Functional & Integrative Genomics (2023) 23:128

https://doi.org/10.1007/s10142-023-01060-w

ORIGINAL ARTICLE

RAD54L promotes progression of hepatocellular carcinoma

via the homologous recombination repair pathway
Hongda Li1 · Haiwen Zhuang2 · Tengfei Gu3 · Guangyu Li1 · Yuhang Jiang1 · Sanrong Xu1 · Qing Zhou1

Received: 8 March 2023 / Revised: 11 April 2023 / Accepted: 12 April 2023

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023

Abstract
Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence worldwide. The underlying mechanisms remain
poorly understood. The DNA metabolic process of homologous recombination repair (HRR) has been linked to a high
probability of tumorigenesis and drug resistance. This study aimed to determine the role of HRR in HCC and identify critical
HRR-related genes that affect tumorigenesis and prognosis. A total of 613 tumor and 252 para-carcinoma tissue samples
were collected from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) to obtain
differentially expressed genes (DEGs). HRR-related genes were assessed using gene enrichment and pathway analyses.
Survival analysis was performed using the Kaplan–Meier method in the Gene Expression Profiling Interactive Analysis portal.
The levels of RAD54L in the HRR pathway were detected by RT-qPCR and western blotting in para-carcinoma and HCC
tissues and in L02 normal human liver cells and Huh7 HCC cells. Immunohistochemistry (IHC) was performed on the clinical
specimens to determine the connection between gene expression and clinical features. Bioinformatics analysis revealed
that the HRR pathway was enriched in HCC tissues. Upregulation of HRR pathway DEGs in HCC tissues was positively
correlated with tumor pathological staging and negatively associated with patient overall survival. RAD54B, RAD54L, and
EME1 genes in the HRR pathway were screened as markers for predicting HCC prognosis. RT-qPCR identified RAD54L
as the most significantly expressed of the three genes. Western blotting and IHC quantitative analyses further demonstrated
that RAD54L protein levels were higher in HCC tissues. IHC analysis of 39 pairs of HCC and para-carcinoma tissue samples
also revealed an association between RAD54L and Edmondson-Steiner grade and the proliferation-related gene Ki67. The
collective findings positively correlate RAD54L in the HRR signaling pathway with HCC staging and implicate RAD54L
as a marker to predict HCC progression.

Keywords  Homologous recombination repair · Hepatocellular carcinoma · RAD54L · Prognosis · Ki67 · Homologous
recombination repair defects

Introduction

Hepatocellular carcinoma (HCC) is a malignant tumor with

high incidence worldwide. The development of HCC is
insidious and difficult to detect at an early stage (Jepsen and
* Qing Zhou West 2021). The incidence and mortality of HCC continue
zhouqingzy@163.com to increase annually worldwide, but the long-term survival
1
and clinical outcomes are not promising (Vogel et  al.
Department of General Surgery, Affiliated Hospital
of Jiangsu University, Zhenjiang, China
2022). Many pathogenic factors, such as viral infections,
2
alcohol, and harmful chemicals can cause DNA damage to
Division of Gastrointestinal Surgery, Department of General
Surgery, The Affiliated Huai’an Hospital of Xuzhou Medical
hepatocytes. Continuous DNA damage to hepatocytes and
University, Huai’an Second People’s Hospital, Huai’an, accumulation of errors in the repair process may lead to
China genetic mutations that cause abnormal cell proliferation. The
3
Department of Anesthesiology, People’s Hospital of Lianshui incidence of HCC increases with genetic mutations and the
County, Huai’an, China severity of cirrhosis. Eventually, hepatocyte proliferation

