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Clinics of Oncology
Research Article ISSN: 2640-1037 Volume 6
IRF5 Promotes the Progression of Hepatocellular Carcinoma and is Regulated by
Trim35
Ying F1
*, Zhihui L2
*, Bangzhong L2
, Nan L3
, Mingzhen Y2
and Ping W2
1
Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China
2
Department of Rehabilitation, Zhongshan Hospital, Fudan University, Shanghai 200032, China
3
Department of Hepatic Surgery I (Ward I), Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, 201805, China
*
Corresponding author:
Ying F and Zhihui L
Department of Rehabilitation,
Zhongshan Hospital, Fudan University, 180
Fenglin Rd, Shanghai 200032, PR China.
Tel: +86-021-64041990,
E-mail: wang.ping8@zs-hospital.sh.cn.
Received: 30 May 2022
Accepted: 07 Jun 2022
Published: 13 Jun 2022
J Short Name: COO
Copyright:
©2022 Ying F and Zhihui L. This is an open access article
distributed under the terms of the Creative Commons At-
tribution License, which permits unrestricted use, distribu-
tion, and build upon your work non-commercially.
Citation:
Ying F and Zhihui L, IRF5 Promotes the Progression of He-
patocellular Carcinoma and is Regulated by Trim35. Clin
Onco. 2022; 6(7): 1-8
Keywords:
IRF5; HCC; TRIM35; Cell growth
clinicsofoncology.com 1
1. Abstract
The IRF family of proteins involves in the tumor progression.
However, but the functions of IRF5 in the tumorigenesis are large-
ly unknown. Here, IRF5 was found to be up-regulated in hepa-
tocellular carcinoma (HCC). Interfering with IRF5 inhibited the
growth and tumorigenic ability of HCC cells. When studying the
molecular mechanism, it was found that TRIM35 interacted with
IRF5, promoting the ubiquitination and degradation of IRF5. In
the clinical specimens of HCC, TRIM35 was negatively correlated
with the expression of IRF5. These observations reveal the onco-
genic function of IRF5 in the progression of HCC, suggesting that
IRF5 is a promising target for the therapy of HCC.
2. Introduction
HCC is frequently observed worldwide [1, 2]. The five-year surviv-
al rate remains low, even with comprehensive treatment involving
chemotherapy, radio-therapy and immunotherapy [3-5]. Further
research on the molecular mechanism of hepatocarcinogenesis is
of great significance in identifying a new target for HCC treatment.
IRF (interferon regulator factor) refers to a group of multifunc-
tional transcription factors which can specifically recognize and
interact with the interferon-stimulated responsive element (ISRE)
in the IFN promoters and the interferon-stimulated gene (ISG) [6].
This activates the relevant signal and regulates the expression of
the target gene [7,8]. The result is that IRF should be able to play
a biological role in antiviral infection, congenital immunity, adap-
tive immunoregulation, cell proliferation, cell apoptosis and cell
homeostasis [9-11].
IRF is often abnormally expressed in tumors. The expression of
IRF1 is down-regulated in colorectal carcinoma (CRC) [12,13].
The overexpression of IRF1 in CRC cells inhibits cell growth, pro-
motes apoptosis and causes radiotherapeutic sensitivity [12,14,15].
More interestingly, the members of the IRF family regulate each
other during the tumorigenesis process. In the esophageal squa-
mous cell carcinoma (ESCC) samples, IRF1 was down-regulated
while IRF2 was overexpressed; IRF2 inhibited IRF1 from entering
the nucleus, and they cooperated to promote the progress of the
esophageal carcinoma [16]. In innate immune, IRF1 promotes in-
nate immune by activating IRF3 [17]. The role of IRF5 in tumors
is poorly understood. Previous study shows that IRF5 can promote
the growth of thyroid carcinoma cells [18]. IRF5 can inhibit HCV
replication and HCV-related hepatocarcinogenesis [19], but in the
HPA (Human Protein Atlas) database, the abundance of IRF5 is
inversely correlated with the outcome of HCC. Therefore, eluci-
dating the role of IRF5 in HCC from a cell-biological perspective
is of great significance.
TRIM35 is an E3 ligase and a suppressor in many types of tumors
[20-23]. In HCC, multiple microRNAs promote the proliferation
of HCC cells by targeting TRIM35 [21]. TRIM35 can interact with
clinicsofoncology.com 2
Volume 6 Issue 7 -2022 Research Article
PKM2 to inhibit the glycolysis and tumorigenic ability of HCC
cells [23,24]. The expression of TRIM35 and PKM2 in HCC pre-
dicts the prognosis for HCC patients [23,24]. To further elucidat-
ing the functions of TRIM35 in HCC, the proteins interacting with
TRIM35 were predicted using the STRING database. IRF5 is one
of the potential proteins to interact with TRIM35. In the present
work, the abundance of IRF5 in HCC was studied. In addition, the
role of IRF5 in HCC cells and the regulating effect of TRIM35 on
IRF5 were also examined.
3. Materials and Methods
3.1. Cell culture
The HepG2, Huh7 and QGY cells were obtained from the Cell
Bank, Chinese Academy of Sciences. The cells were cultured in
DMEM medium with 10% fetal bovine serum (FBS) and antibiot-
ics (100 U/mL of penicillin and 100 µg/ml of streptomycin). Cells
were in a constant temperature incubator (5% CO2
, 37℃). Lipo-
fectamine 8000 was used for the cell transfection.
