Introduction
Hepatocellular carcinoma (HCC) occurs as one of the most prevalent malignancies worldwide [
1]. The symptoms of early HCC are insidious, and the available surveillance tools and biomarkers fail to satisfy the clinical requirements for HCC diagnosis and prognosis prediction [
2]. Meanwhile, the lack of curative treatment and predictive strategies for advanced HCC results in an increase in its mortality rate by 2%~3% per year [
3,
4]. Therefore, the identification of new biomarkers for HCC prognosis is urgent.
Necroptosis is an alternative programmed cell death pattern when the normal apoptotic pathway is inhibited. Necroptosis has mechanistic resemblance to apoptosis and morphological resemblance to necrosis, respectively [
5]. Increasing evidence has shown that necroptosis, as a new promising therapeutic target, functions in the progression of a wide range of human cancers [
6,
7]. In addition, necroptosis may induce a strong adaptive immune response that inhibits tumor development [
8,
9]. Generally, the expressions of the necroptosis pathway-related regulators are dysregulated in cancer cells [
10‐
12]. Unfortunately, the prognostic values of necroptosis-related genes (NRGs) in HCC remain largely unclear.
Herein, a novel prognostic risk signature of 7 NRGs was generated using weighted gene co-expression network analysis (WGCNA), Cox proportional risk regression analysis, and least absolute shrinkage and selection operator (Lasso) analysis in The Cancer Genome Atlas (TCGA) cohort. Subsequently, the prognostic value of this 7-NRG signature was validated in TCGA cohort and the International Cancer Genome Consortium (ICGC) cohort as well as a local cohort. Our results demonstrated that this signature had a good predictive power in HCC prognosis.
Materials and methods
Data source
Transcriptome sequencing data, mutation data, and basic clinical information of 370 patients with HCC were obtained from TCGA database (
https://portal.gdc.cancer.gov ). NRGs were gained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (
https://www.genome.jp/kegg) [
13‐
15]. The complete gene names are shown in Table
S1. Transcriptome sequencing data of 231 donor patients with HCC from Japan and related basic clinical information were downloaded from the ICGC Data Portal database (
https://dcc.icgc.org). We collected a total of 100 surgically resected HCC tissues from the First Hospital of Wenzhou Medical University (FAHWMU), and performed Quantitative Real-Time PCR (qRT-PCR) as the local cohort. Specific clinical parameters for TCGA cohort, ICGC cohort and the local cohort were shown in Table
1.
Table 1
Specific clinical parameters for the TCGA cohort, ICGC cohort and local cohort
age | <=60 | 177(47.84%) | 49(21.21%) | 24(24%) |
> 60 | 193(52.16%) | 182(78.79%) | 76(76%) |
gender | FEMALE | 121(32.7%) | 61(26.41%) | 32(32%) |
MALE | 249(67.3%) | 170(73.59%) | 68(68%) |
stage | Stage I | 171(46.22%) | 36(15.58%) | 13(13%) |
Stage II | 85(22.97%) | 105(45.45%) | 25(25%) |
Stage III | 85(22.97%) | 71(30.74%) | 41(41%) |
Stage IV | 5(1.35%) | 19(8.23%) | 15(15%) |
unknown | 24(6.49%) | 0(0%) | 6(6%) |
grade | G1 | 55(14.86%) | unknown | 17(17%) |
G2 | 177(47.84%) | 29(29%) |
G3 | 121(32.7%) | 36(36%) |
G4 | 12(3.24%) | 14(14%) |
| unknow | 5(1.35%) | | 4(4%) |
Recurrent | Primary | unknown | unknown | 85(85%) |
Recurrent | 15(15%) |
Vascular invasion | Invasion | unknown | unknown | 45(45%) |
No Invasion | 55(55%) |
HBV Infection | Infection | unknown | unknown | 70(70%) |
No Infection | 30(30%) |
Construction of 7-NRG signature
Firstly, differentially expressed necroptosis-related genes (DENRGs) between HCC and adjacent normal tissues were screened out (|log2FC| >1). DENRGs were divided into different modules via WGCNA. Prognostic NRGs were screened by univariate cox regression analysis. The Lasso algorithm was applied to prognostic NRGs to exclude overfitting genes. Finally, multivariate cox analysis was used to construct 7-NRG signature. The necroptosis Riskscore was calculated as Riskscore = \({\sum }_{i=1}^{N}\left(\text{E}\text{x}\text{p}\left(\text{i}\right)\bullet \text{c}\text{o}\text{e}\left(\text{i}\right)\right)\). Exp(i) is the transcriptional value of the genes, and coe(i) is the biased regression coefficient of the genes derived from the multivariate cox analysis.
Independent prognostic factor analysis, nomogram, and calibration plots
Using the univariate and multivariate cox regression analyses, the independent prognostic factors for HCC were identified. R package “rms” was used to develop nomogram. “rms” and “survival” packages were applied to plot calibration curves of the nomogram.
