Introduction
Hepatocellular carcinoma (HCC) is the main type of liver cancer. It has a poor prognosis and ranks as the fourth leading cause of cancer-related death worldwide.1 Due to the lack of effective treatment, the 5-year overall survival of HCC patients is less than 20%.2,3 The high mortality of patients with advanced HCC is often due to metastasis. However, there is little knowledge about the underlying mechanisms of metastasis and invasion in HCC. Thus, a better understanding of the molecular mechanisms of metastasis in HCC may help us explore novel diagnostic and therapeutic strategies for HCC treatment. Collagen has an important role in the migration, invasion and proliferation of cancer cells.4,5 Previous studies have suggested that different covalent collagen cross-links enhance the accumulation of stabilized collagen in many kinds of human cancers, which is related to poor patient survival.6,7 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) is a key enzyme promoting the formation of stabilized collagen cross-links.8 Recent studies have shown that PLOD2 is overexpressed in head and neck squamous carcinomas, breast cancer, biliary tract cancer, cervical cancer and colorectal cancer and that it promotes migration, invasion, and proliferation in these tumor cells.9-13 Previous reports have demonstrated that PLOD2 is regulated by HIF-1α in sarcoma and FOXA1 in non-small-cell lung cancer (NSCLC), which leads to a poor prognosis in these patients.7,14 In terms of HCC, PLOD2 is associated with the recurrence of early-stage HCC after surgery.15 However, the potential molecular mechanisms and regulatory signaling pathway by which PLOD2 is involved in the invasion and metastasis process in HCC remain unknown. Hence, identifying the molecular mechanism by which PLOD2 promotes metastasis and invasion in HCC may help us discover potential targets for treatment.
Baculoviral IAP repeat containing 3 (BIRC3), a member of the antiapoptotic protein (IAP) family, has been found to be highly expressed in a variety of human tumors.16,17 In addition, BIRC3 regulates the nuclear factor-kappa B (NF-κB) signaling pathway, which is involved in the development of cancers.18 In this study, we demonstrated that high expression of PLOD2 predicted an unfavorable prognosis in HCC patients, which makes PLOD2 a potential oncogene. Moreover, we verified that PLOD2 promotes HCC invasion and metastasis both in vitro and in vivo. Mechanistically, we found that inhibition of PLOD2 suppresses HCC cell migration and growth by decreasing BIRC3 expression. Furthermore, we demonstrated that PLOD2 is regulated by interferon regulatory factor 5 (IRF5), which can be responsible for the high expression of PLOD2 in HCC. These data highlighted that PLOD2 plays a critical role in the development of HCC, suggesting that it may serve as a promising novel biomarker for HCC treatment.
Methods
Human tissue specimens and HCC tissue microarray
The HCC and adjacent non-tumor liver tissues prepared into tissue microarray were obtained from 109 patients at the Sun Yat-sen Cancer Center (SYSUCC, Guangzhou, China) between July 2010 and May 2015, and the clinicopathological features are shown in Table 1. The cases selected were based on distinctive pathologic diagnosis of HCC and received curative resection. Average follow-up time was 55.3 months (median: 55.3 months; range: 2.0–105.0 months). Additional HCC tissue from 91 patients received curative resection at SYSUCC were used for immunohistochemical staining study. All samples were obtained with the informed consent of the patients. The study was approved by the Institute Research Ethics Committee at the Cancer Center.
