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Molecular Carcinogenesis
RESEARCH ARTICLE
B-Elemene Inhibits Adrenocortical Carcinoma Cell Proliferation and Migration, and Induces Apoptosis by Up-Regulating miR-486-3p/Targeting NPTX1 Axis
Yan Lin1,2,3,4 | Tailin Guo1,2,3,4 | Lishuang Che5 | Jieqiong Dong1,2,3 | Ting Yu1,2,3 | Chaiming Zeng1,2,3 | Ziyu Wu1,2,3,4
1Provincial Clinical College of Fujian Medical University, Fuzhou, China | 2Department of Geriatric Medicine, Fujian Provincial Hospital, Fuzhou, China | 3Fujian Provincial Center for Geriatrics, Fujian Provincial Hospital, Fuzhou, China | 4Fuzhou University Affiliated Provincial Hospital, Fuzhou, China | 5Department of Nephrology, The Second Affiliated Hospital of Fujian Medical University, Quanzhou, China
Correspondence: Ziyu Wu (dai24768076@163.com)
Received: 29 August 2024 | Revised: 3 December 2024 | Accepted: 20 December 2024
Funding: This research was supported by the Scientific Research, Fujian Medical University (Grant number: 0120210132).
Keywords: - elemene | adrenocortical carcinoma | apoptosis | miR-486-3p | NPTX1
ABSTRACT
B-elemene has a variety of anti-inflammatory, antioxidant, and antitumor effects. Currently, the influence of ß-elemene on adrenocortical carcinoma (ACC) malignant progression and action mechanism remains unclear. This research aims to explore the influence and action mechanism of ß-elemene on ACC progression. The impacts of ß-elemene on ACC cell viability, proliferation, migration, and apoptosis were investigated through CCK-8 assay, clone formation assay, Transwell experiment, Wound healing assay, and flow cytometry. The miR-486-3p expression was analyzed utilizing RT-qPCR. According to different databases, neuronal pentraxin 1 (NPTX1) is the predicted downstream target gene of miR-486-3p. Western blot and RT-qPCR were utilized to examine NPTX1 expression. Silencing miR-486-3p or Overexpression NPTX1 in ACC cells further explored whether ß-elemene affects ACC cells by regulating miR-486-3p/NPTX1. Finally, a subcutaneous graft tumor model was con- structed to investigate how ß-elemene may impact tumor growth in vivo. ß-elemene decreased the cell viability, hindered cell proliferation and migration capacity, and induced apoptosis of ACC cells. miR-486-3p level in ACC cells was notably reduced in comparison to normal cells, but treatment with ß-elemene markedly increased miR-486-3p expression. Additionally, ACC cells showed high level of NPTX1, while miR-486-3p targeted negative regulation of NPTX1. Overexpression miR-486-3p hindered the malignant progression of ACC cells, whereas overexpression NPTX1 reversed the impact of overexpression miR-486-3p. Silencing miR-486-3p or overexpression NPTX1 both attenuated the suppressive influence of ß-elemene on the malignant behavior of ACC cells. Additionally, tumor growth was suppressed and apoptosis was induced in tumor cells in vivo by ß-elemene. In conclusion, ß-elemene reduces ACC cell viability, hinders proliferation and migration, and induces apoptosis through the miR-486-3p/NPTX1 axis.
1 Introduction |
Adrenocortical carcinoma (ACC) is an uncommon cancerous growth that develops in the adrenal cortex, making up
approximately 8%-11% of adrenal tumors, known for its aggressive nature and poor prognosis [1, 2]. The occurrence of ACC is infrequent, about 1-2 cases per 1 million people per year, with a median survival of only 3-4 years [3, 4].
Effective treatments are very limited, and radical surgery is the preferred option for patients with ACC, but the postoperative recurrence rate is high [5, 6]. In addition, it is common for ACC patients to have metastatic tumors at the time of diagnosis, and metastatic ACC has a poorer prognosis, showing a 5-year sur- vival rate of under 20 percent [1, 7]. Therefore, the search for novel and efficient drugs is crucial for the clinical management of ACC patients.
