(2023) 24:14
Wang et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-022-00450-5

BMC Molecular andCell Biology

Open Access

RESEARCHARNTL2 upregulation of ACOT7 promotes
NSCLC cell proliferation through inhibitionof apoptosis and ferroptosis
Tao Wang1, Kai Wang1, Xu Zhu1 and Nan Chen2*

Abstract
Background Recent studies have reported that the circadian transcription factor aryl hydrocarbon receptor nuclear
translocator like 2 (ARNTL2) promotes the metastatic progression of lung adenocarcinoma. However, the molecularmechanisms of ARNTL2 in non-small cell lung cancer (NSCLC) cell growth and proliferation remain to be explored.
Methods The expression of ARNTL2 and acyl-CoA thioesterase 7 (ACOT7) in lung cancer patients was analyzed basedon TCGA database. Gain-of-function of ARNTL2 and ACOT7 was conducted by transfecting the cells with plasmids or
lentivirus. Knockdown assay was carried out by siRNAs. Western blot and qRT-PCR were performed to check the protein and mRNA expression. Dual luciferase and ChIP-qPCR assay was applied to check the interaction of ARNTL2 on
ACOT7’s promoter sequence. Triglyceride level, MDA production, the activity of casapase 3 to caspase 7, and lipid ROS
were measured by indicated assay kit. Cellular function was detected by CCK8, colony formation and flow cytometry
analysis of cell death and cell cycle.
Results We demonstrated that ARNTL2 upregulation of ACOT7 was critical for NSCLC cell growth and proliferation.
Firstly, overexpression of ARNTL2 conferred the poor prognosis of LUAD patients and supported the proliferationof NSCLC cells. Based on molecular experiments, we showed that ARNTL2 potentiated the transcription activity of
ACOT7 gene via direct binding to ACOT7’s promoter sequence. ACOT7 high expression was correlated with the worse
prognosis of LUAD patients. Gain-of-function and loss-of-function experiments revealed that AOCT7 contributed to
NSCLC cell growth and proliferation. ACOT7 regulated the apoptosis and ferroptosis of NSCLC cells, while exhibitedno effect on cell cycle progression. ACOT7 overexpression also potentiated fatty acid synthesis and suppressed lipid
peroxidation. Lastly, we showed that ARNTL2 knockdown and overexpression inhibited and promoted the cellulartriglyceride production and subsequent cell proliferation, which could be reversed by ACOT7 overexpression andknockdown.
Conclusion Our study illustrated the oncogenic function of ARNTL2/ACOT7 axis in the development of NSCLC.
Targeting ARNTL2 or ACOT7 might be promising therapeutic strategies for NSCLC patients with highly expressed
ARNTL2.
Keywords NSCLC, ARNTL2, ACOT7*Correspondence:
Nan Chenchennan3311@yeah.net; chennan331@hotmail.com
1
Affiliated Hospital of Guizhou Medical University, Guiyang, China
2
The Third Affiliated Hospital of Kunming Medical University, YunnanCancer Hospital, Kunming, China

Introduction
Lung cancer is the leading cause of cancer-related deathamong all the malignancies worldwide [1]. With approximately 2 million new cases diagnosed, lung cancers are
histologically classified into two subtypes, including small© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

cell lung carcinoma (SCLC) and non-small cell lung cancer (NSCLC), the latter accounting for 85% of the cases[2]. Benefitting from the intensive genomics, transcriptomics and proteomics studies, promising therapeutictargets and effective drugs have been developed againstto this deadly malignancy [3]. Although the targeted
therapy, including EGFR inhibitors [4], or the immunotherapy, such as PD-1 antibodies [5], have gain prominent success on the treatment of lung cancers, only somepatients show durable response to these drugs. Identifying novel drivers may help us develop specific therapeutic
options which can benefit for the remaining patients.
Recent studies have shown that dysregulation of circadian clock plays an essential role during the progression of various cancers [6]. The most well-known genes
regulating circadian rhythms include Clock, Period 1,
Period 2, Period 3, casein kinase Iε (CKIε), cryptochrome
1 (Cry1), chryptochrome 2 (Cry2), Bmal1, as well as its
paralog Bmal2 (ARNTL2). Comparing with other corecircadian rhythms genes, the relevance of ARNTL2 incarcinogenesis is less reported. Some evidences haveshown that ARNTL2 acts as oncogene in cancers. For
instance, upregulation of ARNTL2 is a worse prognosis
factor for clear cell renal cell carcinoma [7]. Depletion of
ARNTL2 suppresses the malignant growth of colorectal
carcinoma through inhibiting the expression of SMOC2
and the activity of PI3K/AKT signaling pathway [8]. In
addition, oncogenic function of ARNTL2 is reported inother cancer types, including pancreatic ductal adenocarcinoma [9], breast cancer [10], as well as lung cancer [11]. Based on these studies, it seems that ARNTL2
is pivotal for the metastasis and invasion of malignant
tumors. However, more studies should be conducted to
explore the role and underlying mechnisms of ARNTL2
in the growth and proliferation of cancer cells, such aslung cancer.
In this study, we investigated the function of ARNTL2
and its potential mechanism in NSCLC by analyzing
TCGA public database, performing gain-of-function andloss-of-function experiments, as well as dual luciferase
reporter and ChIP-qPCR assay. Our results may provide
novel evidences on the role and molecular mechanismsof ARNTL2 in NSCLC.

