Introduction
Globally, liver cancer is the 6th most common malignancy and the 3rd leading cause of cancer-related fatalities. Hepatocellular carcinoma (HCC) stands as the prevailing form of liver cancer, with around 900,000 new cases and 820,000 fatalities recorded in 2020 alone [
1]. Despite significant advances in therapeutic strategies such as surgical resection, radiofrequency ablation, liver transplantation and immunotherapy, the overall prognosis of HCC is still poor [
2,
3], with the majority of patients suffering from postoperative recurrence, invasion and metastasis [
4]. Extrahepatic metastases have been reported to occur in 13.5-42% of HCC cases [
5], and the most common sites of blood metastasis are the lungs (up to 60% of patients with metastatic disease) and bone (up to 40% of patients) [
5‐
7]. Hence, clarifying the specific mechanisms of cellular metastasis in HCC and finding new targets are critical to improving the survival of HCC patients.
Tumor cell interaction with the extracellular matrix (ECM) is a well-recognized link leading to tumor progression [
8], which forms the scaffolding for tissues and provides structural integrity. The ECM promotes tumor metastasis through the formation of focal adhesions with the integrins on the tumor cell membrane [
9,
10] via glycoproteins such as the laminins [
11]. Laminin-5, encoded by the LAMA3, LAMB3 and LAMC2 genes, is the primary adhesion component of the epidermal basement membrane. Laminin 5 interacts with integrins α3β1 or α6β4 on tumor cells to activate focal adhesion kinase (FAK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which promotes tumor cell migration and invasion [
12‐
14].
Ribosome RNA processing protein 15 (RRP15) is a nucleolar protein required for nucleole formation [
15]. A recent study showed that ribosome biogenesis is accompanied by an increase in RRP15 levels, suggesting that the latter could be a significant factor in carcinogenesis [
16]. Knocking down RRP15 in cancer cells resulted in cell cycle arrest or apoptosis [
15]. Deficiency of RRP15 decreased the proliferation and metastasis of colorectal cancer cells [
17,
18]. Although inhibition of RRP15 has been found to inhibit HCC proliferation and growth [
19], the function of RRP15 in HCC metastasis has not yet been revealed [
20]. Hence, this research sought to elucidate the function of RRP15 in the migration of HCC cells, and explore the underlying mechanisms.
Materials and methods
Patients
Tumors and matched peritumoral specimens were donated by 20 patients with HCC who underwent surgical resection without preoperative treatment at Lishui People’s Hospital (Lishui, Zhejiang, China), who were informed of the objective of the study and gave informed consent. This research was granted approval by the Ethics Committee of the Lishui People’s Hospital (No. LLW-FO-401). 20 pairs of liver tissues were homogenized for total RNA extraction, and 12 pairs were used for protein isolation.
Tissue microarrays and immunohistochemistry assay
The tissue microarray (TMA) was purchased from YEPCOME Biotechnology (YP-LVCSUR1801, Shanghai, China), consists of a total of 79 formalin-fixed and paraffin-embedded tissue sections. The TMA sections were stained with rabbit anti-RRP15 sera were applied at 1: 50 dilution [
17], and the expression level of RRP15 was scored according to the signal intensity and distribution. The specific methods and scoring rules were described previously [
21].
Cell lines and cell culture
The HepG2 cells were purchased from ATCC (Washington D.C., USA), and the Huh7, MHCC-97 H, MHCC-97 L and LM3 cell lines were purchased from the Chinese Academy of Sciences’ Cell Bank (Shanghai, China). MIHA cell line was purchased from the Hunan Fenghui Biotechnology Co., Ltd (CL0469, Hunan, China). Cell lines were cultivated according to the instructions and passaged at 80% fusion.
Small interfering RNA (siRNA) and short hairpin RNA (shRNA) transfection
SiRNAs targeting RRP15, LAMC2 and PATZ1, and the RRP15 shRNA were bought from GenePharma Company (Shanghai, China). The sequences are listed in Supplementary Table
1. ShRNA and negative control were separately cloned into pLKO.1-puro vectors (Sigma-Aldrich, Burlington, MA, USA). Full length RRP15 (NM_016052.4) cDNA was synthesized and cloned into the expression vector pCDH (Sigma-Aldrich). Cells were transfected with the respective constructs (50nM siRNAs) using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA).
Wound healing assay
The transfected cells were cultured in six-well plates, and the monolayers were scraped longitudinally with a sterile 10-µL pipette tip once the cells were 80–90% confluent. After rinsing the cell debris with PBS, the adherent cells were cultivated in serum-free medium, and photographs of the wound area were taken at 12 h intervals over a period of 48 h to assess cell migration.