3.2. Clinical samples
The clinical samples were collected from the Shanghai Eastern
Hepatobiliary Surgery Hospital after the study was approved by
their Ethics Committee. All of the patients have signed the consent
form.
3.3. Immunohistochemistry (IHC)
The array of HCC tissues was provided by the Shanghai Eastern
Hepatobiliary Surgery Hospital. Dewaxing, rehydration and anti-
gen recovery in the 100°C EDTA solution were done. Then, the
activity of endogenous peroxidase was blocked. Before being in-
cubated with the IRF5 antibody (Sigma, HPA046700, 1:100) at
4°C overnight, the tissue sections were washed with PBS. On the
next day, before being incubated with secondary antibody at room
temperature for 1h, the tissue sections were washed with PBS. The
signal was developed using 3,3,0-diaminobenzidine (DAB), and
the nucleus were stained using hematoxylin. Both the staining in-
tensity and protein expression level were automatically scored by
the Vectra 2.0 system.
3.4. qPCR
RNA was extracted with TRIzol (Invitrogen) and reversely tran-
scribed into cDNA using the PrimeScript™ RT kit (Takara) ac-
cording to the instructions. qPCR was performed using The SYBR
Green kit and CFX96 real-time fluorescent quantitative PCR de-
tection system (Bio-Rad, Richmond, CA, USA). The levels of the
transcripts were calculate using the 2−ΔΔ
Ct method. The primer
sequences for IRF5 were: Forward primer, 5’-tgtgcccttaacaagag-
ccg-3’; Reverse primer, 5’-ctctgtgaggctcaggcttg-3’.
3.5. Western blot
The cells were cleaned twice with PBS, and lysed on ice with the
RIPA Lysis buffer, which contains the protease inhibitor and phos-
phatase inhibitor. The supernatant was collected after the cell ly-
sis was centrifuged, and the protein concentration was quantified
using the BCA protein detection kit. An equal amount of protein
was taken for the SDS-PAGE analysis. After separation, the pro-
tein was transferred onto the PVDF membrane and incubated with
a specific primary antibody at 4°C overnight. Then, the membrane
was incubated with the HRP-conjugated secondary antibody for
1-2h. The immune signal was detected with a chemiluminescence
reagent (Milliwell, WBKLS0050), and analyzed with Image Lab
software. The primary antibodies used in this experiment were as
follows: IRF5 (Sigma, HPA046700, 1:100), tubulin (Santa Cruz
Biotechnolog, sc-5286, 1:4000), Flag (Sigma, F9291; 1:3,000),
HA(Sigma, H3663, 1:2000), TRIM35 (Sigma, HPA019647,
1:100), Ubiquitin (proteintech, 10201-2-AP, 1:1000).
3.6. CCK8
Cells were seeded into a 96-well plate, and each well contained
1000 cells. 18 hours later, cells were incubated with a fresh me-
dium containing 10% CCK8 for 2 hours. Then, OD450 nm was
evaluated. The measurement was performed on day 1, 3, 5 and 7,
respectively.
3.7. Edu assay
Cells were seeded into a 96-well plate, and each well contained
1000 cells. 18 hours later, the proliferation was evaluated with
the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, C10310-1).
Photos were taken by the fluorescent microscope for analysis.
3.8. Soft agar assay
When the confluence reached 60-70%, the cells were digested, and
a cell suspension was prepared. Lower-layer gel (20% FBS, 40%
2×RPMI1640 (Basal Medium Eagle) 40% 1.25% Agar) was pre-
pared. 400μL of gel was added to each well in the 24-well plate.
The 24-well plate with the gel was placed in an incubator at 37°C.
The gel was solidified for later use. Upper-layer gel (25% FBS,
37.5% 2×RPMI1640, 37.5% 1% Agar, 0.8% 2mM L-glutamine)
was prepared and mixed evenly with the cell suspension. 400μl
(containing 1000 cells) was added to each well and placed in a
constant temperature incubator (37°C, 5% CO2
) for two weeks. 5
fields were selected randomly under the microscope for colonies
counting.
3.9. Immunoprecipitation
In order to detect the interaction between exogenous IRF5 (Flag-
IRF5) and TRIM35 (HA-TRIM35), the Flag-IRF5 and HA-
TRIM35 plasmids were transferred into 293T. 48h after transfec-
tion, the cells were lysed with an IP lysis buffer (50mM Tris-HCl,
pH 8.0, 150mM NaCl, 1 %NP-40, protease and phosphatase in-
hibitor), with the supernatant collected. The beads coupling an-
ti-Flag antibody (Sigma, A2220) were added to the supernatant
for incubation overnight at 4°C. The next day, the beads were
washed 3 times in wash buffer (50mM Tris-HCl (pH 8.0), 150mM
NaCl, 1%NP-40), with 1×loading buffer added, heated at 100°C
for 5min, and then the supernatant was taken for western blot
clinicsofoncology.com 3
Volume 6 Issue 7 -2022 Research Article
analysis. To detect whether there was any interaction between the
endogenously expressed IRF5 and TRIM35 in HCC cells, an IP
lysis buffer containing protease and a phosphatase inhibitor was
sued for lysis. An equal amount of protein was taken, and 0.25μg
of IRF5 antibody was added for incubation overnight at 4°C. The
next day, 40μL of Protein A/G beads (bimake, B23202) was added
for another incubation overnight at 4°C. The beads were washed
for 3 times with the wash buffer, and then 1×loading buffer was
added for western blotting analysis.