Results
Overexpression of ARNTL2 contributes to the progressionof NSCLC patients and the proliferation of NSCLC cells

TCGA is a public database which could be applied toanalyze the clinical relevance of various genes in cancer
patients. We firstly used TCGA to examine the expressionof ARNTL2 in lung adenocarcinoma (LUAD) and lungsquamous cell carcinoma (LUSC) patients. The results
showed that ARNTL2 was significantly upregulated inPage 2 of 12

LUAD and LUSC tissues compared with normal tissues(Fig. 1A and supplementary Fig. 1A, p < 0.05). We further analyzed the correlation between ARNTL2’s expression and the patients’ survival. The patients were divided
into ARNTL2 high and low expression group. ARNTL2
high expression predicted shorter overall (p = 0.00011)
and disease free (p = 0.019) survival compared with
ARNTL2 low expression in LUAD patients (Fig. 1B),
suggesting that ARNTL2 is a worse prognosis factor for
LUAD patients. However, ARNTL2 expression was not
significantly correlated with the overall (p = 0.67) anddisease free (p = 0.25) survival of LUSC patients (Supplementary Fig. 1B). To illustrate the effect of ARNTL2
on proliferation, we overexpressed and knocked down
ARNTL2 in NSCLC cells. Firstly, we checked the expression of ARNTL2 in normal lung cells Beas-2B and in
three NSCLC cell lines, including A549, H1299 andH1975. The results showed that ARNTL2 expression
was higher in A549, H1299 and H1975 cells as comparedwith Beas-2B cells (Supplementary Fig. 2). Among the
NSCLC cells, ARNTL2 was highest in A549 cells and was
relatively lower in H1299 cells (Supplementary Fig. 2).
Therefore, we overexpressed ARNTL2 in H1299 cells andknocked down ARNTL2 in A549 cells to study the roleof ARNTL2 in NSCLC. Overexpression of ARNTL2 bylentivirus dramatically accelerated the proliferation andcolony formation of H1299 cells (Fig. 1C-1E, p < 0.01). By
contrast, knockdown of ARNTL2 by two siRNAs significantly suppressed the viability of A549 cells (Fig. 1F-1H,
p < 0.01). These results suggest that ARNTL2 functions as
a worse prognosis biomarker and an oncogenic protein in
NSCLC.
ARNTL2 potentiates the transcription activity of ACOT7
via direct interaction on its promoter sequence

Since ARNTL2 is a transcription factor, there must be
downstream effectors which are responsible for thetumor promoting function of ARNTL2 in NSCLC. Circadian has been shown to play an essential role in regulating cellular metabolism [12]. As the central regulatorof circadian, whether ARNTL2 regulates cellular metabolism, such as lipid metabolism, should be investigated.
We focused on acyl-CoA thioesterase (ACOT) family
because the members of this family exhibited important role in regulating fatty acid production. Based on
RT-qPCR and immunoblotting results, we showed that
ARNTL2 overexpression promoted the mRNA and protein expression of ACOT7 (Fig. 2A, p < 0.01). Inverse
results were found in A549 cells after knocking down
ARNTL2 (Fig. 2B, p < 0.05). Dual luciferase reporter assay
demonstrated that the luciferase activity of ACOT7 promoter was increased and decreased in NSCLC cells after
ectopically expressing and downregulating ARNTL2,

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

Page 3 of 12

Fig. 1 Overexpression of ARNTL2 contributes to the progression of NSCLC patients and the proliferation of NSCLC cells. A The mRNA expression of
ARNTL2 was analyzed in LUAD (n = 483) and normal (n = 347) tissues based on the TCGA database p < 0.05. B Overall and disease-free survival was
analyzed for LUAD patients who were divided into ARNTL2 high and low expression group. n = 335 per group. p = 0.00011 and p = 0.018. C RT-qPCR
analysis of ARNTL2 in H1299 cells transfected with Ctrl and ARNTL2 overexpressing lentivirus. **p < 0.01. D and E Cell proliferation was determined
by CCK8 (D) and colony formation (E) assay in H1299 cells transfected with Ctrl and ARNTL2 overexpressing lentivirus. **p < 0.01. F RT-qPCR analysis
of ARNTL2 in A549 cells transfected with siNC, siARNTL2#1 and siARNTL2#2. **p < 0.01. G and H Cell proliferation was determined by CCK8 (G) andcolony formation (H) assay in A549 cells transfected with siNC, siARNTL2#1 and siARNTL2#2. **p < 0.01