ß-elemene is an olefinic monomer extracted from Curcumae Rhizoma, which alleviates cardiac remodeling and heart failure, it possesses a range of properties that combat inflammation, oxida- tion, aging, and tumors [8, 9]. Scholars have shown significant interest in the anticancer properties of ß-elemene in the past few years. ß-elemene has multiple targets of action (e.g., kinases, growth factors, transcription factors, etc.) and enhances the responsiveness of cancer cells to chemotherapy and radiotherapy [10, 11]. A large number of papers reported that ß-elemene has the capacity to block the malignant progression of non-small cell lung cancer [12], colorectal cancer [13], and cholangiocarcinoma [14]. Nevertheless, the influence of ß-elemene treatment on malignant progression in ACC is currently uncertain.
With a length of about 21-22 nt, microRNAs (miRNAs) belong to the noncoding category [15]. Evidence is mounting to suggest that miRNAs are implicated in cell proliferation, apoptosis, and differentiation, are dysregulated in different types of tumors, and act as either oncogenes or tumor suppressors [16, 17]. MiR- 486 is encoded by the MIR486-1 gene located on chromosome 8 (8p11.21) in the human genome [18]. It has been demonstrated in studies that miR-486-3p actions as an oncogene in multiple cancers, including laryngeal squamous cell carcinoma [19], thyroid cancer [20], and oral cavity cancer [21]. Recent research revealed a marked decrease in miR-486-3p levels in ACC tumors, indicating that this reduction could be a crucial factor in ACC malignant progression [22]. However, the specific action mechanism of miR-486-3p in regulating ACC malignant development is currently unclear.
Therefore, the main focus of this research was to explore how ß- elemene affects the biological behavior of ACC cells, and to elucidate how overexpression miR-486-3p impacts the prolif- eration and apoptosis of these cells. Next, different databases were utilized to identify the downstream target gene neuronal pentraxin 1 (NPTX1) of miR-486-3p, and explored whether miR-486-3p impacts the malignant behavior of ACC cell by regulating NPTX1. Finally, it was further explored whether ß- elemene acts through modulating the miR-486-3p/NPTX1 axis. The objective of this research was to clarify the specific action mechanism of ß-elemene in hindering the malignant biological behaviors of ACC, aiming to offer a fresh perspective on the potential clinical use of ß-elemene for treating ACC.
2 Materials and Methods |
2.1 Clinical Tissue Sample |
We randomly collected cancerous and paracancerous normal tissue samples from ACC patients (n = 35) admitted to Fuzhou University Affiliated Provincial Hospital from January to
October 2023, cleaned and stored in liquid nitrogen for backup. None of the patients had preoperative radiotherapy and there were no metastases. The study was approved by the Ethics Committee of Fuzhou University Affiliated Provincial Hospital and, as required, all patients signed an informed consent form for sample acquisition.
2.2 Cell Culture |
Human ACC cells (SW-13 and NCI-H295R) and human em- bryonic kidney cell line (HEK293T) were obtained from Pricella Biotechnology Co. Ltd. (Wuhan, Hubei, China). DMEM medium (Gibco, Grand Island, NY, USA) was enriched with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin double antibody (Gibco) served as the cell culture medium. The cell culture temperatures were all 37°℃ with 5% CO2.
2.3 | CCK-8 Assay
HEK293T and ACC cells were seed into 96-well cell culture plates (1.5 x 104/well), and when the cells were attached to the wall, the original medium was changed to 200 uL of medium that included different doses of ß-elemene (HY-107324, Med- ChemExpress, Monmouth Junction, NJ, USA). Following a 48-h incubation period, 20 µL of CCK-8 reagent (C0038, Beyotime, Shanghai, China) was introduced into every well. After being incubated for 2 h at 37°℃ in a light-protected incubator, the OD450 value of ACC cells was assessed utilizing a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) to screen the optimal acting concentration of ß-elemene and to perform subsequent experiments.
2.4 | Cell Transfection
miR-486-3p mimic (mimic), miR-486-3p inhibitor (inhibitor), NPTX1 overexpression plasmid (OE-NPTX1), and their negative control (mimic NC, inhibitor NC, and OE-NC) were obtained from RiboBio Co. Ltd. (Guangzhou, Guangdong, China). The seeding density for ACC cells in 24-well plates was set at 1 × 104 cells per well, with 500 uL of medium. When the cell confluence was 50%, the mentioned plasmids were transfected into ACC cells utilizing Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), and the transfection procedure was referred to the instructions.