3.10. Data statistics
Data was expressed as mean ± SD. The data were analyzed using
the t test. A survival curve was plotted by the Kaplan-Meier meth-
od, while the log-rank test was used for analysis. GraphPad Prism
8 and SPSS 17.0 were used for statistical analysis.
3. Results
3.1. IRF5 is overexpressed in HCC
To study the abundance of IRF5, the transcripts of IRF5 in HCC
tissues and matched adjacent non-cancerous tissues were mea-
sured. It was found that the levels of IRF5 transcripts in the cancer
tissues were higher than that in normal tissues (Figure 1A). When
the levels of IRF5 mRNA in the tumors was compared with that of
IRF5 in the paired adjacent tissues, higher levels of IRF5 mRNAin
the tumor tissues were observed (Figure 1B). After this, the protein
levels of IRF5 were measured. In the tested HCC tissues, the level
of IRF5 protein was relatively high (Figure 1C). Moreover, the
results were verified using immunohistochemistry staining (Fig-
ure 1D). In the Human Protein Atlas database, IRF5 expression is
inversely correlated with the outcome of HCC (Figure 1E). These
observations indicate that IRF5 might be important for the HCC
development.
Figure 1: IRF5 expression is upregulated in hepatocellular carcinoma.
(A) The levels of IRF5 mRNA in 30 cancer samples (cancer) and 30 adjacent tissues (normal) were examined by qPCR. 18S, internal control. The Ct values
(18S-IRF5) were plotted on the ordinate and analyzed. (B) The mRNA levels of IRF5 in paired cancer tissues (cancer) and non-cancerous tissues (normal)
were analyzed. (C) The levels of IRF5 protein in 5 cancerous tissues (C) and paired non-cancerous tissues (N) were examined by Western blotting. (D) The
levels of IRF5 protein in the tumor samples and adjacent non-cancerous tissue was examined by IHC. (E) Mining the HPA database and analyzing the cor-
relation between IRF5 transcripts and the HCC outcome.
clinicsofoncology.com 4
Volume 6 Issue 7 -2022 Research Article
3.2. IRF5 promotes the growth and colony formation of HCC
cells
To study the IRF5’s functions, exogenous IRF5 was forced ex-
pressed in Huh7 and HepG2 cells, allowing the functions of IRF5
in cell growth to be detected (Figure 2A). In the CCK8 assay, the
promotion of the growth was observed upon the overexpression of
IRF5 in the HCC cells (Figure 2B). After this, the anchorage-in-
dependent growth of HCC cells was evaluated. It shows that the
expression of IRF5 promoted the anchorage-independent growth
(Figure 2C-D). IRF5 in Huh7 and QGY cells was then interfered
with two shRNA sequences (Figure 3A). It was found that the
growth of HCC cells in the liquid medium were inhibited after the
expression of IRF5 was down-regulated (Figure 3B). Moreover,
down-regulation of IRF5 impaired the growth of HCC cells on the
soft agar (Figure 3C-D). Cell growth is the result of apoptosis and
proliferation. The effect of the expression of IRF5 on the prolifer-
ation was detected by means of an EdU assay. The results showed
that silencing IRF5 impaired the proliferation of HCC cells (Fig-
ure 3E-F).
Figure 2: IRF5 promotes the growth of hepatocellular carcinoma cells.
(A) The exogenous Flag-IRF5 was forced expressed in Huh7 and HepG2 cells, and examined using western blot. (B) Cell growth was evaluated with
CCK8. (C-D) The anchorage-independent growth of HCC cells was examined after the overexpression of IRF5. The colonies were counted and statis-
tically analyzed.
*, P<0.05; **, P<0.01.
clinicsofoncology.com 5
Volume 6 Issue 7 -2022 Research Article
Figure 3: Silencing IRF5 inhibits the proliferation.
(A) The IRF5 knockdown efficiency was examined with Western blot. (B) Cell growth was analyzed by CCK8 assay. (C-D) The anchorage-independent
growth of HCC cells was examined after the knockdown of IRF5. The colonies were counted and statistically analyzed. (E-F) Cell proliferation was
analyzed with EdU assay. EdU positively stained cells were counted and statistically analyzed. *, P<0.05; **, P<0.01.
3.3. TRIM35 regulates the stability of the IRF5 protein
In order to study the regulation of IRF5 in HCC, the proteins in-
teracting with IRF5 were searched with the help of STRING da-
tabase. The results showed that IRF5 interacted with the E3 ubiq-
uitin ligase TRIM35 (Figure 4A). The binding between IRF5 and
TRIM35 was evaluated by means of a co-immunoprecipitation
assay (CO-IP). In Huh7 and HepG2 cells, the exogenously ex-
pressed HA-TRIM35 interacted with the exogenously expressed
Flag-IRF5 (Figure 4B). The CO-IP results also showed that the
endogenously expressed IRF5 and TRIM35 in Huh7 cells had
formed a complex (Figure 4C). Moreover, TRIM35 promoted the
ubiquitination and degradation of IRF5 (Figure 4D).