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

Page 4 of 12

Fig. 2 ARNTL2 potentiates the transcription activity of ACOT7 via direct interaction to its promoter sequence. A RT-qPCR and immunoblotting
analysis of ARNTL2 in H1299 cells transfected with Ctrl and ACOT7 overexpressing lentivirus. Quantification of immunoblotting results waspresented. **p < 0.01. B RT-qPCR and immunoblotting analysis of ACOT7 in A549 cells transfected with siNC, siARNTL2#1 and siARNTL2#2.
Quantification of immunoblotting results was presented. *p < 0.05. C Dual luciferase activity of ACOT7 promoter was assessed in H1299 cells with
ARNTL2 overexpressiong and in A549 cells with ARNTL2 knockdown. *p < 0.05. D Immunoblotting analysis of ARNTL2 in H1299 and A549 cells
transfected with pCDNA3.1-Flag (Flag) and pCDNA3.1-ARNTL2-Flag (ARNTL2-Flag). Chip-qPCR was performed to determine the binding of ARNTL2
to the promoter sequence of ACOT7. ***p < 0.001. E Sperman correlation between ARNTL2 and ACOT7 in LUAD patients based on TCGA database.
p < 0.0001. F Overall survival was analyzed for LUAD patients who were divided into ACOT7 high and low expression group. n = 335 per group.
p = 0.00096. The blots were cut prior to hybridisation with antibodies during immunoblotting experiments

respectively (Fig. 2C, p < 0.05). To explore the interaction
between ARNTL2 and the promoter sequence of ACOT7
in H1299 and A549 cells, we transfected the cells with
pCDNA3.1-ARNTL2-Flag vectors and subjected the cells
to Chip-qPCR experiments. Western blot results showedthat ARNTL2 was overexpressed in H1299 and A549
cells transfected with pCDNA3.1-ARNTL2-Flag vectors(Fig. 2D). Chip-qPCR results showed that ARNTL2
directly bound to the promoter sequence of ACOT7(Fig. 2D, p < 0.001). These results suggest that ARNTL2
positively regulates the expression of ACOT7 throughdirect interaction on its promoter. We further analyzed
the relationship between ARNTL2 and ACOT7, as wellas the prognosis value of ACOT7 in LUAD patients. The

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

results showed that ARNTL2 was positively correlatedwith ACOT7 in LUAD patients (Fig. 2E, p < 0.0001). In
addition, ACOT7 high expression predicted poor prognosis of LUAD patients (Fig. 2F, p = 0.00096). Taken
together, ARNTL2 upregulation of ACOT7 confers
worse prognosis of LUAD patients.
ACOT7 promotes the growth and proliferation of NSCLC
cells

To study the role of ACOT7 in NSCLC cell growth, we
checked the expression of ACOT7 in normal lung cells
Beas-2B and in NSCLC cells, including A549, H1975 andH1299. RT-qPCR and immunoblotting results showedthat ACOT7 expression was higher in the NSCLC cells
compared with Beas-2B cells (Fig. 3A). Because ACOT7
was highest in A549 cells, we knocked down ACOT7
in A549 cells by transfecting the cells with siRNAs(Fig. 3B). We found that knockdown of ACOT7 obviously

Page 5 of 12

suppressed the proliferation and colony formation of
A549 cells (Fig. 3C and 3D). By contrast, we overexpressed ACOT7 in H1299 cells (Fig. 3E). As expected,
ectopic expression of ACOT7 promoted the proliferationand growth of H1299 cells (Fig. 3F and G). Collectively,
ACOT7 acts as an oncogenic protein in NSCLC cells.
Next, we analyzed whether ACOT7 regulated cell cycle
by staining the cells with PI and analyzed on flow cytometry. We showed that ACOT7 had no significant effect on
the cell cycle progression of NSCLC cells (Supplementary Fig. 3).
ACOT7 suppresses apoptosis and ferroptosis

Next, we performed PI/Annexin V staining to examine which cell death type was regulated by ACOT7.
As shown by flow cytometry analysis, we found that
ACOT7 downregulation dramatically enhanced thepercentage of Annexin ­V+PI+ cells (Fig. 4A). On theFig. 3 ACOT7 promotes the growth and proliferation of NSCLC cells. A RT-qPCR and immunoblotting analysis of ACOT7 in Beas-2B, A549, H1975
and H1299 cells. *p < 0.05. **p < 0.01. B RT-qPCR and immunoblotting analysis of ACOT7 in siNC, siACOT7#1 and siACOT7#2 A549 cells. **p < 0.01.
C and D Cell proliferation was tested by CCK8 (C) and colony formation (D) assay in siNC, siACOT7#1 and siACOT7#2 A549 cells. **p < 0.01. E
RT-qPCR and immunoblotting analysis of ACOT7 in H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus. **p < 0.01. F and G
Cell proliferation was tested by CCK8 (F) and colony formation (G) assay in H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus.
*p < 0.05. **p < 0.01. The blots were cut prior to hybridisation with antibodies during immunoblotting experiments