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becomes uncontrolled, resulting in carcinogenesis (Roos
et al. 2016; Moon et al. 2020).
DNA damage occurs in several ways, including base
modifications, cross-linking of DNA strands, and single-
strand breaks (SSBs) and double-strand breaks (DSBs).
DSBs are considered one of the most severe types of
damage. These breaks are induced by many exogenous
factors, such as genomic instability caused by drugs or
radiation, transcriptional activation induced by R-loops
and topoisomerase, and transformation of accumulated
SSBs inhibited by poly ADP ribose polymerase inhibitors
(Ui et al. 2020; Waterman et al. 2020; Marnef and Legube
2021). In order to prevent the serious consequences of DSB,
homologous recombination repair (HRR) plays a crucial role.
HRR uses the sister chromatid of the defective DNA strand
as a template (Wagener-Ryczek et al. 2021). When the repair
process is out of control, DSBs can lead to genomic instability,
site mutation breakage, chromosomal rearrangements, cell
death, cell transformation, and tumorigenesis. Although
homologous recombination deficiency (HRD) causes
tumorigenesis, the DNA repair function may be elevated in
cancerous cells. Tumor cells undergo frequent DNA strand
breaks due to various factors. However, their enhanced
ability to repair DNA damage enables them to avoid
death. This ability contributes to cancer cell resistance to
chemotherapeutic agents. HRR is also essential in various
solid tumors. Breast cancer type 1 (BRCA1) and BRCA2 are
critical genes in HRR and are the most classical biomarkers
in breast and ovarian cancers (Tutt et al. 2021; Royfman
et al. 2021; Toh and Ngeow 2021; Launonen et al. 2022).
Pan-cancer analysis has revealed that HRD also occurs in
some cancer types other than common HRD-related tumors,
accounting for 15% of all cases, which may be underestimated
owing to insufficient samples with BRCA1/2 deficiencies
(Nguyen et  al. 2020). HRD caused by dysregulation of
HRR genes is clinically relevant and can provide therapeutic
options for clinically refractory tumors. Olaparib, a PARP
inhibitor which extensively suppressed the DNA damage
repair signaling, has been approved by the Food and Drug
Administration as an antineoplastic drug for patients with
metastatic castration-resistant prostate cancer, which progress
after novel endocrine therapy and carry HRR mutations that
include ATM, BRCA1, BRCA2, BRCA1 associated RING
domain 1 (BARD1), RAD51 paralog B (RAD51B), and
RAD54L (Unlu and Kim 2022). HRR-related agents have also
been used in the treatment of hepatocellular carcinoma. High
expression of the HRR gene PARP1 can be detected in HCC
residual tumors after treatment with the common targeted
drug sorafenib. HRR-targeting agent olaparib can effectively
eliminate residual HCC tumors and improve the treatment
efficiency (Yang et al. 2021).
RAD54L, one of the critical genes in HRR, plays an
important role in the DNA repair process. The protein
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Functional & Integrative Genomics (2023) 23:128
RAD54L is a DEAD-like decapping enzyme and a member
of the Swi2/Snf2 family of proteins (Mason et al. 2015).
RAD54L is also known to act in concert with other DNA
repair proteins, such as RAD51, BRCA1, and BRCA2
(Zhang et  al. 2007). Therefore, abnormal function of
RAD54L may lead to dysregulation of DNA double-strand
break repair, which could be a potential value in the diagno-
sis and treatment of HCC.
Much research attention has focused on BRCA1/2, with
less attention to many other genes in the HRR pathway that
may have the same important role as BRCA1/2. In this study,
analyses of HCC data and clinical samples and in  vitro
experiments with HCC cell lines confirm that RAD54L is
elevated in HCC and correlates with tumor development.
Materials and methods
Data collection
Gene expression profiles and clinical information were
obtained from The Cancer Genome Atlas Liver Hepatocel-
lular Carcinoma (TCGA-LIHC) and Liver Cancer-RIKEN-
JP (LIRI-JP) databases. The data from 373 tumor tissues
and 50 para-carcinoma tissues were selected from TCGA
(Gao et al. 2022). Data from 240 tumors and 202 para-car-
cinoma tissues were selected from the International Cancer
Genome Consortium (ICGC) database. For data normaliza-
tion, the reads per kilobase of exon per million reads mapped
(RPKM) normalization method provided on the SangerBox
website (http://​sange​rbox.​com/​Tool) was used.
Cell lines and culture
L02 human liver cell line and Huh7 human HCC cell line
were obtained from the Chinese Academy of Sciences
(Shanghai, China). L02 cells and Huh7 cells were cultured
in Dulbecco’s modified Eagle’s medium (Hyclone) contain-
ing 10% fetal bovine serum (FBS; Biological Industries, Beit
Haemek, Israel). The cells were cultured in a ­CO2 incubator
at 37°C in an atmosphere of 5% C ­ O2. The cells were sub-
cultured when they reached approximately 80% confluence.
Clinical sample collection
Thirty-nine pairs of HCC tumor and para-carcinoma tis-
sue samples were collected from the Affiliated Hospital of
Jiangsu University (Jiangsu, China). The patients had not
received preoperative radiotherapy or chemotherapy. All
human samples were obtained with written informed con-
sent from the patients. Sample collection was approved by
the Ethics Committee of the Affiliated Hospital of Jiangsu
University (approve number: KY2022K0912). Based on
Functional & Integrative Genomics (2023) 23:128 Page 3 of 13  128