Transwell migration and invasion assay
8 × 104 cells were inoculated into the upper chamber of each well of a transwell plate (24 wells, 8 μm pore size; Corning, New York, USA) with 200 µL of serum-free medium and the lower chamber with medium containing 10% FBS. For the invasion assay, the cells were seeded in transwell filters pre-coated with 30 µL diluted (1:9) Matrigel (Corning). After incubating for 36 h, the cells remaining on the surface of the filter were removed, and those that had migrated/invaded were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet (Beyotime Biotechnology, Shanghai, China). The count of migrated or invaded cells was counted in 5 random fields per well.
Cell adhesion assay
MHCC-97 H and LM3 cells were seeded into 96-well plates pre-coated with Matrigel at the density of 8 × 103 cells/well. After 1 h, the plates were rinsed with PBS, the attached cells were fixed with 4% PFA and stained with Wright’s Giemsa (Beyotime Biotechnology). The cells were photographed and counted under a microscope.
Cell proliferation assay
MHCC-97 H and LM3 cells were inoculated into 96-well plates at a density of 2 × 103 cells/well, and then transfected with different siRNAs (siNT/siRRP15-1/siRRP15-2) or treated with 5 µM Sorafenib (Solarbio Life Sciences, Beijing, China), and cultured for 24, 48, 72, 96 and 120 h respectively. 10 uL of Cell Counting Assay Kit 8 Reagent (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added to each well for incubation. Spectrophotometers (Varioskan Flash, Thermo Fisher Scientific, Waltham, MA, USA) were used to measure absorbance at 450 nm and to calculate the percentage of living cells.
The cells were inoculated into a 6-pore panel at a 1000 cells/well. After 14 days, the colonies were washed twice with PBS, fixed with 4% PFA, stained with crystal violet (Beyotime Biotechnology), and counted.
Apoptosis assay
MHCC-97 H and LM3 cells were inoculated in 24-well plates at a density of 2 × 104 cells/well and transfected with different siRNAs (siNT/siRRP15-1/siRRP15-2). Apoptosis was assessed by flow cytometry (Becton Dickinson FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA) analysis according to the instructions of Annexin V-PE / 7-AAD Apoptosis Detection Kit (KeyGEN BioTECH, Nanjing, China).
Cell cycle assay
MHCC-97 H and LM3 cells were seeded in 24-well plates at the density of 2 × 104 cells/well, and transfected with different siRNAs (siNT/siRRP15-1/siRRP15-2). After culturing for 48 h, the cells were collected into single cells, rinsed with ice-cold PBS and fixed overnight with chilled 70% ethanol at -20℃. The cells were washed twice with PBS and incubated with 50 µg/mL propidium iodide (Solarbio Life Sciences, Beijing, China) and 0.1 mg/mL RNase A (Qiagen, Hilden, Germany) in PBS. The stained cells were analyzed in a flow cytometer.
Establishment of in vivo tumor models
Male BALB/c nude mice (age: 5–6 weeks; weight: 18–22 g; SLAC Laboratory Animal Co. Ltd., Shanghai, China) were randomly divided into the RRP15 knockdown and control groups. The mice were injected with 3 × 10
6 MHCC-97 H cells in 100 µL PBS and 100 µL Matrigel to create subcutaneous tumors in their right axillary fossa. The tumors were measured every 3 days using calipers, and tumor volume was calculated as (length × width^2)/2. The migration model involved the injection of 1 × 10
7 MHCC-97 H cells in 100 µL PBS into the mice’s tail vein. Eight weeks after inoculation, mice were euthanized by CO
2 asphyxiation, and the lungs were dissected [
22]. The tissues were fixed with 4% PFA, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Animal experiments were approved by the Wenzhou Medical University Animal Experiment Committee (No. wydw2022-0164).
RNA-sequencing analysis
TRIzol™ reagent (Invitrogen) was utilized to extract total RNA from MHCC-97 H cells. LC-Bio Technology CO. Ltd. (Hangzhou, China) conducted RNA-sequencing. The entirety of the sequencing data produced in this research has been stored in the GEO database (GSE228416).
mRNA and protein detection
Total RNA and protein were extracted from cells using TRIzol
™ reagent (Invitrogen) and RIPA lysis buffer (Millipore, Billerica, Massachusetts, USA), respectively. Real-time PCR analyses were performed according to manufacturers’ instructions. Primers used for real-time PCR were shown in Supplementary Table
S2. The expression of proteins was determined with immunoblotting. Briefly, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride or polyvinylidene difluoride membranes (Millipore). Then, the membrane was cut into strips and incubated overnight with primary antibodies at 4 °C (Supplementary Table
3). Blots were detected by chemiluminescence (Bio-Rad, Hercules, CA, USA) and visualized using Image Lab software (version 6.1, Bio-Rad Laboratories).