Enrichment analysis
Gene Set Enrichment Analysis (GSEA) [
16] was performed to analyze all TCGA patients through “c5.go.v7.5.1.symbols.gmt” and “c2.cp.kegg.v7.5.1.symbols.gmt” gene sets via “limma”, “org.Hs.eg.db”, “DOSE”, “clusterProfiler” and “enrichplot” packages. R packages “clusterProfiler”, “org.Hs.eg.db”, “enrichplot” and “ggplot2” were used for Gene Ontology (GO) enrichment analysis [
17].
Immune infiltration analyses
Single-sample gene set enrichment analysis (ssGSEA) [
18] was utilized to estimate the relative infiltration characteristics of 16 immune cells and 13 immune related functions in the TCGA cohort. The ssGSEA was performed via “GSVA” package. R packages “limma”, “reshape2” and “ggpubr” were used to analyze the difference of immune-infiltrating cells between the high- and low-risk subgroups.
Tumor mutational burden (TMB) analysis
Upstream analysis of whole-genome sequencing and whole-exome sequencing data was conducted using “mattool” R package. Somatic mutation analysis was used to perform a systematic analysis of TMB for individual HCC patients in TCGA cohort. The “getsamplsummary” and “getgenesumprice” functions were used to retrieve patient information and genetic information, respectively. Package “maftools” was used to analyze TMB differences between the high- and low-Riskscore subgroups.
Immunohistochemistry
Immunohistochemistry was performed as previously described [
19]. Briefly, the tissues were immersed in 4% formalin for fixation, and then the formalin-fixed tissue was degreased and rehydrated. Next, the sections, blocked in 10% BSA, were in the incubation with primary antibody at 4 °C for at least 12 h. Then, the sections were incubated with a horseradish secondary antibody for 30 min.
Cell culture
The cell line Huh7 and HL-7702 were purchased from ATCC. Huh7 was cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% antibiotics. HL-7702 was cultured in RPMI-1640 medium with 10% FBS and 1% antibiotics. Cells were maintained in a 37℃ incubator with 5% CO
2 [
20].
Cell transfection
Huh7 and HL-7702 cells were cultured in a 6-well plate with 8 × 10
4 cells per well. When the cell density was near to 80%, si-NC, si-USP21, si-NRF1 packaged byLipofectamine™ 2000 ( Invitrogen) were transfected into cells at 37℃ for 6 h. Then fresh medium was replaced and cells were collected for subsequent experiments after 48 h of transfection [
20].
qRT-PCR analysis
Total RNA was isolated from Huh7 cells and HCC tissues as well as adjacent normal tissues using the Tiangen RNA extraction reagent kit. Each sample was reversely transcribed into complementary DNA (cDNA) using a reverse-transcription (RT) reagent kit (Takara Biotechnology Co., Ltd., Dalian, China). Then, Real-time PCR was performed using SYBR Premix ExTaq (Takara). GAPDH was used as endogenous controls for mRNAs [
20]. HSP90AA1 forward, 5’- AGGAGGTTGAGACGTTCGC − 3’; HSP90AA1 reverse, 5’- AGAGTTCGATCTTGTTTGTTCGG − 3’. PPIA forward, 5’- CCCACCGTGTTCTTCGACATT − 3’; PPIA reverse, 5’- GGACCCGTATGCTTTAGGATGA − 3’. SQSTM1 forward, 5’- GCACCCCAATGTGATCTGC − 3’; SQSTM1 reverse, 5’- CGCTACACAAGTCGTAGTCTGG − 3’. HSP90AB1 forward, 5’- AGAAATTGCCCAACTCATGTCC − 3’; HSP90AB1 reverse, 5’- ATCAACTCCCGAAGGAAAATCTC − 3’. FAF1 forward, 5’- GAGATGATCCTGGCGGATTTTC − 3’; FAF1 reverse, 5’- AGGTCCTGGTATGGTCTCACC − 3’. PGAM5 forward, 5’- TCGTCCATTCGTCTATGACGC − 3’; PGAM5 reverse, 5’- GGCTTCCAATGAGACACGG − 3’. USP21 forward, 5’-GAATCCTCGTGCTCCATCTGA − 3’; USP21 reverse, 5’-CAGCTGGTATACAGGACTTCCG-3’. GAPDH forward, 5’- AAAGCCTGCCGGTGACTAAC − 3’; GAPDH reverse, 5’- GCCCAATACGACCAAATCAGA − 3’. The full information of primer used for qRT-PCR were listed in Table S2.
Western blot analysis
The proteins from Huh7 cells were extracted using RIPA extraction buffer. The protein samples of interested were separated by 10% SDS-PAGE electrophoresis, and then transferred to PVDF membranes. The primary anti-USP21 (Invitrogen, PA5-11055) and anti-GAPDH (CST, #2118) were added in PVDF membranes and incubated overnight at 4 °C. Then, the second antibody was added and incubated at room temperature for 1 h [
19].
Cell proliferation assays
Cell Counting Kit-8 (CCK8) (Dojindo, Japan) was used for the assessment of cell proliferation. Cells were seeded into 96-well plate at a density of 2 × 10
3/100 µl per well to incubate for 48 h. Then, 10 µl CCK8 solution were added to each well and maintained in a 37 °C incubator for 1 h. Finally, the absorbance of each well was measured at 450 nm [
20].