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

contrary, ACOT7 overexpression suppressed the percentage of Annexin V­ +PI+ cells (Fig. 4B). In addition, theactivity of caspase 3 to caspase 7 was negatively regulated by ACOT7 (Supplementary Fig. 4). These results
suggest that ACOT7 suppresses the apoptosis of NSCLC
cells. Since the Annexin V­ −PI+ cells were also regulated
by ACOT7 knockdown and overexpression (Fig. 4A andPage 6 of 12B), we predicted that ACOT7 might influence other cell
death type, such as ferroptosis. To dissect this question,
we stained the cells with C-11 BODIPY and subjected
them to lipid ROS detection on flow cytometer. The
results showed that ACOT7 overexpression suppressed,
while ACOT7 knockdown enhanced the lipid ROS in
NSCLC cells (Fig. 4C and D). Furthermore, we treatedFig. 4 ACOT7 suppresses apoptosis and ferroptosis. A siNC, siACOT7#1 and siACOT7#2 A549 cells were stained with PI and Annexin V and thensubjected to flow cytometry analysis. ­A+P−, Annexin ­V+PI−; ­A+P+, Annexin ­V+PI+; ­A−P+, Annexin ­V−PI+. *p < 0.05. B H1299 cells transfected with
Ctrl and ACOT7 overexpression lentivirus were stained with PI and Annexin V and then subjected to flow cytometry analysis. A­ +P−, Annexin­V+PI−; ­A+P+, Annexin ­V+PI+; ­A−P+, Annexin ­V−PI+. *p < 0.05. C and D Lipid peroxidation was examined by using BODIPY 581/591C11 in H1299
cells transfected with Lenti-Ctrl and Lenti-ACOT7, and in A549 cells transfected with siNC, siACOT7#1 and siACOT7#2. *p < 0.05. **p < 0.01. E siNC,
siACOT7#1 and siACOT7#2 A549 cells were treated with DMSO, ferrostatin-1 (Ferr-1, 2 uM), Z-VAD-FMK (Z-VAD, 8 ug/ml) and Ferr-1 + Z-VAD and cell
viability was tested by trypan blue staining. *p < 0.05. **p < 0.01. n.s., not significant. F H1299 cells transfected with Ctrl and ACOT7 overexpression
lentivirus were treated with DMSO, erastin (5 uM), apoptosis activator 2 (AA2, 4 uM) and erastin + AA2 and cell viability was tested by trypan blue
staining. *p < 0.05. **p < 0.01

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

siNC, siACOT7#1 and siACOT7#2 A549 cells with ferroptosis inhibitor Ferr-1, apoptosis inhibitor Z-VAD
or their combination. We showed that either Ferr-1 or
Z-VAD partly reversed the cell death induced by ACOT7
knockdown, and their combination could completely
reverse the phenotype (Fig. 4E). Then we treated Ctrl
and ACOT7 overexpressing H1299 cells with ferroptosis
inducer erastin, apoptosis inducer AA2 or their combination. The results showed that ACOT7 overexpression
significantly rescued the cell death induced by erastin,
AA2 and their combination (Fig. 4F). Taken together,
ACOT7 suppresses both apoptosis and ferroptosis in
NSCLC cells.
ACOT7 promotes the production of monounsaturated fatty
acids and lipid peroxidation

ACOT7 is an important regulator of fatty acid metabolism. Dysregulated fatty acid synthesis and lipid peroxidation play essential roles in cell ferroptosis. Thus, wetested whether ACOT7 regulated fatty acid synthesis
and lipid peroxidation. The results showed that ACOT7
knockdown enhanced malondialdehyde (MDA) production, triglyceride abundance, and the synthesis ofoleic acid and palmitoleic acid (Fig. 5A-C). By contrast,

Page 7 of 12

ACOT7 overexpression suppressed MDA production,
triglyceride abundance, and the synthesis of oleic acidand palmitoleic acid (Fig. 5D-F). These results suggestthat ACOT7 play important role in regulating monounsaturated fatty acid metabolism.
ACOT7 acts as a pivotal contributor for the proliferationof NSCLC cells with ARNTL2 overexpression