2.5 Clone Formation Assay |
ACC cells from each group were taken, PBS washed, and di- gested separately with 0.25% trypsin (Gibco) and blown into individual cells, which were then counted. Five hundred cells were seeded into every well of a 6-well plate and incubated for 14 d (37°℃, 5% CO2). The cell culture medium with or without ß-elemene was changed every 2-3 days. The culture ceased when clonal cell clusters could be seen by the naked eye. Aspirated the culture solution and rinsed it twice with PBS, then exposed it to 4% paraformaldehyde (Solarbio, Beijing, China)
for fixation for 20 min. The fixative was discarded, exposed to crystal violet (Solarbio) for 10 min, then imaged utilized an inverted microscope, and the amount of clone formation was quantified.
2.6 | Transwell Assay
Cells were routinely digested, collected, and suspended in serum-free medium added or without ß-elemene. The upper chamber was filled with cell suspension (200 µL, 1 x 105 cells/ mL), followed by the addition of the right amount of DMEM medium added 15% fetal bovine serum in the lower section, and subsequently incubated for 24 h. The Transwell was taken out, rinsed two times with PBS, exposed to 4% paraformaldehyde for 30 min, subsequently dyed with 0.1% aqueous crystal violet for 10 min. Using a cotton swab to delicately wipe away un- migrated cells from the upper section of the chambers. The field of view was randomly selected and photographed with an inverted microscope to count the amounts of migratory cells.
2.7 Wound Healing Assay |
The SW-13 and NCI-H295R cells were taken, trypsin-digested, collected, and 1 mL of cell suspension (3× 105cells/mL) was aspirated with a sterile lance tip and inoculated into 6-well plates with the horizontal lines drawn in advance. When the cells had completely attached to the wall and grew to more than 80% density, after aspirating and discarding the medium, a 20 uL sterile tip was employed to make a perpendicular scratch at the bottom of the 6-well plate. The cells at the scratches were removed using PBS, and serum-free medium with or without ß- elemene was added. The observation of scratch healing oc- curred at two time points: 0 and 24 h. To evaluate the cell migration rate, the widths of scratches were measured utilizing Image J software (version 1.54 h, Wavne Resband, National Institute of Mental Health, USA).
2.8 | Flow Cytometry
The cells from various treatment groups were rinsed twice with PBS, and gently mixed by adding 500 uL of Binding Buffer. Following that, 5 uL of Annexin-V-FITC (HY-K1073, Med- ChemExpress) and 5 uL of propidium iodide (ST1569, Beyo- time) were introduced and left to incubate for 15 min in the dark. Flow-specific supersampling tubes were used to transfer the samples, and apoptosis was then identified through flow cytometry.
2.9 | Bioinformatics Analysis
To obtain the target gene of miR-486-3p, we utilized miRDB (https://mirdb.org/), miRWalk (http://mirwalk.umm.uni- heidelberg.de/), TargetScan (https://www.targetscan.org/vert_ 80), and GEPIA (http://gepia.cancer-pku.cn/) databases, and discovered NPTX1 is downstream target gene. Additionally, the
binding site between NPTX1 and miR-486-3p was predicted using TargetScan.
2.10 Dual-Luciferase Reporter Assay |
NPTX1wild-type (Wt) and NPTX1 mutant (Mut) dual luciferase reporter plasmids were synthesized by RiboBio Co. Ltd. NPTX1 Wt and mimic NC, NPTX1 Wt and mimic, NPTX1 Mut and mimic NC, and NPTX1 Mut and mimic were cotransfected into ACC cells, respectively, using Lipofectamine 3000. Following a 48-h incubation period, cells were gathered and the luciferase activity of ACC cells was evaluated utilizing the Dual-Lucy Assay Kit (Solarbio).
2.11 | Subcutaneous Tumor Model in Nude Mice
Twenty BALB/c nude mice were purchased from Vitalriver (Beijing, China) and placed in a standard animal feeding room for 1 week for acclimatization. The rearing temperature was 22 ±2℃, relative humidity 45% and light cycle 12/12 h. The nude mice were randomly divided into four groups of five mice each. Nude mice in the i-NC + O-NC + PBS group were sub- cutaneously injected with 0.2 mL of SW-13 cell suspension transfected with miR-486-3p inhibitor NC and OE-NC (2x 106 cells/each). The i + O-NC + PBS group was then injected with cell suspension transfected with miR-486-3p inhibitor and OE- NC. The i+O-NPTX1 + PBS group and i+O-NPTX1+ß- elemene group were injected with cell suspensions transfected with miR-486-3p inhibitor and OE-NPTX1. Referring to the administered dose of Wang et al. [23], the administration of the drug was started on the 7th day after inoculation. Nude mice in the i-NC + O-NC + PBS group, i+O-NC+ PBS group, and i + O-NPTX1 + PBS group were injected intraperitoneally with 0.1 mL of saline, and the i + O-NPTX1+ß-elemene group were injected intraperitoneally with 0.1 mL of ß-elemene (45 mg/kg), administered once a day for 28 days. The size of subcutaneous tumors in nude mice was measured every 7d with vernier calipers, and the tumor volume was calculated. Nude mice were anaesthetized and executed 1 h after the last dose, tumors were excised and photographed for documentation. This experiment was approved by the Experimental Animal Ethics Committee of Fuzhou University Affiliated Provincial Hospital.