Table 1Clinical features and PLOD2 protein expression in HCC patients
| Characteristics | N | PLOD2 expression
| p |
|---|
| High | Low |
|---|
| Sex | | | | 0.750 |
| Male | 94 | 46 (48.94%) | 48 (51.06%) | |
| Female | 15 | 8 (53.33%) | 7 (46.67%) | |
| Age in years | | | | 0.640 |
| >50 | 65 | 31 (47.69%) | 34 (52.31%) | |
| ≤50 | 44 | 23 (52.27%) | 21 (47.73%) | |
| BCLC stage | | | | 0.030 |
| I | 86 | 38 (44.19%) | 48 (55.81%) | |
| II–III | 23 | 16 (69.57%) | 7 (30.43%) | |
| Histologic grade | | | | <0.001 |
| I–II | 57 | 15 (26.32%) | 42 (73.68%) | |
| III–IV | 52 | 39 (75.00%) | 13 (25.00%) | |
| AFP in µg/L | | | | 0.640 |
| ≥400 | 46 | 24 (52.17%) | 22 (47.83%) | |
| <400 | 63 | 30 (47.62%) | 33 (52.38%) | |
| Vascular invasion | | | | 0.823 |
| Yes | 17 | 8 (47.06%) | 9 (52.94%) | |
| No | 92 | 46 (50.00%) | 46 (50.00%) | |
| Tumor size in cm | | | | 0.001 |
| ≤5 | 54 | 20 (37.04%) | 34 (62.96%) | |
| >5 | 55 | 34 (61.82%) | 21 (38.18%) | |
| Tumor number | | | | 0.180 |
| Solitary | 94 | 49 (52.13%) | 45 (47.87%) | |
| Multiple | 15 | 5 (33.33%) | 10 (66.67%) | |
| HBsAg | | | | 0.540 |
| Negative | 14 | 8 (57.14%) | 6 (42.86%) | |
| Positive | 95 | 46 (48.42%) | 49 (51.58%) | |
| MVI | | | | 0.730 |
| Yes | 36 | 17 (47.22%) | 19 (52.78%) | |
| No | 73 | 37 (50.68%) | 36 (49.32%) | |
| Cirrhosis | | | | 0.630 |
| Yes | 79 | 38 (48.10%) | 41 (51.90%) | |
| No | 30 | 16 (53.33%) | 14 (46.67%) | |
| Tumor encapsulation | | | | 0.490 |
| Complete | 33 | 18 (54.55%) | 15 (45.45%) | |
| None | 76 | 36 (47.37%) | 40 (52.63%) | |
Immunohistochemical (IHC) staining
HCC and adjacent tissues were incubated with anti-PLOD2 antibody (Origene, Rockville, USA), anti-IRF5 (Abcam, Shanghai, China) antibody and anti-BIRC3 antibody (Proteintech, Wuhan, China) respectively at 4°C for 10 h. The details of antibody dilution were shown in Supplementary Table 1. The tissues were then incubated with horseradish peroxidase-conjugated anti-rabbit/mouse antibodies (Dako, Copenhagen, Denmark) at 37°C for 30 m. After that, diaminobenzidine chromogen (Dako) was applied to the samples for reaction followed by counterstaining with hematoxylin (Leagene, Beijing, China). Immunoreactivity for PLOD2, IRF5 and BIRC3 proteins was tested with a semiquantitative method by evaluating the number of positive tumor cells within the total population of tumor cells. Scores were assigned using 5% increments from 0–100% as in our previous study.19,20 The expression levels of these proteins were divided into four groups based on the IHC scores: negative group, 0–10%; weak group, 11–50%; moderate group, 51–80%; and strong group, 80–100%. We referred to proteins associated with moderate or strong intensities as the high expression group, while proteins associated with tumors of negative or weak intensities were identified as the low expression group.
Cell lines and cell culture
Human HCC cell lines (PLC-8024, Huh7, Hep3B, MHCC-97H and HepG2) and MIHA normal liver cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) with short tandem repeat appraisal certificates. All cells were cultured as described previously.21 Huh7 and MHCC-97H cells were infected with lentiviral pLKO.1 particles containing short hairpin RNA (shRNA) and were selected with 3 mg/mL puromycin (ThermoFisher, Waltham, MA, USA) for 4 days. Lentiviral pLKO.1 plasmids for shPLOD2 (Jidan, Guangzhou, China) were packaged with Lenti-Pac HIV mix (GeneCopoeia, Guangzhou, China) and Endo-Fectin Lenti (GeneCopoeia, Guangzhou, China) in 293T cells to produce lentiviral particles. For BIRC3 overexpression experiment, the BIRC3 expression plasmid (Jidan, Guangzhou, China) was packaged with Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA) and then infected PLOD2 knockdown (KD) Huh7 and MHCC-97H cells.
Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting
Total RNA was extracted from cell lines using a TRIzol reagent kit (Life Technologies, Carlsbad, CA, USA). After treatment with DNase I (TaKaRa, Dalian, China), 2 µg of total RNA was used for cDNA synthesis with random hexamers and Superscript III (Invitrogen, Waltham, MA, USA). The primer sequences are shown in Supplementary Table 2. The cDNA templates were subject to PCR amplification. qRT–PCR analysis of PLOD2, IRF5 and BIRC3 was performed on an ABI PRISM 7900 Sequence Detector using SYBR Green PCR Kits (Applied Biosystems, Carlsbad, CA, USA). All reactions were run in triplicate. Cycle threshold values should not differ by more than 0.5 among triplicate reactions. PLOD2, IRF5, and BIRC3 expression levels were normalized to RNU6B, which yielded a 2−ΔΔCt value.
Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight. Mouse anti-β-actin monoclonal antibody (Sigma–Aldrich, Shanghai, China) was used to estimate protein loading. The details of antibody dilution are shown in Supplementary Table 3.
In vitro cell migration and invasion assays
Cell migration and invasion assays were performed in Transwell chambers (8 µm pore size; CoStar, Washington DC, USA) according to the manufacturer’s instructions. Huh7 cells (1.2×105) and MHCC-97H cells (2.0×105) were placed in the top chamber of each insert (BD Biosciences, Franklin Lakes, NJ, USA) and cultured at 37°C for 24 h. The migrated and invaded cells were stained with crystal violet and photographed at 200× magnification. All measurements were performed in triplicate.
Cell proliferation assay
Cell proliferation was measured using Cell Counting Kit-8 (CCK-8) assay kits (Dojindo Corp., Kumamoto, Japan) according to the manufacturer’s instructions. Huh7 cells and MHCC-97H cells (5×103) were placed in 96-well plates (1,000 cells per well) and incubated at 37°C for 24 h.
Animal studies
Male BALB/c nude mice at 4 weeks of age were purchased from the Medical Experimental Animal Center of Guangdong Province (China) and fed at the Animal Experimental Center of Sun Yat-Sen University. Experiments involving animals were used randomly. To establish the subcutaneous tumor model, Huh7 cells (4×106 cells in 200 µL of serum-free medium) were injected into the right flanks of each group (n=6). Tumor volumes were measured every 3–4 days using a caliper until the day of sacrifice. Tumor volume (V, mm3) was measured by using the following formula: V=L×W2/2 where L was the largest diameter in mm, and W the smallest diameter. In the lung metastasis model, Huh7 cells (2×106 cells suspended in 200 µL DMEM) were injected through the tail veins (n=4). The lungs were dissected after 9 weeks.
Luciferase reporter assay
Luciferase reporter plasmids (full-length and mutant PLOD2 promoters) were constructed by GeneCopoeia (Rockville, IL, USA). Huh7 cells (8.0×104) and MHCC-97H cells were seeded in each well of 24-well plates and incubated at 37°C for 18 h. After that, siIRF5 or negative controls were cotransfected into HCC cells together with PLOD2 promoters. The Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia, Guangzhou, China) were used to measure the Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP) luciferase activities after 48 h. Gluc activity was normalized to the SEAP activity and each group was analyzed in triplicate experiments.
Cancer genome atlas data analysis
The data for all box plots and correlation plots were retrieved from The Cancer Genome Atlas Data Analysis (https://portal.gdc.cancer.gov/ ), as RNAseq data in level 3 HTSeq-fragments per kilobase per million (FPKM) format from the liver hepatocellular carcinoma (LIHC) project. Then, the RNAseq data in FPKM were converted into log2 format. R software (version 3.6.3) was used to create boxplots and correlation plots.