Lastly, we tested whether ACOT7 upregulation contributed the oncogenic function of ARNTL2 in
NSCLC cells. To address this question, we overexpressed ACOT7 in A549 cells transfected with siNC or
siARNTL2 and knocked down ACOT7 in H1299 cells
transfected with Lenti-Ctrl or Lenti-ARNTL2. Firstly, weobserved that ACOT7 overexpression promoted, while
ACOT7 knockdown suppressed the triglyceride production and proliferation ability of NSCLC cells transfected with control lentivirus or siRNAs (Fig. 6AD). In
addition, we showed that ACOT7 overexpression also
increased the production of triglyceride in ARNTL2
knockdown cells (Fig. 6A). Opposite results wereobserved in H1299 cells with ARNTL2 overexpression and ACOT7 knockdown (Fig. 6B). Importantly,
ACOT7 overexpression rescued the cell proliferation ofFig. 5 ACOT7 promotes the production of oleic acid and lipid peroxidation. A and B MDA (A) and triglyceride (B) abundance was tested byindicated assay kit in siNC, siACOT7#1 and siACOT7#2 A549 cells. **p < 0.01. C The level of palmitic acid, palmitoleic acid, stearic acid, and oleic acidwas measured in siNC, siACOT7#1 and siACOT7#2 A549 cells. *p < 0.05. D and E MDA (D) and triglyceride (E) abundance was tested by indicatedassay kit in H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus. *p < 0.05. **p < 0.01. F The level of palmitic acid, palmitoleic acid,stearic acid, and oleic acid was measured in H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus. *p < 0.05

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

Page 8 of 12

Fig. 6 ACOT7 acts as a pivotal contributor for the proliferation of NSCLC cells with ARNTL2 overexpression. A A549 cells transfected with siNC,
siNC + ACOT7 lentivirus, siARNTL2 + Ctrl lentivirus and siARNTL2 + ACOT7 lentivirus subjected to RT-qPCR analysis of ACOT7 and detection of
triglyceride. *p < 0.05. **p < 0.01. B H1299 cells transfected with Ctrl lentivirus, Ctrl lentivirus + siACOT7, ARNTL2 lentivirus + siNC and ARNTL2
lentivirus + siACOT7 were subjected to RT-qPCR analysis of ACOT7 and detection of triglyceride. *p < 0.05. **p < 0.01. ***p < 0.001. C A549 cells
described in A were subjected to CCK-8 analysis of cell proliferation. **p < 0.01. D H1299 cells described in B were subjected to CCK-8 analysis of cellproliferation. *p < 0.05. **p < 0.01

A549 cells which was suppressed by ARNTL2 knockdown (Fig. 6C). On the contrary, ACOT7 knockdown
suppressed the proliferation of H1299 cells which waspromoted by ARNTL2 overexpression (Fig. 6D). In
summary, ARNTL2 promotes NSCLC cell proliferation
through upregulation of ACOT7.

Discussion
During the past decades, a wide range of studies haveshown that dysregulations of circadian rhythms genes,
including Clock, Period 1, Period 2 and Period 3, andBmal1, play essential role in the development of various cancers [13]. Comparing to these proteins, theWang et al. BMC Molecular and Cell Biology(2023) 24:14

involvement and molecular mechanisms of another circadian rhythms gene, named Bmal2 (ARNTL2), was lessclear in the development of malignant tumors. Basedon recent studies, the significance of ARNTL2 in carcinogenesis has been addressed. ARNTL2 upregulation
promotes pancreatic ductal adenocarcinoma development via potentiation of TGF-β signaling pathway [9].
ARNTL2 is identified as a susceptibility gene for themetastasis of breast cancer with estrogen receptor-negative characteristic [10]. In addition, overexpression of
ARNTL2 contributes to the metastatic ability of lungcancer through regulating pro-metastatic secretome [11].
These studies suggest that ARNTL2 exhibits importantfunctions during carcinogenesis and metastasis. In thisstudy, we illustrated that ARNTL2 was overexpressed in
LUAD and LUSC patients. Overexpression of ARNTL2
conferred poor prognosis of LUAD, but not LUSC
patients. Based on gain-of-function and loss-of-function
experiments, we demonstrated that ARNTL2 promoted
NSCLC cell proliferation and growth. Consistent withprevious studies, our study showed that ARNTL2 overexpression was critical to maintain the function of NSCLC
cells.
Since ARNTL2 is a transcription factor, the downstream effectors which are essential for the oncogenic
function of ARNTL2 should be investigated. Increasing
evidence has reported that dysregulation of ARNTL2 isinvolved in regulating the metabolism of fatty acid [12,14, 15]. Nevertheless, whether ARNTL2 regulates theexpression of fatty acid-associated genes should be determined. The acyl-CoA thioesterase (ACOT) genes encode
the enzymes that catalyze the production of free fatty
acids and CoA derived from fatty acyl-CoA [16]. Abnormal expression of different ACOT proteins has beenshown to participate in the development of cancers. For
example, ACOT4 expression is potentiated in pancreatic
ductal carcinoma (PDAC). Depletion of ACOT4 blunts
PDAC tumorigenesis [17]. In clear cell renal cell carcinoma (ccRCC), the majority of ACOT members, including ACOT1, ACOT2, ACOT3, ACOT8 and ACOT11,
are downregulated in the cancer samples [18]. On thecontrary, the role of ACOT11 in lung cancer is different
from its role in ccRCC. ACOT11 is upregulated in lungadenocarcinoma. Knockdown of ACOT11 alleviates theproliferation, migration and tumorigenesis of lung cancer cells [19]. These results suggest that the role of ACOT
proteins is distinct among various cancer types. Comparing with other ACOTs, ACOT7 is more frequently
reported as cancer promoter by various studies. ACOT7
promotes cell cycle progression by regulating p53-p21
signaling pathway in breast cancer [20]. In acute myeloid
leukemia patients, higher expression of ACOT7 predicts
the poorer prognosis of the patients [21]. Furthermore,