2.12 : | Immunohistochemistry
Tumor tissue wax blocks were routinely sectioned and dewaxed for microwave antigen repair. Sections were incubated in 3% H2O2 solution for 25 min. Subsequently, the tissues were evenly covered with drops of 5% bovine serum albumin (V900933, Sigma-Aldrich), and closed for 30 min. Ki67 primary antibody (ab15580, 1:1000, Abcam, Cambridge, MA, USA) was gently added and incubated at 37℃ for 90 min. Following that, the sections were covered with HRP-labeled goat anti-rabbit IgG (1:10,000) for 20 min at 37°C. DAB (DA1010, Solarbio) color development, tap water terminated color development. Mayer Hematoxylin (MHS16, Sigma-Aldrich) was re-stained and neutral gum was sealed and placed under the microscope (DM IL LED, Leica, Heidelberg, Germany).
After fixation with 4% paraformaldehyde, tumor tissues were routinely dehydrated, sectioned after paraffin embedding (thickness of 4 ~ 5 um), and deparaffinized with xylene (247642, Sigma-Aldrich). Gradient ethanol (100%, 95%, 75% and 50%) for hydration. DNase-free proteinase K (20 µg/mL, ST532, Beyo- time) was added slowly, and left to incubate for 30 min. After that, TUNEL assay solution (C1086, Beyotime) was slowly ad- ded and left to incubate for 1.5 h away from light. Subsequently, incubated with DAPI staining solution (D9542, Sigma-Aldrich) for 10 min away from light. Inverted fluorescence microscope was used to observe and photograph.
2.14 | RT-qPCR
Total RNA was isolated from different cells and tissues utilizing Trizol reagent (Invitrogen), and subsequently reverse tran- scription was performed by adding AMV reverse transcriptase (TAKARA, Tokyo, Japan) to obtain cDNA. Then, PCR ampli- fication was carried out with TB Green Premix Ex Taq II (TAKARA). The U6 and GAPDH serving as internal references for miR-486-3p and NPTX1, respectively.
The following are the primer sequences utilized in this experi- ment: miR-486-3p: F: 5’-GCCGAGCGGGGCAGCTCAGTA-3’; R: 5’-CGCAGGGTCCGAGGTATTC-3’. U6: F: 5’-CTGGTGAAG GGGAGGGGATA-3’; R: 5’-ACAGGATAGGGGGACCACTC-3’. NPTX1: F: 5’-CGATCTACTGGACCGCAGAC-3’; R: 5’-AGAG GATGGCTCATGGTCCT-3’. GAPDH: F: 5’-TCGGCAGGATGT AGGGCTAAAAGC-3’; R: 5’-GTAGCCCATGGGTTTTAGCCC-3’.
2.15 Western Blot |
After different treatments, HEK293T, SW-13, and NCI-H295R cells were lysed utilizing RIPA lysis solution (P0013B, Beyotime) to obtain proteins, and the BCA kit (P0012, Beyotime) was for assessing protein concentrations. After separation using SDS- PAGE (12%, Invitrogen), the samples were shifted to a PVDF membrane (Invitrogen), followed by blocking with skimmed milk for 3 h. After rinsing the membranes, placed at 4℃ for an overnight incubation with NPTX1 primary antibody (PA5-25954, 1:2000, Invitrogen). On the next day, sheep anti-rabbit secondary IgG (31460, 1:10,000, Invitrogen) was incubated after washing the membrane 3 times, developed, and exposed. GAPDH (MA5- 15738, 1:1000, Invitrogen) serving as the internal reference, and the grayscale values of each protein band were obtained after processing the images with Image J software.