UALCAN analysis
UALCAN (http://ualcan.path.uab.edu ) is an interactive web portal for performing in-depth analyses of RNA-seq and clinical data from 31 cancer types of The Cancer Genome Atlas (TCGA) database.22 In this study, we used UALCAN to analyze the mRNA expression differences in PLOD2 between primary HCC tissues and normal samples. UALCAN was also used to analyze the mRNA expression differences among various subgroups based on cancer stages and tumor grades. Student’s t test was used to compare the difference in transcriptional expression, and p<0.05 was considered statistically significant.
Statistical analysis
The Kaplan–Meier method was used to compute survival curves and survival curves were analyzed by the log-rank test. The multivariate analysis was used to analyze significant prognostic factors found in univariate analysis (p<0.05) by the Cox proportional hazards model. Student’s t test was used to compare any two groups, and one-way analysis of variance was used for multiple comparisons. GraphPad Prism 8.0 software (GraphPad, Inc., La Jolla, CA, USA) and R program (R version 3.6.3; R Foundation for Statistical Computing, Vienna, Austria) were performed for the statistical analysis.
Results
PLOD2 was highly expressed in HCC, and high expression of PLOD2 in HCC predicted poor prognosis
TCGA pan-cancer analysis revealed that PLOD2 was highly expressed in various types of cancers, including kidney renal clear cell carcinoma, lung adenocarcinoma, LIHC and prostate adenocarcinoma compared with expression in normal tissues (Fig. 1A). To investigate the expression of PLOD2 in HCC, we used the TCGA database and found that the mRNA level of PLOD2 was significantly increased in HCC tissues compared with paired adjacent liver tissues (Fig. 1B, C). Moreover, the mRNA expression of PLOD2 was correlated with patients’ cancer stages and tumor grades, indicating that higher mRNA expression of PLOD2 was found in advanced cancer stages and increased tumor grades according to the TCGA database (Fig. 1D, E). TCGA database analysis also revealed that higher PLOD2 expression was positively correlated with poor prognosis in human HCC patients (Fig. 1H, I). HCC tissue microarrays including 109 cases of HCC and adjacent liver tissues also demonstrated that PLOD2 protein levels were significantly higher in HCC tissues than adjacent liver tissues (Fig. 1F, G). Additionally, patients with higher PLOD2 expression had shorter overall survival (OS) and recurrence-free survival (RFS) (Fig. 1J and K). Apart from clinical stages and histologic grades, we also found that high PLOD2 expression was significantly associated with tumor size (Table 1). Finally, the analysis of the Cox proportional hazards regression showed that high PLOD2 expression was an independent unfavorable prognostic factor for both OS and RFS (Tables 2 and 3). These results suggest that elevated PLOD2 expression might correlate with aggressive clinicopathological characteristics and poor prognosis in HCC patients.