postoperative pathology and immunohistochemistry results,
the samples were categorized using the Edmondson-Steiner
grade system by experienced pathologists.
Differential expression of mRNA
The Ensembl ID in the TCGA database was converted to
a gene symbol. To screen differentially expressed genes
(DEGs) between tumor and para-carcinoma tissues in the
TCGA-LIHC and LIRI-JP datasets, data analysis was per-
formed using the “limma” package in the R software. DEGs
from the two different datasets were intersected using VENN
tools (Ritchie et al. 2015).
Functional enrichment and functional pathway
enrichment analyses of HCC samples
To confirm the influence of the HRR pathway on HCC, we
investigated the functions of the DEGs using Gene Ontol-
ogy (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) enrichment analyses. GO analysis was performed
to annotate the genes in terms of biological processes (BP),
molecular functions (MF), and cellular components (CC)
using the DAVID website (https://​david.​ncifc​rf.​gov/), and
demonstrate the molecular and cellular biological functions
of the DEGs. KEGG enrichment analysis was used to screen
HRR-relative DEGs. Gene set enrichment analysis (GSEA)
of TCGA-LIHC data was performed using the Gene Expres-
sion Profiling Interactive Analysis (GSEA) software (version
3.0; https://w
​ ww.g​ seams​ igdb.o​ rg/g​ sea/i​ ndex.j​ sp) (Subrama-
nian et al. 2005).
Kaplan–Meier survival curve analysis
TCGA-LIHC patient data were divided into two groups
based on the median cutoff values of gene expression to
explore the relationship between these DEGs and patient
prognosis. The differences in survival between the high-risk
(n=182) and low-risk (n=182) groups were assessed using
Kaplan–Meier curves. Prognostic analyses were performed
with regard to overall survival (OS). The follow-up period
was 120 months. Correlations between the expression of
crucial DEGs and pathological staging were ascertained
using GEPIA (http://​gepia.​cancer-​pku.​cn/) (Tang et  al.
2017). Pathological diagnoses were determined using the
International Union Against Cancer staging system.
Reverse transcription and quantitative polymerase
chain reaction (RT‑qPCR)
Total RNA was extracted from the liver tissue using TRIzol
reagent (Applygen, Beijing, China) and purified using a Spin
Column RNA Concentration Kit (Sangon Biotech, Shang-
hai, China). Reverse transcription was performed using the
HiScript® II SuperMix (Vazyme, Nanjing, China). Accord-
ing to the kit instructions of AceQ Universal SYBR qPCR
Master Mix (Vazyme), template cDNA, primers, and other
reagents were added to 20 μL of the reaction system in PCR
tubes. Three replicate wells were set up for each sample.
The primer sequences are shown in Table 1. The results
were normalized to those of β-actin and analyzed using the
­2−ΔΔCT method.
Western blot analysis
Tumor and para-carcinoma tissue samples were homog-
enized using a TGrinder 3rd Gen Homogenizer (Tiangen,
Beijing, China). Tissue and cell samples were lysed in RIPA
buffer containing protease inhibitor cocktail (Cell Signaling
Technology, Beverly, MA, USA). Protein samples (20 μg)
were resolved by 12.5% SDS-PAGE. The transmembrane
process was performed at a constant flow of 300 mA for 2 h
under low-temperature conditions. The following antibod-
ies were used: anti-RAD54L (1:1000, ImmunoWay, Plano,
TX, USA), anti-GAPDH (1:5000, Abcam, Cambridge, UK),
and secondary antibody (1:5000, Affinity Biosciences, Cin-
cinnati, OH, USA). The signals were measured using an
enhanced chemiluminescence kit (Abbkine, Wuhan, China).
Immunohistochemistry (IHC) analysis
The analysis was performed according to routine proto-
cols. The sections of tissue were incubated with antibodies
to RAD54L (1:300; ImmunoWay), Ki67 (1:200; Abcam,
Cambridge, UK), and alfa-fetoprotein (AFP; 1:200; Abcam).
Images were captured by optical microscopy and processed
using ImageJ software (NIH, Bethesda, MD, USA).