2.16 Statistical Analysis |
Every experiment was conducted a minimum of three times, with the results were documented as the mean value ± corresponding standard deviation. Student’s t-test was utilized to evaluate the distinctions between the two groups. To evaluate differences among multiple groups, we conducted a one-way analysis of variance. SPSS 26.0 software (IBM SPSS Statistics 26)
was utilized to process and analyze the data statistically. Prism software (Graphpad 9.0) was utilized for plotting. * p <0.05 signifying that there was a significant difference.
3 | Results
3.1 ß-Elemene Suppresses ACC Cell Proliferation and Migration, While Inducing ACC Cell Apoptosis |
CCK-8 assay was employed to evaluate the cell viability of HEK293T, SW-13, and NCI-H295R cells following different doses of ß-elemene treated. The findings revealed that ß- elemene (0-40 µg/mL) had no significant effect on HEK293T cell viability, suggesting that it has no toxic effect on normal cells (Figure 1A). Notably, ß-elemene treatment at 5, 10, 20, and 40 µg/mL caused a marked decrease in cell viability (p <0.001), with the impact increasing with higher doses (Figures 1B,C). The cell viability was notably reduced with 5 µg/mL of ß- elemene, leading us to select this concentration for further ex- periments. The clone formation experiment results indicated that the clone formation rate of ACC cells was significantly reduced after ß-elemene treatment (p <0.001, Figure 1D). By Transwell assay, we noted a marked decrease in the migration amount of ACC cells after ß-elemene treatment (p <0.001, Figure 1E). Wound healing assay results similarly showed that the migration rates of SW-13 and NCI-H295R cells were sig- nificantly reduced after @-elemene treatment (p <0.001, Figure 1F). Additionally, the apoptosis rate of ACC cells was markedly elevated following ß-elemene treatment (p <0.001), as indicated by the flow cytometry results (Figure 1G). These data revealed that ß-elemene treatment decreased the cell via- bility, hindered proliferation and migration, and increased apoptosis in SW-13 and NCI-H295R cells.
3.2 miR-486-3p Shows Low Levels in ACC Cells, While -Elemene Upregulates miR-486-3p Expression |
The miR-486-3p expression in different tissues and cells were assessed using RT-qPCR. The data revealed a notable difference in miR-486-3p expression between ACC tissue and adjacent normal tissue samples, miR-486-3p level in adjacent normal tissues was notably higher than that in ACC tissues (p <0.001, Figure 2A). Not only that, HEK293T cells also exhibited higher miR-486-3p level (p <0.001, Figure 2B). Following ß-elemene treatment, miR-486-3p expression showed a notable increase in ACC cells (p<0.001), indicating that ß-elemene upregulates miR-486-3p expression (Figure 2C,D).
3.3 Overexpression miR-486-3p Hinders Proliferation, Migration, and Promotes Apoptosis in ACC Cells |
Next, we transfected mimic into SW-13 and NCI-H295R cells to explore the impact of Overexpression miR-486-3p on the malignant behavior of ACC cells. Following transfection with
A
HEK293T
B
C
D
Control
ß-elemene
SW-13
NCI-H295R
Relative cell viability(%)
ns
200
Relative cell viability(%)
Relative cell viability(%)
SW-13
ns
200
200
ns
150
ns
150
150
ns
100-
100
100
50
50-
50
NCI-H295R
0
0
0
Oug/mL
2.5µg/mL
5µg/mL
10µg/mL
20µg/mL
40µg/mL
Oug/mL
2.5µg/mL
5µg/mL
10µg/mL
20µg/mL
40µg/mL
Oug/mL
2.5µg/mL
5µg/mL
10µg/mL
20µg/mL
40µg/mL
E
F
Control
ß-elemene
Colony formation rate (% of Control)
Control
Control
ß-elemene
150-
SW-13
Migration cell numbers
ß-elemene