Table 2Univariate and multivariate Cox regression analyses for overall survival
| Variable | Univariate analysis
| Multivariate analysis
|
|---|
| HR (95% CI) | p | HR (95% CI) | p |
|---|
| PLOD2, high vs. low | 2.47 (1.24–4.95) | 0.011 | 2.12 (1.01–4.46) | 0.046 |
| Sex, M vs. F | 5.62 (0.77–41.01) | 0.089 | | |
| Age, ≥50 vs. 50 years | 0.64 (0.33–1.23) | 0.180 | | |
| BCLC stage, I vs. II–III | 4.27 (2.22–8.24) | <0.001 | 2.51 (1.11–5.68) | 0.028 |
| Histologic grade, I–II vs. III–IV | 1.53 (0.79–2.54) | 0.206 | | |
| HBsAg, positive vs. negative | 0.72 (0.30–1.73) | 0.721 | | |
| Cirrhosis, yes vs. no | 1.34 (0.66–2.76) | 0.422 | | |
| MVI, present vs. absent | 1.75 (0.89–3.42) | 0.103 | | |
| Tumor capsule, none vs. complete | 1.26 (0.63–2.52) | 0.514 | | |
| Tumor size, ≥5 cm vs. <5 cm | 2.24 (1.12–4.48) | 0.023 | | |
| Tumor number, multiple vs. solitary | 1.31(0.31–5.47) | 0.712 | | |
| AFP, ≥400 vs. <400 µg/L | 1.22 (0.63–2.37) | 0.55 | | |
| Vascular invasion, present vs. absent | 2.99 (1.53–5.85) | 0.001 | | |
Table 3Univariate and multivariate Cox regression analyses for recurrence-free survival
| Variable | Univariate analysis
| Multivariate analysis
|
|---|
| HR (95% CI) | p | HR (95% CI) | p |
|---|
| PLOD2, high vs. low | 2.73 (1.60–4.66) | <0.001 | 2.43 (1.29–4.56) | 0.006 |
| Sex, M vs. F | 1.65 (0.71–3.83) | 0.247 | | |
| Age, ≥50 vs. <50 years | 0.74 (0.45–1.24) | 0.255 | | |
| BCLC stage, I vs. II–III | 2.88 (1.69–4.91) | <0.001 | 1.93 (1.00–3.73) | 0.049 |
| Histologic grade, I–II vs. III–IV | 1.68 (1.00–2.79) | 0.046 | | |
| HBsAg, positive vs. negative | 1.54 (0.66–3.58) | 0.318 | | |
| Cirrhosis, yes vs. no | 1.00 (0.59–1.69) | 0.994 | | |
| MVI, present vs. absent | 1.59 (0.93–2.71) | 0.087 | | |
| Tumor capsule, none vs. complete | 0.912 (0.54–1.53) | 0.726 | | |
| Tumor size, ≥5 cm vs. <5 cm | 2.04 (1.21–3.44) | 0.007 | | |
| Tumor number, multiple vs. solitary | 1.06 (0.33–3.40) | 0.918 | | |
| AFP, ≥400 vs. <400 µg/L | 1.49 (0.89–2.48) | 0.127 | | |
| Vascular invasion, present vs. absent | 1.80 (1.01–3.20) | 0.045 | | |
KD of PLOD2 reduced metastasis and invasion of human HCC cells in vitro and in vivo
We examined the expression of PLOD2 in HCC cell lines and found that PLOD2 was highly expressed in HepG2, Huh7, MHCC-97H and Hep3B cell lines (Fig. 2A). To investigate the functions of PLOD2 in HCC cells, we used shRNA to KD PLOD2 in two HCC cell lines, namely, MHCC-97H and Huh7. Western blotting and qRT-PCR showed that PLOD2 was successfully knocked down by the two shRNAs in both cell lines (Fig. 2B). Transwell and wound healing assays showed that KD of PLOD2 significantly decreased the migration and invasion of HCC cells (Fig. 2C–E). Moreover, KD of PLOD2 slowed down cell growth in both MHCC-97H and Huh7 cells (Fig. 2F).
In addition, we established subcutaneous xenograft and lung metastatic tumor models with Huh7 cells in mice to determine whether PLOD2 deficiency inhibits tumor growth and metastasis in vivo. In subcutaneous models, PLOD2 KD reduced the growth of tumors (Fig. 2G), and tumor volume and tumor weight significantly decreased in the PLOD2 KD groups (Fig. 2H, I). Furthermore, the expression of PLOD2 in the KD groups was lower than that in the PLOD2 negative control group (Fig. 2J). The number of Ki-67-positive cells was significantly reduced in cancer tissues with PLOD2 KD (Fig. 2J, K), indicating that the KD of PLOD2 significantly decreased the capacity of tumor growth in vivo. Moreover, smaller and fewer metastatic lung nodules were observed in the PLOD2 KD groups than in the control group in our tail vein lung metastasis mouse model (Fig. 2L). Collectively, these results indicate that KD of PLOD2 reduced human HCC cell migration and proliferation.