Table 1  Genes and primers list
for RT-qPCR
Gene symbol Forward primer (5′-3′) Reverse primer (5′-3′)
RAD54B TCC​AGG​TCT​GAA​TGA​AGA​GAT​TAC​ TCT​AGT​ACT​TTC​TTC​ACT​AGG​CAG​
RAD54L GAG​CCC​AGA​GGA​CCT​TGA​TA AAC​CAC​CTT​GTC​TGG​ACA​GC
EME1 CTC​ATC​CCT​GAG​GGC​TAG​AA AGT​TGA​AAG​AGT​GGC​GGG​A
β-actin AGA​GCT​ACG​AGC​TGC​CTG​AC AGC​ACT​GTG​TTG​GCG​TAC​AG

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Statistical analyses
All data are presented as the mean ± SD of three independ-
ent experiments. Comparisons between the two groups were
performed using the paired Student’s t-test or unpaired Stu-
dent’s t-test. Clinical data were analyzed using SPSS version
22.0 (IBM, Armonk, NY, USA). Pearson χ2 test was used
to assess the association between RAD54L immunohisto-
chemical results and clinicopathological parameters of the
patients. The Kruskal-Wallis test was used to evaluate ordi-
nal variables among clinical characteristics. Statistical sig-
nificance was determined using a two-tailed P<0.05. DEGs
were screened using the standard |log2 fold change| >2 and
FDR <0.05.
Results
Identification of DEGs
A flow chart of the study design is shown in Fig.  1. To
investigate whether the HRR pathway was involved in
tumor characteristics of HCC, such as liver tumorigenesis
and drug resistance, DEGs were screened from TCGA-LIHC
Fig. 1  Flow chart of study
design
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Functional & Integrative Genomics (2023) 23:128
and ICGC-LIRI-JP liver cancer databases using the criteria
of |log2 fold change| >2 and FDR <0.05. The screening
revealed a set of 1533 TCGA-LIHC dataset DEGs, including
498 upregulated and 1035 downregulated genes (Fig. 2a)
and 1697 ICGC-LIRI-JP DEGs, including 783 upregulated
and 914 downregulated genes (Fig. 2b). A Venn diagram
visualized 783 overlapping DEGs between the two datasets
(Fig.  2c). A heatmap of the top 20 upregulated and
downregulated (blue) DEGs is shown in Fig. 2d in red and
blue, respectively.
GSEA
To obtain a deeper insight into the biological roles of these
DEGs, top GO annotation (Fig. 3a–c) and KEGG pathway
(Fig.  3d) enrichment analyses were performed. The top
KEGG pathway and GO terms with the highest P values
focused on proliferation-related oncological features, such
as cell replication and cell cycle. KEGG pathway analysis
revealed the enrichment of the HRR pathway in HCC tissues.
To reduce the interference caused by setting the difference
threshold, genomic enrichment analysis (GSEA) was used
to verify the enrichment of the HRR pathway (Fig. 3e). The
results demonstrated the presence of significant enrichment
Functional & Integrative Genomics (2023) 23:128 Page 5 of 13  128
Fig. 2  DEGs between HCC and para-carcinoma tissues screened
using TCGA and ICGC databases. a, b Volcano plot of DEGs from
TCGA and ICGC databases. c Venn diagram analysis for screening
in homologous recombination (P=0.002). Seven DEGs
were identified in this pathway: RAD54B, BLM, RAD51,
RAD54L, XRCC2, XRCC3, and EME1. As shown in Fig. 4,
these DEGs mainly constituted RAD51 paralogs or affected
DSB repair and synthesis-dependent strand annealing
processes.
Prognostic prediction of HRR pathway–related
genes and survival analysis
To better understand these genes, the three most
significant DEGs (RAD54B, EME1, and RAD54L)
the intersection of the two databases. d Heatmap of the top 20 upreg-
ulated (red) and 20 downregulated (blue) genes
that have not been previously reported in HCC stud-
ies were selected from the seven DEGs (Fig.  5a).
The expression levels of these DEGs at different
tumor stages were significantly different (P<0.05;
Fig.  5b). The expression of these DEGs increased
with tumor staging and reached the highest point at
stages II and III. Kaplan–Meier analysis indicated
that the OS rate in the low-risk group of RAD54B,
RAD54L, and EME1 was significantly higher than
that in the high-risk group (Fig.  5c). High expres-
sion of RAD54B, RAD54L, and EME1 was associated
with poor prognosis.
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Fig. 3  GO, KEGG, and GSEA analyses of the critical DEGs. a Biological process (BP). b Molecular function (MF). c Cellular component (CC).
d KEGG pathway analysis findings. e GSEA findings

