC3 indeed plays a tumor suppressive role in the progression of HCC.
TUSC3 is a subunit of the oligosaccharide transferase complex with oxidoreductase activity. And it plays a very important role in catalyzing the N-terminal glycosylation of proteins [
21]. Aberrant expression of TUSC3 leads to alternations in N-glycosylation, which is closely related to tumorigenesis and malignance [
22]. TUSC3 has been reported in a variety of cancers, such as prostate cancer, ovarian cancer, and pancreatic cancer, and its effects on cell proliferation, migration, and invasion have also been validated [
6,
7,
23]. However, the mechanism of TUSC3 in HCC has not been previously reported.
Through the analysis of the TUSC3 microarray, LIPC was the most related gene to TUSC3. Synthesized in the endoplasmic reticulum of liver parenchymal cells, LIPC containing N-linked high mannose type was transferred to Golgi and secreted as an active form [
24]. Based on previous studies, LIPC plays a disparate function in different cancers [
12‐
16]. In our study, the protein and mRNA levels of LIPC were both downregulated in HCC, and it was important to note that there was a significant correlation between the downregulation of LIPC expression and the clinicopathological traits of HCC patients, including tumor size, tumor amounts, differentiation grade, AFP level, and BCLC stage. And vitro studies showed that the downregulation of LIPC promoted the proliferation and migration of HCC cells. It was shown that the downregulation of LIPC expression was significantly associated with unfavorable progression of HCC. Moreover, the function of LIPC in the development of HCC was first demonstrated in this study.
In order to elaborate the relationship between TUSC3 and LIPC, we performed IHC, co-immunoprecipitation, immunofluorescence co-localization, and western blotting assays, and these results showed that TUSC3 can act on LIPC and positively regulate its expression. As TUSC3 can change the process of N-linked glycosylation, we speculate that TUSC3 regulates LIPC by the function of oligosaccharyltransferase. Unfortunately, the detailed mechanisms of how TUSC3 regulates LIPC need to be further studied.
The epithelial–mesenchymal transition refers to the biological process in which cells with an epithelial phenotype are transformed into cells of mesenchymal origin under the modulation of certain cytokines [
25]. It was reported that TUSC3 enhances EMT progress in colorectal cancer [
8] and non-small cell lung cells [
26]. On the contrary, it was also reported that TUSC3 prevents the EMT process in ovarian cancer [
7]. Lipid metabolism is reportedly involved in EMT in cancer, according to recent studies. Fatty acid synthetic enzymes regulated the EMT process in breast cancer [
27‐
29]. In this study, we found that the expression of EMT-related genes was changed after the alternation of TUSC3 and LIPC expression. The results implied that TUSC3 and LIPC may be involved in EMT. The AKT signaling pathway has been demonstrated to have a substantial influence on the EMT process [
30‐
32]. As a tumor suppressor gene, it was reported that TUSC3 is related to the progression of glioblastoma by inhibiting the activity of the Akt signaling pathway [
33,
34]. In addition, previous research has demonstrated that suppressing TUSC3-dependent AKT signaling pathway may affect the progression of melanoma cells [
35], prostate cancer cells [
6], and cervical squamous cell carcinoma cells [
36]. Therefore, the current work looked at whether the downregulation of TUSC3 may affect the AKT signaling pathway in HCC cells. In this study, we found that the activity of AKT phosphorylation was changed after the alternation of TUSC3 and LIPC expression. Furthermore, treatment of the cell with the AKT inhibitor MK2206 inhibited the expression of p-AKT and eliminated the effect of shTUSC3 or siLIPC, leading to the reversal of the EMT process. Therefore, knockdown TUSC3 resulted in the activated of AKT signalling pathway.
Altogether, downregulation of TUSC3 promoted the EMT process and HCC progression via LIPC/AKT axis.
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