BIRC3 was the downstream factor of PLOD2 in HCC
To explore the molecular mechanisms by which PLOD2 promotes HCC cell metastasis and invasion, we performed RNA sequencing analysis using the shPLOD2 Huh7 cell line vs. the control, and pathway enrichment was performed via DAVID and gene set enrichment analysis (GSEA). Focal adhesion signaling was one of the most significantly downregulated gene pathways that resulted from PLOD2 KD (Fig. 3A). GSEA revealed that focal adhesion signaling was prominently inhibited in HCC with the KD of PLOD2 (Fig. 3B). Focal adhesion signaling is a pathway that interacts with cells and extracellular matrix which plays essential roles in biological processes, including cell motility, cell proliferation, cell differentiation, regulation of gene expression and cell survival. Moreover, previous study showed that PLOD2 promoted the migration and invasion of cervical cancer cells by focal adhesion formation.12,23 Because PLOD2 is an oncogene related to extracellular matrix, we hypothesized that KD of PLOD2 inhibits HCC cell proliferation by inhibiting the focal adhesion signaling pathway. We used real-time polymerase chain reaction assays to confirm that PLOD2 KD decreased the mRNA expression of genes of the focal adhesion signaling pathway, including COMP, CAV1, CAV2, EGF, BIRC3 and CAPN2 (Fig. 3C).
Focal adhesion signaling receptor BIRC3 is an oncogene that promotes cell invasion and proliferation via NF-κB signaling in HCC.24,25 Moreover, BIRC3 is regulated by COL11A1 and enhances ovarian cancer cell resistance to cisplatin thereby promoting ovarian cancer progression.26 Given that BIRC3 is an oncogene that related to collagen, we hypothesized that PLOD2 promotes HCC cell migration and proliferation via BIRC3. To verify the hypothesis, we performed western blotting to demonstrate that the expression of BIRC3 was decreased by KD of PLOD2 in HCC cells (Fig. 3D). To test the relationship between PLOD2 and BIRC3, we examined the protein levels of PLOD2 and BIRC3 in a total of 200 HCC specimens using tissue microarrays by IHC (Fig. 3E). The expression of BIRC3 was significantly correlated with the levels of PLOD2 in human HCC specimens (Fig. 3F). We further analyzed the TCGA database and found that the relationship between BIRC3 and PLOD2 mRNA levels was positively correlated (Fig. 3G), which was consistent with our results. Taken together, our data illustrated that the expression of BIRC3 was the downstream factor of PLOD2 in HCC.
KD of PLOD2 inhibited HCC cell metastasis and invasion by decreasing BIRC3 expression
Given that BIRC3 is regulated by PLOD2, we focused on whether loss of PLOD2 inhibits HCC metastasis and invasion by decreasing BIRC3 expression. We first overexpressed BIRC3 in PLOD2 KD Huh7 and MHCC-97H cell lines (Fig. 4A). Overexpression of BIRC3 promoted cell proliferation, migration, and invasion, which rescued the phenotype caused by PLOD2 KD in HCC cells (Fig. 4B–D). Moreover, overexpression of BIRC3 promoted cell migration and restored the decreased wound healing ability caused by PLOD2 KD in HCC cells (Fig. 4E). In subcutaneous models, the expression of BIRC3 was reduced in the KD groups compared with the negative control group (Fig. 4F–G), indicating that BIRC3 is regulated by PLOD2 in vivo. Overall, our results demonstrate that KD of PLOD2 inhibited HCC cell migration and growth by decreasing BIRC3 expression.