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Functional & Integrative Genomics (2023) 23:128 Page 7 of 13  128
Fig. 4  KEGG pathway map of the homologous recombination signaling pathway
High expression of RAD54L in HCC cells
To verify these results, we selected the human normal hepat-
ocyte line L02 cells and human hepatoma cell line Huh7
cells for the assay. RT-qPCR was used to analyze changes in
critical genes at the mRNA level (Fig. 6a). Only the mRNA
expression of RAD54L was significantly upregulated, while
the other genes did not show significant differences. West-
ern blotting also showed that the expression of RAD54L in
Huh7 cells was higher than that in L02 cells (Fig. 6b), which
was consistent with the RT-qPCR results.
RAD54L expression is increased in HCC tissues
The results of cellular experiments indicated that RAD54L
might be an essential factor influencing the function of HCC
cells involved in the HRR pathway. To further explore the
clinical implications of RAD54L, surgical specimens were
used to evaluate its expression of RAD54L. Thirty-nine pairs
of HCC tumor and para-carcinoma tissue specimens were
evaluated by pathologists using the Edmondson-Steiner
grading system. There were four stage I patients, 18 stage
II patients, 13 stage III patients, and four stage IV patients.
Based on previous bioinformatics analysis results showing
that RAD54L was significantly elevated in the mid-stage
of HCC, we randomly selected five pairs of fresh HCC
tissue and adjacent non-tumor tissue from the 31 cases of
mid-stage HCC (stage II and stage III). RT-qPCR analyzed
the changes in critical genes in these five pairs of samples
(Fig. 7a). Only the mRNA expression of RAD54L was sig-
nificantly upregulated in HCC tissues compared to their
paired non-tumor tissues. The expressions of other vital
genes did not differ significantly. IHC and western blotting
analyses were performed to further validate the changes in
RAD54L at the protein level (Fig. 7b, c). Ratios of integrated
optical density (IOD) to picture area were used to assess
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Fig. 5  Gene expressions and clinical significance of RAD54B, HCC by tumor stage plots. c Kaplan–Meier analysis of overall sur-
RAD54L, and EME1. a Box plots of the distributions of RAD54B, vival of HCC based on the expression of RAD54B, RAD54L, and
RAD54L, and EME1. b Comparisons among different staging of EME1

the protein expression in the IHC. RAD54L expression was individual tumor samples. Moreover, RAD54L was located
higher in the tumor samples than in the paired para-carci- mainly in the nucleus of hepatocytes, although some were
noma tissues, although some variation existed among the also found in the cytoplasm.