Statistical analyses
Data were analyzed using GraphPad Prism 8 software (San Diego, CA). The experimental data were presented as the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicate. Student’s t test and one-way analysis of variance (ANOVA) were used to compare data between groups, and p-value < 0.05 was considered statistically significant.
Discussion
Zhao et al. showed that ablation of RRP15 prevented HCC proliferation and growth both in a p53-dependent manner in vitro and in vivo [
19]. However, whether and how RRP15 affects the migration of HCC has not been studied so far. Since the high migration rates are responsible for the high mortality rate of patients with HCC [
5‐
7], it is momentous to unearth new molecular markers that can accurately predict HCC migration. We innovatively found that high expression of RRP15 was associated with lower survival in HCC patients. Knocking down RRP15 expression prevented the proliferation, migration and invasion of HCC cells, independent of P53 expression, and also suppressed lung migration in a mouse model.
Laminin-5 is considerably associated with the growth, metastasis and prognosis of HCC tumors [
27,
28]. It consists of the laminin α3, γ2 and β3 subunits, which are encoded by
LAMA3,
LAMC2 and
LAMB3 respectively [
25]. LAMC2 is overexpressed in multiple cancers and drives tumorigenesis by interacting with α6β4 and α3β1 integrins, epidermal growth factor receptor (EGFR), and other surface receptors [
26,
27,
29‐
32]. In addition, deletion of LAMC2 suppressed the EMT and lymph node metastasis of cholangiocarcinoma via inactivation of the EGFR signaling pathway [
33,
34]. LAMC2 is also overexpressed in ovarian cancer and can regulate tumor cell proliferation and metastasis [
35]. We similarly found that RRP15 overexpression promoted the migration of HCC cells in vitro, and its oncogenic effect was abrogated by the simultaneous knockdown of LAMC2. Furthermore, the lack of RRP15 also decreased the expression of LAMC2 in HCC cells.
LAMC2 binds to integrins α6β4 and phosphorylates FAK, and activates downstream signaling pathways, such as those already reported in esophageal squamous cell carcinoma and breast cancer [
26,
36‐
38]. FAK protein overexpression is highly associated with aggressive behavior and undesirable outcome in HCC [
39,
40]. In colon cancer cells, FAK phosphorylation regulates E-cadherin expression by activating the Src signaling pathways [
41]. Our results indicate that knockdown of RRP15 inhibited the FAK signaling pathway, whereas overexpression increased the level of phosphorylated FAK. LAMC2 knockdown attenuated FAK signaling in the RRP15-overexpressing cells. Furthermore, NF-κB, a downstream effector of FAK [
42,
43], was also downregulated by RRP15 knockdown. Altogether, these findings suggest that knockdown of RRP15 suppresses HCC migration via attenuation of LAMC2/FAK/NF-κB signaling.
In addition, we also found an interesting phenomenon that loss of RRP15 up-regulates the expression of fibrogenic genes, including
COL1A1,
COL4A5 and
COL4A6, suggesting that RRP15 may inhibit fibrosis. The role of tumor-associated fibrosis in cancer progression is inconclusive, and increasing evidence has revealed that tumor-associated fibrosis inhibits the development, proliferation and metastasis of cancer. Alkasalias et al. found that fibroblast fusion into monolayers effectively inhibited tumor cell proliferation in vitro [
44]. Mechanistically, hedgehog, as a key signaling pathway that promotes fibrosis, may play an important role in stromal cells inhibiting tumor progression. For example, genetic and pharmacological inhibition of hedgehog accelerates the progression of pancreatic and bladder cancers [
45,
46]; hedgehog agonists induce stromal hyperplasia but reduce epithelial cell proliferation, thereby inhibiting cancer development [
45]. However, the role of RRP15 in fibrosis has not been reported, and we speculate that RRP15 may also promote the progression of HCC by inhibiting fibrosis, but this speculation and the underlying mechanism need to be further studied.
Further, the predominantly nuclear location of RRP15 suggests that it may regulate gene expression [
15]. Dong et al. showed that RRP15 promotes colorectal cancer metastasis through regulating leucine zipper tumor suppressor 2-mediated β-catenin signaling [
17]. We found that RRP15 regulates downstream target genes at the transcriptional level, and RRP15 depletion attenuated LAMC2 expression, at least in part, through the inactivation of PAZT1 promotor. Nevertheless, we were unable to conclusively identify the mechanisms through which RRP15 regulates LAMC2, and the purported interaction between RRP15 and the transcription factor PATZ1 needs to be investigated further.
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