Page 9 of 12

ACOT7 is positively regulated by transcription factor
krüppel-like factor 13 (KLF13). Activation of KLF13/
ACOT7 axis supports the progression of hepatocellular
carcinoma (HCC) through potentiation of oleic acid [22].
These results illustrate that ACOT7 exhibits oncogenic
potential in different cancers. However, the upstream
controller and the significance of ACOT7 in NSCLC areless clear. Here, we demonstrated that ACOT7 expression
was stimulated by circadian rhythms transcription factor
ARNTL2. ARNTL2 and ACOT7 expression showed positive spearman correlation and their high expression was
worse predictor for LUAD patients. Importantly, ACOT7
expression was critical to maintain the proliferation ability of NSCLC cells. The oncogenic function of ARNTL2
could also be reversed by changing the expression of
ACOT7. Therefore, ACOT7 serves as an oncogene in
NSCLC patients. ARNTL2 promotes NSCLC progression through positive regulation of ACOT7.
Ferroptosis is a specific type of cell death which is
closely correlated with fatty acid metabolism, such as
lipid peroxidation [23]. Suppression of ferroptosis playsa pivotal role in the contribution of cancer development[24, 25]. Unlike apoptosis, which is positively stained byAnnexin V, ferroptosis is specifically stained by PI [25].
Initially, we observed that ACOT7 downregulation promoted, while ACOT7 overexpressed suppressed thepercentage of Annexin V positive cells. The activity of
caspase 3 to caspase 7 was repressed by ACOT7, indicating that ACOT7 inhibits the apoptosis of NSCLC cells.
Additionally, ACOT7 knockdown also enhanced the percentage of PI positive cells, revealing that ACOT7 could
regulate other types of cell death besides apoptosis. Various studies reported that lipid ROS was another hallmark
of ferroptosis [24, 26]. By staining the cells with C-11
BODIPY, we found that ACOT7 negatively regulated thelipid ROS. To further validate the role of ACOT7 on ferroptosis, NSCLC cells transfected with different siRNAs
were treated with ferroptosis or apoptosis inhibitors,
the cell death triggered by ACOT7 knockdown could bereversed by the inhibitors alone or their combination.
Furthermore, ACOT7 overexpression dramatically rescued the cell death triggered by ferroptosis, apoptosis
inducer, or their combination. These results demonstratethat ACOT7 suppresses apoptosis and ferroptosis in
NSCLC cells. Consistent with its role in HCC cells [22],ACOT7 positively regulated the production of oleic acid,as well as the cellular abundance of oleic acid, palmitoleic acid and triglyceride, suggesting that ACOT7 plays
an important role in the synthesis of monounsaturated
fatty acids. However, whether ACOT7 modulates the
production of polyunsaturated fatty acids (PUFAs), suchas arachidonic acid, should be explored in the future.
Moreover, ACOT7 suppressed lipid peroxidation, asWang et al. BMC Molecular and Cell Biology(2023) 24:14

shown by reduced MDA level in ACOT7 overexpressed
cells and by enhanced MDA level in ACOT7 knockdown
cells. These results reveal that ACOT7 has important role
in regulating fatty acid synthesis and lipid peroxidation.
We also observed that ARNTL2 promoted the production of triglyceride, which could be reversed by changing
the expression of ACOT7. Therefore, ARNTL2 upregulation of ACOT7 suppresses NSCLC cell apoptosis andferroptosis by regulating fatty acid synthesis and lipid
peroxidation.