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Functional & Integrative Genomics (2023) 23:128 Page 9 of 13  128
Fig. 6  Relative expression of
RAD54L between L02 and
Huh7 cells. a Relative mRNA
expression of the critical genes
examined by RT-qPCR. b
Relative protein expression of
RAD54L examined by western
blot. All experiments were
repeated three times. *P<0.05,
**P<0.01
Relationship between RAD54L expression
and clinicopathological parameters
Our previous results indicated the possible association with
RAD54L and HCC staging. To further explore the associa-
tion between RAD54L and other clinical features, we sum-
marized the clinical information of the HCC cases (Table 2).
In view of the non-normal distribution of RAD54L expres-
sions, IOD/area=10% was set as a threshold. Patients were
divided into high expression (n=27) and low expression
(n=12) groups according to the IHC results of RAD54L.
RAD54L was associated with the Edmondson-Steiner
grade and the level of the proliferation-related marker Ki67
(Fig. 7d). However, there was no significant relationship
with sex, age, tumor size, condition of hepatitis, AFP, and
microvascular invasion (MVI) (Long et al. 2023). And all
patients do not have hepatitis B and C co-infection. Since
RAD54L was positively correlated with the Edmondson-
Steiner grade, we hypothesized that the HRR pathway is
involved in tumor progression. This hypothesis is consistent
with previous tumor staging data from TCGA-LIHC. We
presently observed that the expression of RAD54L was not
evident in the early stage of the tumor (stage I), similar to
its expression in the cirrhosis region, but was significantly
increased in the mid-stage of HCC (stage II + III). The cur-
rent view is that RAD54L participates in promoting cell
viability, cell proliferation, and tumor progression (Choi
et al. 2017). The tumor proliferation marker Ki67 has been
extensively studied. Based on the trend in Ki67 expression, it
is conceivable that RAD54L might influence tumor behavior
in HCC by promoting cell proliferation.
Discussion
DNA damage results from endogenous and exogenous
sources. The DNA damage response is a process in which
cells respond to threats to their genomic integrity. DNA
damage triggers DNA damage repair (DDR) which blocks
the cell cycle and activates specific repair pathways.
Furthermore, DDR activates the senescent or apoptotic sign-
aling pathway when DNA damage overwhelms its repair
capacity (De Zio et al. 2013; Bruix et al. 2017). DNA repair
is necessary to maintain genomic integrity. Dysregulations
of DDR and checkpoint signaling are relevant to cancer
predisposition and impact tumor resistance to genetically
damaging anticancer therapies (Curtin 2012; Xue et  al.
2021; Sharma et al. 2022). DSBs are the major type of DDR
by HRR. In the HRR process, sister chromatid sequences
act as DNA repair templates (Jiang et al. 2021). However,
DNA repair is an error-prone process when individuals with
HRD prevent HRR from proceeding correctly. Increasing the
expression of relevant repair proteins in HRR can also cause