PLOD2 transcription was regulated by IRF5
According to previous reports, the differential expression of PLOD2 is regulated at the transcriptional level in multiple human carcinomas.7,14 Thus, we analyzed the promoter region of PLOD2 (+99 to −2,000 kb) in JASPAR (http://jaspar.genereg.net/ ). Using 10 scores as a cutoff point and 80% as the relative profile score threshold, four potential genes were selected as the underlying transcription factors. Interferon regulatory factor 5 (IRF5) is a transcription factor with diverse functions, including modulation of cell growth, differentiation, apoptosis, and immune system activity. Moreover, the mRNA level of IRF5 was higher in HCC tissues than in normal tissues and correlated with poor prognosis according to TCGA analysis (Fig. 5A, B). We therefore hypothesized that PLOD2 is regulated by IRF5 at the transcriptional level. To verify our hypothesis, we silenced IRF5 with siRNA in the Huh7 and MHCC-97H cell lines. The mRNA and protein expression levels of PLOD2 were significantly decreased after specifically silencing IRF5 (Fig. 5C, D). In addition, IHC of tissue microarray including 200 HCC samples indicated that PLOD2 was positively correlated with IRF5 at the protein level (Fig. 5E), consistent with the mRNA level confirmed by TCGA database analysis (Fig. 5F). According to the JASPAR results, two binding sites of IRF5 were predicted in the putative promoter region of PLOD2 (Fig. 5G). To further explore the binding site, we performed a promoter activity assay using a plasmid constructed by linking the PLOD2 promoter to a luciferase gene. Promoter luciferase assays showed that the transcriptional activity of the full-length PLOD2 promoter was significantly reduced when IRF5 was silenced with siRNA (Fig. 5H). Next, we identified the functional IRF5 binding region in the PLOD2 promoter by mutating two potential binding regions (Fig. 5J, K). IRF5 silencing significantly reduced the luciferase activity of mutant site A, while the luciferase activity of mutant site B construct remained similar to that of the control group (Fig. 5I–L). The results show that site B was the binding site of IRF5, regulated the transcription and PLOD2 expression in HCC cells.
Altogether, our results suggest that PLOD2 acts as a tumor promoter regulated by IRF5 and promotes progression of HCC via BIRC3, providing a molecular mechanism for the increased aggressiveness in PLOD2 highly expressed tumors (Fig. 6).
Discussion
The poor behaviors of HCC may be attributed to tumor metastasis and invasion. In multiple cancers, these aggressive behaviors may be attributed to accumulated collagen deposition and cross-linking, which lead to poor prognosis in tumor patients.27-30 Tougher tumor stroma was generated by cross-linking via PLOD2-dependent collagen modification and organization,7 which could be regarded as an ‘expressway’ for tumor cells migrating to remote organs and blood vessels in tumor patients, resulting in metastasis. These reports have revealed the relationship between PLOD2 and the degree of malignancy, indicating that PLOD2 might be a potential molecular marker for several cancers. In this study, we found that increased PLOD2 expression in HCC tissues is correlated with poor prognosis in HCC patients. Moreover, the expression of PLOD2 was related to HCC tumor size, grade, and cancer BCLC stage. Furthermore, PLOD2 could be regarded as an independent unfavorable prognostic factor for both OS and RFS in HCC patients. By in vitro and in vivo experiments, we confirmed that KD of PLOD2 inhibits HCC cell metastasis and invasion, suggesting that PLOD2 may be a promising oncogene for HCC.
Based on the results, we explored the potential molecular mechanism by which PLOD2 promotes HCC development. However, the signaling pathways and target genes regulated by PLOD2 have not been discovered. In our study, RNA sequencing revealed that PLOD2 participates in the focal adhesion signaling pathway. Due to the high expression of BIRC3 found in a variety of human malignant tumors and BIRC3 involved in multiple biological processes, including cell proliferation, migration and apoptosis,18,31,32 we selected BIRC3 as the target gene of PLOD2 among various alternative genes. Furthermore, we demonstrated that HCC cells promoted metastasis and invasion via PLOD2-mediated activation of BIRC3. In summary, our study first discovered the target gene regulated by PLOD2.