Figure 4: TRIM35 promotes the degradation of IRF5 in HCC cells.
(A) Mining the STRING database for the binding proteins of TRIM35. (B) The binding of ectopically expressed HA-TRIM35 and Flag-IRF5 was
examined using immunoprecipitation assay in Huh7 cells. (C) The binding of endogenously expressed TRIM35 and IRF5 was examined using immu-
noprecipitation assay in Huh7 cells. (D) The levels of the ubiquitinated IRF5 by TRIM35 were elevated.
clinicsofoncology.com 6
Volume 6 Issue 7 -2022 Research Article
3.4. TRIM35 is down-regulated in HCC and negatively cor-
related with the expression of IRF5
The expression of TRIM35 in HCC was analyzed using a publicly
available database. The cBioportal database shows that TRIM35
is deleted in HCC (Figure 5A). The HPA database shows that the
expression of TRIM35 is positively correlated with the HCC out-
come (Figure 5B). The levels of TRIM35 mRNA remained lower
in cancerous tissues (Figure 5C). The level of IRF5 protein and
TRIM35 protein in 20 cases of HCC was then measured by means
of immunocytochemistry (IHC). The negative correlation between
their expression was observed (Figure 5D). The level of IRF5 pro-
tein was high in the HCC tissue with low-expressed TRIM35 (Fig-
ure 5D).
Figure 5: The expression of TRIM35 and IRF5 was negatively correlated in HCC.
(A) Mining the cBioportal database for the mutation, amplification and deletion of TRIM35 in HCC. (B) Mining the HPA database to analyze the cor-
relation between TRIM35 mRNA level and survival. (C) The levels of TRIM35 mRNA were evaluated by qPCR. 18S, internal control. The Ct values
(18S-IRF5) were plotted on the ordinate and analyzed. (D) The correlation between the expression of IRF5 and TRIM35 in the clinical HCC tissues
was analyzed. The protein levels of IRF5 and TRIM35 in the 20 HCC tissues were examined using IHC and scored.
4. Discussion
It has been reported that IRF5 inhibits the replication of HCV,
suggesting that it may also inhibit HCV-related HCC progression
[19]. But in the HPA database, IRF5 is inversely correlated with
the outcome of HCC patients. Therefore, further evidence is need-
ed to clearly support the oncogenic roles of IRF5 in HCC. It was
seen that the levels of IRF5 mRNA and protein were up-regulat-
ed in HCC, and that IRF5 expression accelerated the growth and
colony formation of HCC cells. TRIM35 was low-expressed in
the HCC specimen, and it promoted the degradation of IRF5 by
binding to IRF5; in the clinical tissues, IRF5 expression was re-
versely correlated with TRIM35 expression. These data indicate
that IRF5 is essential for the HCC progression. The most import-
ant finding from this study is that IRF5 promotes cell growth. The
IRF family of proteins, such as IRF1 and IRF2, have been proven
to have completely opposite effects on the growth of ESCC [16].
IRF8 promotes the proliferation of acute myelocytic leukemia
(AML) cells [25]. This shows that the IRF protein family have a
regulating effect on the growth of tumor cells. In addition, the IRF
family interferes with tumor progression by reshaping the tumor
immunity microenvironment. For example, the GM/CSF/IRF5
pathway enhances antitumor immunity by activating the Type 1
T-cell response [26]. This indicates that IRF proteins can regulate
tumorigenesis via different mechanisms. This study reveals the
ubiquitination of IRF5 by TRIM35. Up to date, IRF5 is rarely to
be post-transcriptionally modified. IRF5 can be acetylated by his-
tone acetyltransferase (HAT)[27]. The ubiquitination of IRF5 can
be catalyzed by pellino-1, which helps M1 macrophages regulate
obesity [28]. The results of this study show that TRIM35 mediates
the degradation of IRF5. Coincidentally, many of the existing stud-
ies show that TRIM35 can also regulate the interferon signaling
pathways. The findings of this study further support the previous
clinicsofoncology.com 7
Volume 6 Issue 7 -2022 Research Article
conclusions. In conclusion, this work reveals the promoting func-
tion of IRF5 in hepatocarcinogenesis, and the regulation of IRF5
by TRIM35, suggesting IRF5 as a promising targe.
5. Acknowledgement
Not applicable.
6. Disclosure of Conflict of Interests
No potential conflict of interest was reported by the author(s).
7. Funding
This work was supported by foundation of Shanghai Institute of
Rehabilitation with Integrated Western and Chinese Traditional
Medicine (082)
8. Data Availability
The data supporting the results reported in the manuscript can be
acquired by the corresponding author.
9. Ethics Statement
All patients participated in this study have signed informed con-
sent, and this research was approved by the Ethics Committee of
Shanghai Eastern Hepatobiliary Surgery Hospital.
10. Author Contribution
Fang Ying and Lu Zhihui collected the clinical tissues, performed
the IHC staining, and studied the molecular mechanisms; Liu
Bangzhong performed the CCK8 assay; Yang Mingzhen per-
formed the soft agar assay; Li Nan provided the clinical tissues;
Wang Ping conceived and designed the experiments, analyzed the
data, reviewed drafts of the paper, and approved the final draft.
11. Consent for Publication
All authors agreed to the publication of the manuscript.