this condition, which disrupts the repair process and leads to
genomic instability (Richardson et al. 2004).
By screening for relevant repair proteins in HRR, we
discovered elevated RAD54L expression in HCC. RAD54L
is similar to Saccharomyces cerevisiae RAD54, a protein
involved in DNA repair. This protein helps repair DSBs
via the HRR pathway. When RAD54L binds to double-
stranded DNA (dsDNA), it alters DNA topology and stimu-
lates homologous DNA pairing. RAD54L has a main motor
domain that allows ATP-dependent tracking of dsDNA, but
does not separate strands (Gorbalenya and Koonin 1993).
Moreover, RAD54L is involved in unloading the RAD51
filaments from D-loops and allowing recombination to pro-
ceed more efficiently (Heyer et al. 2006). RAD54L is one
of many genes involved in the HRR pathway that influences
cancer progression. BRCA1 and BRCA2 are the most criti-
cal genes in HRR and are the most classical biomarkers in
breast and ovarian cancers, which has been well known
for decades (Nicoletto et al. 2001). But in recent years, an
improved understanding of the complex network of HRR
pathways has been associated with various tumors. RAD51
and RAD54 are critical HR factors for preventing tumori-
genesis due to gene mutations (Choi et al. 2017). RAD51
is an essential biomarker for patients with HCC; its expres-
sion is significantly associated with the presence of immune
infiltration (Xu et al. 2021). RAD52 showed similar results.
Huh7 cells overexpressing RAD52 display significantly
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Fig. 7  Expression of RAD54L and Ki67 in HCC tumor and para-car-
cinoma tissues. a Relative mRNA expression of RAD54L of tumor
and para-carcinoma tissues. b Protein expression of RAD54L in five
pairs of representative HCC tissue samples. c Immunohistochemistry
increased proliferation and migration. In addition, some
proteins interacting with RAD52 are upregulated and are
involved in the biological processes of HCC (Li et al. 2019).
For instance, RAD54B promotes the progression of HCC
by modulating the Wnt/β-catenin signaling pathway, and
DNA amplification leads to elevated levels of RAD54B
(Feng et al. 2021). Knockdown of RAD54L in HepG2 HCC
cells can significantly inhibit cell proliferation (Wang et al.
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Functional & Integrative Genomics (2023) 23:128
analyses of RAD54L in representative tumor and para-carcinoma tis-
sues. d Ki67 expression in RAD54L low expression group (n=12)
and high expression group (n=27). All experiments were repeated
three times. *P<0.05, **P<0.01
2021). A prediction model of immune checkpoint inhibi-
tors indicates an increased chance of survival after immu-
notherapy in patients with high expression levels of EME1,
RAD51AP1, and RAD54L (Li et al. 2022).
Unfortunately, few clinically available drugs are avail-
able for the treatment of HCC, which remains a global
threat to human health. In 2007, the Food and Drug Admin-
istration approved sorafenib for the first-line treatment of
Functional & Integrative Genomics (2023) 23:128 Page 11 of 13  128