PLOD2 has been reported to be regulated at the transcriptional level by numerous transcription factors, such as FOXA1, HIF-1α and TGFβ1, in multiple tumors.7,14,33 Interferon regulatory factor 5 (IRF5) is a member of the IRF family and plays a critical role in diverse immunomodulatory activities.34 In our study, we not only found that IRF5 and PLOD2 are positively correlated but also demonstrated that IRF5 can bind to the promoter of PLOD2 and regulate the expression of PLOD2 in HCC. Although IRF5 is expressed at low levels in a variety of tumors and is positively correlated with the prognosis of these tumors, we demonstrated that IRF5 is overexpressed in HCC tissue and correlated with poor prognosis in HCC patients. These results clarified that IRF5 could transcriptionally regulate the expression of PLOD2 and explained why PLOD2 is upregulated in HCC tissues.
In addition to these results, there are some limitations in this study. First, the detailed molecular interaction mechanism between PLOD2 and BIRC3 in HCC has not been elucidated. Moreover, due to technical limitations, we failed to perform a chromatin immunoprecipitation assay to prove that PLOD2 is directly regulated by IRF5. In addition, we need to perform rescue experiments in vivo to verify that PLOD2 promotes HCC cell metastasis and invasion via BIRC3.
Conclusions
In summary, our present work identified that PLOD2 plays a critical role in HCC invasion and metastasis by activating BIRC3. KD of PLOD2 significantly suppresses the malignant phenotype of HCC. Thus, our findings provide valuable evidence that PLOD2 may be a prognostic biomarker and a novel therapeutic target for HCC treatment.
Supporting information
Supplementary Table 1
Antibodies used for immunohistochemical staining.
(DOCX)
Supplementary Table 2
Primers used for polymerase chain reaction assays.
(DOCX)
Supplementary Table 3
Antibodies used for western blotting.
(DOCX)
Abbreviations
- AFP:
α-fetoprotein
- BCLC:
Barcelona Clinic Liver Cancer
- BIRC3:
Baculoviral IAP repeat containing 3
- CI:
confidence interval
- DMEM:
Dulbecco’s modified Eagle medium
- GSEA:
Gene Set Enrichment Analysis
- HBsAg:
hepatitis B surface antigen
- HCC:
hepatocellular carcinoma
- HR:
hazard ratio
- IHC:
immunohistochemical
- IRF5:
interferon regulatory factor 5
- KD:
knockdown
- KEGG:
Kyoto Encylopaedia of Genes and Genomes
- MVI:
microvascular invasion
- OS:
overall survival
- NC:
Negative control
- PLOD2:
procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2
- RFS:
recurrence-free survival
- SYSUCC:
Sun Yat-sen Cancer Center
- TCGA:
The Cancer Genome Atlas
Declarations
Acknowledgement
The authors would like to thank all the members who made contributions to this research.
Ethical statement
The Institutional Review Board of Sun Yat-Sen University Cancer Center approved this study (B2022-095-01). The Institutional Animal Care and Use Committee of Sun Yat-Sen University Cancer Center approved this study. The Laboratory Animal-Guideline for ethical review of animal welfare (GB/T 35892-2018) were adhered to regarding the use of animals in this research.
Data sharing statement
All data generated or analyzed during this study are included in this article.
Funding
This work was supported by grants from the National Natural Science Foundation of China (Nos. 81902473, 82172815, and 82103601).
Conflict of interest
The authors have no conflict of interests related to this publication.
Authors’ contributions
Study conception and design (KrL, YN, KL, CZ, ZQ, YhY, YS, ZL, ZH, DZ, YfY, BL), performance of experiments, analysis of the data and writing of the manuscript (KrL, YN), collection of the clinical samples (CZ, ZQ), assistance with the immunohistochemistry assays and animal experiments (YhY, YS, ZL, ZH, DZ), and revision of the manuscript (YfY, BL). All authors read and approved the final manuscript.