Cell migration assays
Migration assays were performed in a Transwell chemotaxis 24-well chamber with 8.0 μm pore polycarbonate membrane insert (CORNING, 3422). Briefly, 3 × 104 cells were plated in the upper chamber with a non-coated membrane. After 24 h of incubation at 37 °C, migrating cells were fixed and stained with 20% methanol and 0.1% crystal violet dye. Migrated cells were counted and imaged with an inverted microscope.
Statistical analysis
At the present study, R software (version 4.1.0) was utilized for statistical analysis. Data were presented as mean ± SD of at least three independent experiments, and differences between two groups were compared using student’s t-test. Rank correlations were assessed by the performance of spearman’s correlation coefficient test among different variables. The R package “survival” was used for survival analysis, and Kaplan-Meier (K-M) survival curves were used to display survival differences between different groups. Statistical p-values were subjected to two tailed tests, and p < 0.05 was considered as significance.
Discussion
It is known that HCC imposes significant economic and medical burdens on societies worldwide. Effective biomarkers for monitoring HCC progression as well as treatment guidance are urgently needed [
21‐
23]. In recent years, scholars have dedicated substantial efforts to exploring prognostic and therapeutic molecular markers for HCC. In addition, increasing studies have demonstrated the involvement of necroptosis in HCC progression [
24‐
27]. In this study, we developed a 7-NRG prognostic signature and assigned Riskscores to HCC patients based on the prognostic signature computation formula
via cox regression analysis. HCC patients were divided into the high- and low-risk subgroups according to the median of Riskscore. K-M survival analysis revealed that patients in high-risk subgroup had a shorter OS in comparison with those in low-risk subgroup (p < 0.05). It was observed that patients with advanced tumor grades and stages exhibited elevated Riskscores (p < 0.05). In addition, univariate and multivariate cox regression analysis identified Riskscore as an independent prognostic factor for HCC (p < 0.001). The differences in immune cell infiltration scores, PD-1 and PD-L1 expression levels, as well as tumor mutation burden between the high-risk and low-risk subgroups suggested that high-risk patients may be more suitable candidates for immunotherapy. Additionally, USP21, one of the 7-NRGs, was demonstrated to play a promotional role in HCC cell proliferation and migration. Drug sensitivity analysis revealed that patients with low USP21 expression were more sensitive to sorafenib compared to those with high USP21 expression. In summary, our signature may potentially improve HCC prognosis prediction and guidance in immunotherapy and drug treatment strategies tailored to different patients.
The immune system plays a pivotal role in eliminating tumor cells and distinct immune cells exert varying functions during this process. In this study, we found that patients with low Riskscores exhibited higher NK cell infiltration and better prognosis, which was consistent with the fact that higher levels of NK cell infiltration contribute to anti-tumor immunity [
28,
29]. Conversely, patients with high-risk showed higher M2 macrophage infiltration, which has been reported to promote tumor growth, invasion, and metastasis by secreting various active substances [
30]. In line with it, we found that patients with high M2 macrophage infiltration had a shorter OS. In addition, high-risk patients expressed higher levels of immune checkpoint PD-1 and CTLA4, suggesting that high-risk patients may benefit from immunotherapy targeting PD-1 or CTLA4 checkpoint inhibitors. However, it should be noted that our analysis results were derived from the TCGA cohort, which requires validation in more datasets and samples to ensure consistency.
This prognostic signature comprises 7 NRGs, namely HSP90AA1, HSP90AB1, PPIA, PGAM5, FAF1, USP21, and SQSTM1. Previous studies have demonstrated that HSP90A, PPIA, PGAM5, USP21, and SQSTM1 are involved in regulating HCC progression through distinct mechanisms [
31‐
37], which supports the relevance of our prognostic signature to HCC progression. Given that USP21 exhibits the highest correlation with NRF1 (a TF), the preliminary verification of the regulatory relationship between NRF1 and USP21 was performed in HCC. We found that silencing NRF1 suppressed USP21 expression in HCC cells. Knockdown of NRF1 or USP21 inhibited HCC cell proliferation, whereas it had no impact on normal human liver cell (HL-7702) proliferation. Knockdown of USP21 was observed to suppress cell migration and reduce the mRNA expressions of MMP9, VIM, and CCL2 in Huh7 cells. Our findings suggest that NRF1 may influence HCC progression by regulating USP21. However, further validation are needed in more cell lines and animal experiments.
Previously, the prognostic value of necroptosis in cancers has been explored. For instance, Zhao et al. analyzed the prognostic values of necroptosis-associated lncRNA in stomach cancer [
38]. Ren et al. constructed a 13-NRG signature for predicting HCC prognosis using univariate cox and lasso cox regression analyses [
39]. However, these prognostic signatures were established only through analysis of public databases and lacked validation in clinical cohorts. Notably, our signature was not only validated in the TCGA and ICGC databases but also in the local cohort. Moreover, we validated the high expression of this 7-NRG signature in HCC tissues through immunohistochemical experiments.
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