Table 2  Relationship between Total RAD54L expression P-value

RAD54L expression level and
clinical features in HCC patients High expression Low expression
group (n=27) group (n=12)

Ki67 0.020294
 <25% 13 5 8
  ≥25% and <50% 10 9 1
  ≥50% and <75% 9 7 2
 ≥75% 7 6 1
The Edmondson-Steiner grade 0.034630
 I 4 1 3
 II 18 12 6
 III 13 10 3
 IV 4 4 0
MVI 0.492224
 M0 20 13 7
 M1 6 4 2
 M2 13 10 3
Gender 0.643615
 Male 31 22 9
 Female 8 5 3
Age 0.200037
 <65 20 12 8
 ≥65 19 15 4
AFP 0.284307
 Positive 21 13 8
 Negative 18 14 4
Tumor size (cm) 0.323436
 <5 24 18 6
 ≥5 15 9 6
Types of hepatitis 0.538457
 Non-hepatitis 7 5 2
  Hepatitis B 26 19 7
  Hepatitis C 6 3 3

HCC. However, patients with advanced HCC treated with
sorafenib usually do not survive for more than 1 year (Luo
et al. 2021). A phase 3 study conducted at 152 sites in 21
countries between 2013 and 2015 demonstrated that patients
with progressive HCC treated with sorafenib had a median
survival of 7.8 months (Bruix et  al. 2017). OS among
patients with HCC remains low despite the application of
such first-line drugs. Drug resistance is one reason. Poly
ADP ribose polymerase (PARP) inhibitors are the first clini-
cally available drugs. They act by targeting the DNA damage
response and cause synthetic lethality through DNA repair
gene mutations. These drugs have been approved for treating
BRCA-mutated ovarian and breast cancer (Slade 2020). The
PARP inhibitor olaparib inhibits DDR signaling, which can
enhance the role of sorafenib in eliminating residual cancer
cells in HCC and improving therapeutic efficiency (Yang
et al. 2021). Inducing HRD is a viable strategy. Depletion of
bromodomain containing 9 (BRD9) in ovarian cancer limits
the interaction between RAD54 and RAD51, which can sen-
sitize cancer cells to olaparib and enhance the therapeutic
effect (Zhou et al. 2020).
In this study, TCGA and ICGC databases were used
to search for DEGs. RAD54L was identified as the most
significant gene involved in the HRR pathway. Analysis of
clinical data from TCGA-LIHC indicated that RAD54L
expression increased with tumor staging and was associ-
ated with poor OS. RAD54L was highly expressed in HCC
at both cellular and tissue levels. IHC analysis of clinical
samples also revealed an association of RAD54L with the
Edmondson-Steiner grade and Ki67. Since RAD54L plays
a role in the G2-M transition, we attributed the elevation
of Ki67 to the impact of HR on the cell cycle (Choi et al.
2017). In fact, there were large inter-individual differences
in RAD54L in HCC tissue. Some cirrhotic tissues adjacent

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to the carcinoma also showed elevated RAD54L, with a
degree of positivity similar to early-stage HCC. Therefore,
we preferred to use RAD54L to determine the progres-
sion of HCC in a single individual, rather than in different
individuals.
RAD54L is expressed differently in different types of
tumors. It is upregulated in acute myeloid leukemia and
thyroid cancer, and downregulated in lung, gastric, and
colon cancer. There are two possible mechanisms under-
lying the role of RAD54L in HCC. On the one hand, the
increase in RAD54L may indicate that the enhancement of
HRR protects cancer cells from dying from drug-induced
DSBs. In contrast, RAD54L expression may be related to
HRD, which affects genomic stability and causes tumori-
genesis. According to our experimental results, elevated
RAD54L expression was mainly concentrated in the mid-
stage of HCC, while the tumor tissue in the early stage
approximated the cirrhotic region. Based on these observa-
tions, we speculate that the first mechanism is a plausible
mechanism of action of RAD54L. Due to rapid prolifera-
tion or drug interactions, HCC cells have reduced genetic
stability. However, high expression of RAD54L may coun-
teract this instability by enhancing HRR. HCC patients
with high RAD54L expression exhibit increased resistance
to chemotherapy, which results in decreased OS.
In conclusion, we found that RAD54L, a representative
gene of HRR in HCC, had elevated expression in tumors
and positively correlated with HCC staging and level of
Ki67. RAD54L was also associated with poor prognosis of
the patients and could be used as a marker to predict HCC
progression. This study has several limitations. These
include an insufficient clinical sample size, which may not
adequately reflect RAD54L distribution, and the absence
of knockdown experiments to demonstrate the regulatory
mechanism of the HRR pathway in HCC by RAD54L.
In the future, we hope to increase the sample size and
explore how RAD54L affects specific functions of tumor
development. Further research has significant implications
for improving the mechanism of HRR in the progression
of HCC. It will contribute to the diagnosis of HCC and
determine the prognosis of patients. Furthermore, it will
guide chemotherapy and immunotherapy in some patients.
Acknowledgements  We acknowledge the Central Laboratory of the
Affiliated Hospital of Jiangsu University for providing the experimental
facilities.
Funding  The study was funded by the Excellent Talents Fund of
Xuzhou Medical University (No. XYFY202244) and the Doc-
toral Initiation Fund of Affiliated Hospital of Jiangsu University
(jdfyRC2021009).
Declarations 
Ethics approval  This study was approved by the Ethics Committee of
the Affiliated Hospital of Jiangsu University and the ethical approval
number is KY2022K0912. All patients provided written informed
consent to participate in the study. Written informed consent was
obtained from each individual(s) for the publication of potentially
identifiable images or data included in this article.
Conflict of interest  The authors declare no competing interests.
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