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IRF5 Promotes the Progression of Hepatocellular Carcinoma and is Regulated by Trim35

  • 1. Clinics of Oncology Research Article ISSN: 2640-1037 Volume 6 IRF5 Promotes the Progression of Hepatocellular Carcinoma and is Regulated by Trim35 Ying F1 *, Zhihui L2 *, Bangzhong L2 , Nan L3 , Mingzhen Y2 and Ping W2 1 Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China 2 Department of Rehabilitation, Zhongshan Hospital, Fudan University, Shanghai 200032, China 3 Department of Hepatic Surgery I (Ward I), Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai, 201805, China * Corresponding author: Ying F and Zhihui L Department of Rehabilitation, Zhongshan Hospital, Fudan University, 180 Fenglin Rd, Shanghai 200032, PR China. Tel: +86-021-64041990, E-mail: wang.ping8@zs-hospital.sh.cn. Received: 30 May 2022 Accepted: 07 Jun 2022 Published: 13 Jun 2022 J Short Name: COO Copyright: ©2022 Ying F and Zhihui L. This is an open access article distributed under the terms of the Creative Commons At- tribution License, which permits unrestricted use, distribu- tion, and build upon your work non-commercially. Citation: Ying F and Zhihui L, IRF5 Promotes the Progression of He- patocellular Carcinoma and is Regulated by Trim35. Clin Onco. 2022; 6(7): 1-8 Keywords: IRF5; HCC; TRIM35; Cell growth clinicsofoncology.com 1 1. Abstract The IRF family of proteins involves in the tumor progression. However, but the functions of IRF5 in the tumorigenesis are large- ly unknown. Here, IRF5 was found to be up-regulated in hepa- tocellular carcinoma (HCC). Interfering with IRF5 inhibited the growth and tumorigenic ability of HCC cells. When studying the molecular mechanism, it was found that TRIM35 interacted with IRF5, promoting the ubiquitination and degradation of IRF5. In the clinical specimens of HCC, TRIM35 was negatively correlated with the expression of IRF5. These observations reveal the onco- genic function of IRF5 in the progression of HCC, suggesting that IRF5 is a promising target for the therapy of HCC. 2. Introduction HCC is frequently observed worldwide [1, 2]. The five-year surviv- al rate remains low, even with comprehensive treatment involving chemotherapy, radio-therapy and immunotherapy [3-5]. Further research on the molecular mechanism of hepatocarcinogenesis is of great significance in identifying a new target for HCC treatment. IRF (interferon regulator factor) refers to a group of multifunc- tional transcription factors which can specifically recognize and interact with the interferon-stimulated responsive element (ISRE) in the IFN promoters and the interferon-stimulated gene (ISG) [6]. This activates the relevant signal and regulates the expression of the target gene [7,8]. The result is that IRF should be able to play a biological role in antiviral infection, congenital immunity, adap- tive immunoregulation, cell proliferation, cell apoptosis and cell homeostasis [9-11]. IRF is often abnormally expressed in tumors. The expression of IRF1 is down-regulated in colorectal carcinoma (CRC) [12,13]. The overexpression of IRF1 in CRC cells inhibits cell growth, pro- motes apoptosis and causes radiotherapeutic sensitivity [12,14,15]. More interestingly, the members of the IRF family regulate each other during the tumorigenesis process. In the esophageal squa- mous cell carcinoma (ESCC) samples, IRF1 was down-regulated while IRF2 was overexpressed; IRF2 inhibited IRF1 from entering the nucleus, and they cooperated to promote the progress of the esophageal carcinoma [16]. In innate immune, IRF1 promotes in- nate immune by activating IRF3 [17]. The role of IRF5 in tumors is poorly understood. Previous study shows that IRF5 can promote the growth of thyroid carcinoma cells [18]. IRF5 can inhibit HCV replication and HCV-related hepatocarcinogenesis [19], but in the HPA (Human Protein Atlas) database, the abundance of IRF5 is inversely correlated with the outcome of HCC. Therefore, eluci- dating the role of IRF5 in HCC from a cell-biological perspective is of great significance. TRIM35 is an E3 ligase and a suppressor in many types of tumors [20-23]. In HCC, multiple microRNAs promote the proliferation of HCC cells by targeting TRIM35 [21]. TRIM35 can interact with
  • 2. clinicsofoncology.com 2 Volume 6 Issue 7 -2022 Research Article PKM2 to inhibit the glycolysis and tumorigenic ability of HCC cells [23,24]. The expression of TRIM35 and PKM2 in HCC pre- dicts the prognosis for HCC patients [23,24]. To further elucidat- ing the functions of TRIM35 in HCC, the proteins interacting with TRIM35 were predicted using the STRING database. IRF5 is one of the potential proteins to interact with TRIM35. In the present work, the abundance of IRF5 in HCC was studied. In addition, the role of IRF5 in HCC cells and the regulating effect of TRIM35 on IRF5 were also examined. 3. Materials and Methods 3.1. Cell culture The HepG2, Huh7 and QGY cells were obtained from the Cell Bank, Chinese Academy of Sciences. The cells were cultured in DMEM medium with 10% fetal bovine serum (FBS) and antibiot- ics (100 U/mL of penicillin and 100 µg/ml of streptomycin). Cells were in a constant temperature incubator (5% CO2 , 37℃). Lipo- fectamine 8000 was used for the cell transfection. 