Conclusion
In summary, we identified that ARNTL2 transcriptionally upregulation of ACOT7 suppressed the apoptosis
and ferroptosis, resulting in their promting function on
the growth and proliferation of NSCLC cells. Mechanistically, ACOT7 modulation of fatty acid synthesis and lipid
peroxidation could explain its dual role in apoptosis andferroptosis. Our study provided an important clue that
ARNTL2/ACOT7 contributed to NSCLC progression
and could be targeted for the treatment of this deadly
malignancy.
Methods
Analyzing ARNTL2 and ACOT7 in LUAD from TCGA​

Gene Expression Profiling Interactive Analysis (GEPIA:
http://​gepia.​cancer-​pku.​cn/) is a public database whichanalyzes the data from TCGA. We analyzed the expression of ARNTL2 and ACOT7, correlation between
ARNTL2/ACOT7’s expression and patients’ survival, andthe spearman correlation between ARNTL2 and ACOT7
in LUAD or LUSC patients based on TCGA database.
Cell culture

Human immortalized normal lung cells Beas-2B and
NSCLC cells A549, H1299 and H1975 were purchasedfrom ATCC and were grew in RPMI-1640 complete
medium, in which 10% fetal calf serum and 1% antibiotics were added. The cells were maintained in a 37 °C cellincubator in which the C­ O2 concentration was kept at 5%.
RNA extraction and real‑time quantitative polymerase
chain reaction (RT‑qPCR)

RNA was extracted from whole cell lysates using Trizol regent, following the manufacturers’ protocols. The
RNA was reversely transcribed into cDNA using M-MLV
reverse transcriptase (Promega). Quantification of thecDNA of indicated genes was conducted using SYBR
master mixture (Takara) on Bio-rad real-time PCR system. The sequences of the primers were listed below:
ARNTL2 forward, 5’-ACT​TGG​TGC​TGG​TAG​TAT​
TGGA-3’, and reverse, 5’-TGT​TGG​ACT​CGA​ATC​ATC​
AAGG-3’. ACOT7 forward, 5’-GGC​CGG​AAG​CGG​TAT​

Page 10 of 12

GAA​G-3’, and reverse, 5’-GAC​TGG​CTG​TAG​CTG​ACA​
GTG-3’. β-actin forward, 5’-GAG​CTG​CGT​GTG​GCT​
CCC​-3’, and reverse, 5’-CCA​GAG​GCG​TAC​AGG​GAT​
AGCA-3’.
Immunoblotting

To assess the protein abundance in cells, we collected
whole cell lysates from indicated cells using RIPA lysis
buffer (Beyotime). Protein concentration was determined by BCA assay kit (Beyotime). 30–50 μg of thetotal proteins were separated on SDS-PAGE gels andimmunoblotted onto PVDF membranes, which wereactivated by methanol. To avoid non-specific protein signal, the membranes were incubated with 5%
skim milk dissolved in 0.1% PBST (99.9% PBS and 1%
TWEEN-20). After incubating with secondary antibodies at room temperature for 2 h, protein signals
were detected using chemiluminescence assay kit,according to the manufacturers’ protocols. Primary
antibody against ARNTL2 was from Sigma-Aldrich(SAB2100154). ACOT7 (15,972–1-AP) and GAPDH(60,004–1-Ig) antibodies were from Proteintech. Antimouse (sc-2005) and rabbit (sc-2004) secondary antibodies were from SantaCruz.
Cell proliferation and cell viability

Cell proliferation was analyzed by using CCK-8 assay kit(Beyotime). A549 cells transfected with siNC, siACOT7(siACOT7#1 and siACOT7#2) or siARNTL2 (siARNTL2#1
and siARNTL2#2), and H1299 cells transfected with lentiCtrl, lenti-ACOT7 or lenti-ARNTL2, were seeded in 96-wellplates at a density of 3000 cells per well. Each well contained 100 μl RPMI-1640 complete medium. For the indicated time, 10 μl of CCK-8 buffer was added in each welland the plates were maintained in the cell incubator for 3 h.
After vibrating the plates for 10 min, OD450 was measured
on a microplate reader. The siRNAs were obtained from
Hippo Biotechnology (Huzhou, China) and the sequenceswere as following: siNC: 5’-UUC​UCC​GAA​CGU​GUC​ACG​
U-3’; siARNTL2#1: 5’-GAU​G GU​GAA​C AC​C AA​GUU​
A-3’; siARNTL2#2, 5’-GGA​C AA​G AC​C AA​C AG​C UA​
U-3’; siACOT7#1, 5’-GCA​UGA​CCU​UCA​CGA​GCA​A-3’;
siACOT7#2, 5’-CGC​UGA​AGA​AUG​UGG​ACA​A-3’.
For cell viability analysis, indicated cells were incubatedwith DMSO, ferrostatin-1 (ferr-1, 2 μM, Selleck), Z-VADFMK (Z-VAD, 8 μg/ml, Selleck), erastin (5 μM, Selleck),
apoptosis activator 2 (AA2, 4 μM, Selleck) for 6–10 hand cell viability was tested by trypan blue staining. Cell
death (%) = 100%—cell viability (%).
Colony formation

A total of 1000 siNC, siACOT7#1 and siACOT7#2
A549 cells and a total of 2000 siNC, siARNTL2#1 andWang et al. BMC Molecular and Cell Biology(2023) 24:14

Page 11 of 12

siARNLT2#2 A549 cells were seeded in 6-well plates. A totalof 500 lenti-Ctrl, lenti-ARNTL2 and lenti-ACOT7 H1299
cells were seeded in 6-well plates. 10 days later, colonies
were formed. The plates were washed by PBS and the colonies were fixed by methanol and stained by crystal violet.

and subjected to chromatin immunoprecipitation assay
by using SimpleChIP enzymatic chromatin IP kit (Cell
Signaling), according to the manufacturer’s instructions.
qPCR was applied to examine the DNA amplification
samples in Chip-IgG and Chip-Flag group.