3.2. Clinical samples The clinical samples were collected from the Shanghai Eastern Hepatobiliary Surgery Hospital after the study was approved by their Ethics Committee. All of the patients have signed the consent form. 3.3. Immunohistochemistry (IHC) The array of HCC tissues was provided by the Shanghai Eastern Hepatobiliary Surgery Hospital. Dewaxing, rehydration and anti- gen recovery in the 100°C EDTA solution were done. Then, the activity of endogenous peroxidase was blocked. Before being in- cubated with the IRF5 antibody (Sigma, HPA046700, 1:100) at 4°C overnight, the tissue sections were washed with PBS. On the next day, before being incubated with secondary antibody at room temperature for 1h, the tissue sections were washed with PBS. The signal was developed using 3,3,0-diaminobenzidine (DAB), and the nucleus were stained using hematoxylin. Both the staining in- tensity and protein expression level were automatically scored by the Vectra 2.0 system. 3.4. qPCR RNA was extracted with TRIzol (Invitrogen) and reversely tran- scribed into cDNA using the PrimeScript™ RT kit (Takara) ac- cording to the instructions. qPCR was performed using The SYBR Green kit and CFX96 real-time fluorescent quantitative PCR de- tection system (Bio-Rad, Richmond, CA, USA). The levels of the transcripts were calculate using the 2−ΔΔ Ct method. The primer sequences for IRF5 were: Forward primer, 5’-tgtgcccttaacaagag- ccg-3’; Reverse primer, 5’-ctctgtgaggctcaggcttg-3’. 3.5. Western blot The cells were cleaned twice with PBS, and lysed on ice with the RIPA Lysis buffer, which contains the protease inhibitor and phos- phatase inhibitor. The supernatant was collected after the cell ly- sis was centrifuged, and the protein concentration was quantified using the BCA protein detection kit. An equal amount of protein was taken for the SDS-PAGE analysis. After separation, the pro- tein was transferred onto the PVDF membrane and incubated with a specific primary antibody at 4°C overnight. Then, the membrane was incubated with the HRP-conjugated secondary antibody for 1-2h. The immune signal was detected with a chemiluminescence reagent (Milliwell, WBKLS0050), and analyzed with Image Lab software. The primary antibodies used in this experiment were as follows: IRF5 (Sigma, HPA046700, 1:100), tubulin (Santa Cruz Biotechnolog, sc-5286, 1:4000), Flag (Sigma, F9291; 1:3,000), HA(Sigma, H3663, 1:2000), TRIM35 (Sigma, HPA019647, 1:100), Ubiquitin (proteintech, 10201-2-AP, 1:1000). 3.6. CCK8 Cells were seeded into a 96-well plate, and each well contained 1000 cells. 18 hours later, cells were incubated with a fresh me- dium containing 10% CCK8 for 2 hours. Then, OD450 nm was evaluated. The measurement was performed on day 1, 3, 5 and 7, respectively. 3.7. Edu assay Cells were seeded into a 96-well plate, and each well contained 1000 cells. 18 hours later, the proliferation was evaluated with the Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, C10310-1). Photos were taken by the fluorescent microscope for analysis. 3.8. Soft agar assay When the confluence reached 60-70%, the cells were digested, and a cell suspension was prepared. Lower-layer gel (20% FBS, 40% 2×RPMI1640 (Basal Medium Eagle) 40% 1.25% Agar) was pre- pared. 400μL of gel was added to each well in the 24-well plate. The 24-well plate with the gel was placed in an incubator at 37°C. The gel was solidified for later use. Upper-layer gel (25% FBS, 37.5% 2×RPMI1640, 37.5% 1% Agar, 0.8% 2mM L-glutamine) was prepared and mixed evenly with the cell suspension. 400μl (containing 1000 cells) was added to each well and placed in a constant temperature incubator (37°C, 5% CO2 ) for two weeks. 5 fields were selected randomly under the microscope for colonies counting. 3.9. Immunoprecipitation In order to detect the interaction between exogenous IRF5 (Flag- IRF5) and TRIM35 (HA-TRIM35), the Flag-IRF5 and HA- TRIM35 plasmids were transferred into 293T. 48h after transfec- tion, the cells were lysed with an IP lysis buffer (50mM Tris-HCl, pH 8.0, 150mM NaCl, 1 %NP-40, protease and phosphatase in- hibitor), with the supernatant collected. The beads coupling an- ti-Flag antibody (Sigma, A2220) were added to the supernatant for incubation overnight at 4°C. The next day, the beads were washed 3 times in wash buffer (50mM Tris-HCl (pH 8.0), 150mM NaCl, 1%NP-40), with 1×loading buffer added, heated at 100°C for 5min, and then the supernatant was taken for western blot
  • 3. clinicsofoncology.com 3 Volume 6 Issue 7 -2022 Research Article analysis. To detect whether there was any interaction between the endogenously expressed IRF5 and TRIM35 in HCC cells, an IP lysis buffer containing protease and a phosphatase inhibitor was sued for lysis. An equal amount of protein was taken, and 0.25μg of IRF5 antibody was added for incubation overnight at 4°C. The next day, 40μL of Protein A/G beads (bimake, B23202) was added for another incubation overnight at 4°C. The beads were washed for 3 times with the wash buffer, and then 1×loading buffer was added for western blotting analysis. 3.10. Data statistics Data was expressed as mean ± SD. The data were analyzed using the t test. A survival curve was plotted by the Kaplan-Meier meth- od, while the log-rank test was used for analysis. GraphPad Prism 8 and SPSS 17.0 were used for statistical analysis. 3. Results 3.1. IRF5 is overexpressed in HCC To study the abundance of IRF5, the transcripts of IRF5 in HCC tissues and matched adjacent non-cancerous tissues were mea- sured. It was found that the levels of IRF5 transcripts in the cancer tissues were higher than that in normal tissues (Figure 1A). When the levels of IRF5 mRNA in the tumors was compared with that of IRF5 in the paired adjacent tissues, higher levels of IRF5 mRNAin the tumor tissues were observed (Figure 1B). After this, the protein levels of IRF5 were measured. In the tested HCC tissues, the level of IRF5 protein was relatively high (Figure 1C). Moreover, the results were verified using immunohistochemistry staining (Fig- ure 1D). In the Human Protein Atlas database, IRF5 expression is inversely correlated with the outcome of HCC (Figure 1E). These observations indicate that IRF5 might be important for the HCC development. Figure 1: IRF5 expression is upregulated in hepatocellular carcinoma. (A) The levels of IRF5 mRNA in 30 cancer samples (cancer) and 30 adjacent tissues (normal) were examined by qPCR. 18S, internal control. The Ct values (18S-IRF5) were plotted on the ordinate and analyzed. (B) The mRNA levels of IRF5 in paired cancer tissues (cancer) and non-cancerous tissues (normal) were analyzed. (C) The levels of IRF5 protein in 5 cancerous tissues (C) and paired non-cancerous tissues (N) were examined by Western blotting. (D) The levels of IRF5 protein in the tumor samples and adjacent non-cancerous tissue was examined by IHC. (E) Mining the HPA database and analyzing the cor- relation between IRF5 transcripts and the HCC outcome.
  • 4. clinicsofoncology.com 4 Volume 6 Issue 7 -2022 Research Article 3.2. IRF5 promotes the growth and colony formation of HCC cells To study the IRF5’s functions, exogenous IRF5 was forced ex- pressed in Huh7 and HepG2 cells, allowing the functions of IRF5 in cell growth to be detected (Figure 2A). In the CCK8 assay, the promotion of the growth was observed upon the overexpression of IRF5 in the HCC cells (Figure 2B). After this, the anchorage-in- dependent growth of HCC cells was evaluated. It shows that the expression of IRF5 promoted the anchorage-independent growth (Figure 2C-D). IRF5 in Huh7 and QGY cells was then interfered with two shRNA sequences (Figure 3A). It was found that the growth of HCC cells in the liquid medium were inhibited after the expression of IRF5 was down-regulated (Figure 3B). Moreover, down-regulation of IRF5 impaired the growth of HCC cells on the soft agar (Figure 3C-D). Cell growth is the result of apoptosis and proliferation. The effect of the expression of IRF5 on the prolifer- ation was detected by means of an EdU assay. The results showed that silencing IRF5 impaired the proliferation of HCC cells (Fig- ure 3E-F). Figure 2: IRF5 promotes the growth of hepatocellular carcinoma cells. (A) The exogenous Flag-IRF5 was forced expressed in Huh7 and HepG2 cells, and examined using western blot. (B) Cell growth was evaluated with CCK8. (C-D) The anchorage-independent growth of HCC cells was examined after the overexpression of IRF5. The colonies were counted and statis- tically analyzed. *, P<0.05; **, P<0.01.
  • 5. clinicsofoncology.com 5 Volume 6 Issue 7 -2022 Research Article Figure 3: Silencing IRF5 inhibits the proliferation. (A) The IRF5 knockdown efficiency was examined with Western blot. (B) Cell growth was analyzed by CCK8 assay. (C-D) The anchorage-independent growth of HCC cells was examined after the knockdown of IRF5. The colonies were counted and statistically analyzed. (E-F) Cell proliferation was analyzed with EdU assay. EdU positively stained cells were counted and statistically analyzed. *, P<0.05; **, P<0.01. 3.3. TRIM35 regulates the stability of the IRF5 protein In order to study the regulation of IRF5 in HCC, the proteins in- teracting with IRF5 were searched with the help of STRING da- tabase. The results showed that IRF5 interacted with the E3 ubiq- uitin ligase TRIM35 (Figure 4A). The binding between IRF5 and TRIM35 was evaluated by means of a co-immunoprecipitation assay (CO-IP). In Huh7 and HepG2 cells, the exogenously ex- pressed HA-TRIM35 interacted with the exogenously expressed Flag-IRF5 (Figure 4B). The CO-IP results also showed that the endogenously expressed IRF5 and TRIM35 in Huh7 cells had formed a complex (Figure 4C). Moreover, TRIM35 promoted the ubiquitination and degradation of IRF5 (Figure 4D). Figure 4: TRIM35 promotes the degradation of IRF5 in HCC cells. (A) Mining the STRING database for the binding proteins of TRIM35. (B) The binding of ectopically expressed HA-TRIM35 and Flag-IRF5 was examined using immunoprecipitation assay in Huh7 cells. (C) The binding of endogenously expressed TRIM35 and IRF5 was examined using immu- noprecipitation assay in Huh7 cells. (D) The levels of the ubiquitinated IRF5 by TRIM35 were elevated.