Cell cycle

Fatty acid metabolism

A549 cells transfected with siNC and siACOT7(siACOT7#1 and siACOT7#2), and H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus,
were seeded in 6-well plates. 48 h later, cells were harvested and maintained in 70% cold alcohol for 8–12 h.
Subsequently, cell cycle was analyzed by PI staining(YEASEN, 40301ES50) on flow cytometry.
Annexin V and PI staining

A549 cells transfected with siNC and siACOT7(siACOT7#1 and siACOT7#2), and H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus, were seeded in 6-well plates. 48 h later, cells were
harvested and subjected to PI/Annexin-V (YEASEN,
40302ES20) staining and analysis on flow cytometry.
Lipid ROS detection on flow cytometry

Resuspended cells were stained with 3uM of C-11
BODIPY dye (Invitrogen) for 30 min, according to the
manufacturers’ protocols. Fluorescence intensity was
analyzed on flow cytometry.
Caspase 3 to caspase 7 activity measurement

The activity of caspase 3 to caspase 7 was detected byusing caspase-Glo reagent (Promega), according to the
manufacturers’ protocols. In brief, after incubating the
caspase-Glo reagent for 2 h, the activity of caspase 3 to
caspase 7 was examined on a microplate reader.
Dual luciferase reporter activity

Dual luciferase reporter activity was analyzed by using
assay kit from Promega, according to the manufacturers’ protocols. The coding sequence of ARNTL2 wasinserted into pCDNA3.1 vectors. The promoter sequenceof ACOT7 (-2000 ~ 0 bp) was inserted into pGL3.basic
vectors. Dual luciferase reporter activity was examined
48 h after co-transfecting siRNAs (siNC or siARNTL2)/
pCDNA3.1 (pCDNA3.1-Ctrl or pCDNA3.1-ARNTL2),
pGL3.basic vectors and TK vectors into NSCLC cells.
Relative luciferase activity was adjusted to TK activity.
Chromatin immunoprecipitation‑qPCR assay

To assess whether ARNTL2 binds to the promoter
sequence of ACOT7, we inserted the coding sequenceof ARNTL2 into the pCDNA3.1-Flag plasmid. A549
and H1299 cells were transfected with the plasmids

The cellular abundance of MDA in siNC, siACOT7#1
and siACOT7#2 A549 cells, and in lenti-Ctrl and lentiACOT7 H1299 cells was measured by using the assay kit
from Beyotime (Shanghai, China), according to the manufacturer’s instructions. The cellular abundance of triglyceride in A549 and H1299 cells was assessed by using
the assay kit from Nanjing Jiancheng, according to the
manufacturer’s instructions.
Statistical analysis

Statistical data were analyzed on GraphPad Prism 8 software and presented as mean ± standard error of mean
(SEM). Students’t test was applied to determine the difference between two groups. One-way ANOVA followed
by a Tukey’s post hoc test was used to determine the difference among groups. Statistical difference was considered significantly when p < 0.05.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​022-​00450-5.
Additional file 1: Supplementary Figure 1. The clinical relevance of
ARNTL2 in LUSC patients. Supplementary Figure 2. The expressionof ARNTL2 in normal and cancer cell lines. Supplementary Figure 3.
The effect of ACOT7 on the cell cycle of NSCLC cells. Supplementary
Figure 4. The effect of ACOT7 on the activity of caspase 3 to caspase 7 in
NSCLC cells. Original immunoblotting results.

Acknowledgements
N/A.
Authors’ contributions
T.W. and N.C. designed this study. T.W. carried out the majority of the experiments. K.W. and X.Z. provided experiment assistance. T.W., K.W., X.Z. and N.C.
analyzed the data and prepared the figures. N.C. wrote the draft and revised
the manuscript. All of the authors read and approved the final manuscript.
Funding
This study was supported by The Science and Technology Fund Project ofGuizhou Provincial Health Commission (gzwkj2021-201).
Availability of data and materials
The data generated during this work were available in this article.

Declarations
Competing interests
The authors declare no competing interests.
Ethics approval and consent to participate
N/A.

Wang et al. BMC Molecular and Cell Biology(2023) 24:14

Consent for publication
N/A.
Competing interest
These authors declared no conflicts of interest.
Received: 2 July 2022 Accepted: 5 October 2022

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