  • 6. clinicsofoncology.com 6 Volume 6 Issue 7 -2022 Research Article 3.4. TRIM35 is down-regulated in HCC and negatively cor- related with the expression of IRF5 The expression of TRIM35 in HCC was analyzed using a publicly available database. The cBioportal database shows that TRIM35 is deleted in HCC (Figure 5A). The HPA database shows that the expression of TRIM35 is positively correlated with the HCC out- come (Figure 5B). The levels of TRIM35 mRNA remained lower in cancerous tissues (Figure 5C). The level of IRF5 protein and TRIM35 protein in 20 cases of HCC was then measured by means of immunocytochemistry (IHC). The negative correlation between their expression was observed (Figure 5D). The level of IRF5 pro- tein was high in the HCC tissue with low-expressed TRIM35 (Fig- ure 5D). Figure 5: The expression of TRIM35 and IRF5 was negatively correlated in HCC. (A) Mining the cBioportal database for the mutation, amplification and deletion of TRIM35 in HCC. (B) Mining the HPA database to analyze the cor- relation between TRIM35 mRNA level and survival. (C) The levels of TRIM35 mRNA were evaluated by qPCR. 18S, internal control. The Ct values (18S-IRF5) were plotted on the ordinate and analyzed. (D) The correlation between the expression of IRF5 and TRIM35 in the clinical HCC tissues was analyzed. The protein levels of IRF5 and TRIM35 in the 20 HCC tissues were examined using IHC and scored. 4. Discussion It has been reported that IRF5 inhibits the replication of HCV, suggesting that it may also inhibit HCV-related HCC progression [19]. But in the HPA database, IRF5 is inversely correlated with the outcome of HCC patients. Therefore, further evidence is need- ed to clearly support the oncogenic roles of IRF5 in HCC. It was seen that the levels of IRF5 mRNA and protein were up-regulat- ed in HCC, and that IRF5 expression accelerated the growth and colony formation of HCC cells. TRIM35 was low-expressed in the HCC specimen, and it promoted the degradation of IRF5 by binding to IRF5; in the clinical tissues, IRF5 expression was re- versely correlated with TRIM35 expression. These data indicate that IRF5 is essential for the HCC progression. The most import- ant finding from this study is that IRF5 promotes cell growth. The IRF family of proteins, such as IRF1 and IRF2, have been proven to have completely opposite effects on the growth of ESCC [16]. IRF8 promotes the proliferation of acute myelocytic leukemia (AML) cells [25]. This shows that the IRF protein family have a regulating effect on the growth of tumor cells. In addition, the IRF family interferes with tumor progression by reshaping the tumor immunity microenvironment. For example, the GM/CSF/IRF5 pathway enhances antitumor immunity by activating the Type 1 T-cell response [26]. This indicates that IRF proteins can regulate tumorigenesis via different mechanisms. This study reveals the ubiquitination of IRF5 by TRIM35. Up to date, IRF5 is rarely to be post-transcriptionally modified. IRF5 can be acetylated by his- tone acetyltransferase (HAT)[27]. The ubiquitination of IRF5 can be catalyzed by pellino-1, which helps M1 macrophages regulate obesity [28]. The results of this study show that TRIM35 mediates the degradation of IRF5. Coincidentally, many of the existing stud- ies show that TRIM35 can also regulate the interferon signaling pathways. The findings of this study further support the previous
  • 7. clinicsofoncology.com 7 Volume 6 Issue 7 -2022 Research Article conclusions. In conclusion, this work reveals the promoting func- tion of IRF5 in hepatocarcinogenesis, and the regulation of IRF5 by TRIM35, suggesting IRF5 as a promising targe. 5. Acknowledgement Not applicable. 6. Disclosure of Conflict of Interests No potential conflict of interest was reported by the author(s). 7. Funding This work was supported by foundation of Shanghai Institute of Rehabilitation with Integrated Western and Chinese Traditional Medicine (082) 8. Data Availability The data supporting the results reported in the manuscript can be acquired by the corresponding author. 9. Ethics Statement All patients participated in this study have signed informed con- sent, and this research was approved by the Ethics Committee of Shanghai Eastern Hepatobiliary Surgery Hospital. 10. Author Contribution Fang Ying and Lu Zhihui collected the clinical tissues, performed the IHC staining, and studied the molecular mechanisms; Liu Bangzhong performed the CCK8 assay; Yang Mingzhen per- formed the soft agar assay; Li Nan provided the clinical tissues; Wang Ping conceived and designed the experiments, analyzed the data, reviewed drafts of the paper, and approved the final draft. 11. Consent for Publication All authors agreed to the publication of the manuscript. References 1. Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Nature reviews Disease primers. 2021; 7(1): 6. 2. Craig AJ, Von Felden J, Garcia-Lezana T, Sarcognato S, Villanueva A. Tumour evolution in hepatocellular carcinoma. Nature reviews Gastroenterology & hepatology. 2020; 17(3): 139-152. 3. Dickson I. Liver cancer: A new drug target for hepatocellular car- cinoma. Nature reviews Gastroenterology & hepatology. 2016; 13: 375. 4. Duffy AG, Greten TF. 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