100-
100-
0h
50-
50
NCI-H295R
Y
5
24h
0
SW-13
NCI-H295R
0
SW-13
NCI-H295R
Control
ß-elemene
Control
ß-elemene
SW-13
NCI-H295R
G
104
Q1
Q2
10
Q1
Q2
1.63
2.55
17.6
13.5
,
103
102
4
Migration rate (% of Control)
SW-13
1.2-
NO
10’
Apoptosis rate (%)
30
0.8
Q4
03
Q4
03
10º
94.8
1.06
100
68.1
0.83
20
0.4-
10°
10’
10ª
10ª
0
10º
10’
102
10ª
10
L
10-
Q5
06
05
06
0.0
1.21
2.72
7.54
16.0
SW-13
NCI-H295R
10
102
0
Control
ß-elemene
NCI-H295R
SW-13
NCI-H295R
PI FL-2
Control
ß-elemene
10’
101
Q8
07
08
Q7
89.1
6.93
68.9
7.58
80º
10
102
10
10º
10
102
10
Annexin V(APC) FL-4
A
B
C
D
Relative miR-486-3p level
Relative miR-486-3p level
Relative miR-486-3p level
SW-13
Relative miR-486-3p level
1.5-
NCI-H295R
1.5
2.0
2.0
1.0
1.0
1.5
1.5-
1.0-
1.0-
0.5-
0.5
0.5
0.5-
0.0
0.0
0.0
0.0
Adjacent
ACC
HEK293T
SW-13
NCI-H295R
Control ß-elemene
Control ß-elemene
A
B
C
mimic-NC
mimic
mimic-NC
mimic
Relative miR-486-3p level
8-
mimic NC
mimic
SW-13
Colony formation rate (% of NC)
150-
mimic NC
mimic
SW-13
Migration cell numbers
150
mimic NC
mimic
6-
100
100
4-
T
2
NCI-H295R
50-
NCI-H295R
50
0
SW-13
NCI-H295R
0
SW-13
NCI-H295R
0
SW-13
NCI-H295R
1
D
E
mimic-NC
mimic
1.74
02
9
02
mimic NC
mimic
mimic NC
mimic
2.36
16.2
137
1.5-
mimic NC
mimic
SW-13
₩
40-
mimic NC
mimic
0h
Migration rate (% of NC)
Apoptosis rate (%)
12
₩
1.0
T
T
30
10”
-
20
L
04
03
03
0.5
T
L
10ª
0.94
1
24h
1º
19”
1ª
12
5ª
-
1
Nº
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·
10-
SW-13
NCI-H295R
0.0
os
1.41
2.90
7.38
17.1
SW-13
NCI-H295R
r
0
NCI-H295R
Is2
12
SW-13
NCI-H295R
PI FL-2
1ª
1ª
20
ar
7.00
or
8.14
1ª
M’
2
ª
1ª
18
w2
2
Annexin V(APC) FL-4
mimic, the RT-qPCR results indicated a notable rise in miR-486-3p level in ACC cells (p<0.001), which satisfied the requirements of subsequent experiments (Figure 3A). After Overexpression miR- 486-3p, clone formation assay results revealed a marked decline in the clone formation rate of ACC cells (p <0.001, Figure 3B). Moreover, Transwell assay results indicated a notable decrease in the migration amount of ACC cells and migration rate following Overexpression miR-486-3p (p <0.001, Figure 3C,D). Additionally, we investigated apoptosis through flow cytometry and demon- strated that Overexpression miR-486-3p caused a significant ele- vation in the apoptosis rate of ACC cells (p <0.001, Figure 3E). These findings indicated that Overexpression miR-486-3p decreased proliferation and migration ability and induced apo- ptosis in ACC cells, suggesting that ß-elemene may exert an inhibitory effect on the malignant progression of ACC via upre- gulating miR-486-3p.
3.4 | miR-486-3p Targets Negative Regulation of NPTX1
By utilizing the TargetScan, miRWalk, miRDB, and GEPIA databases, NPTX1 was obtained as the target gene of miR- 486-3p, and we used TargetScan to predict the binding site of NPTX1 and miR-486-3p (Figure 4A). GEPIA database indicated a notable increase in NPTX1 expression in ACC compared to normal tissues (Figure 4B). Following that, we validated the targeted binding between NPTX1 and miR-486-3p through Dual-Luciferase reporter assay. The findings demonstrated that overexpression miR-486-3p markedly hindered the luciferase signal of NPTX1 Wt (p <0.001), with no significant influence on
Mut, thereby confirming that miR-486-3p targeted regulation of NPTX1 (Figure 4C,D). Western blot results indicated a notable decline in NPTX1 protein level in NCI-H295R and SW-13 cells following Overexpression miR-486-3p (p <0.001), suggesting that miR-486-3p negative regulation of NPTX1 expression (Figure 4E). Correlation analysis of miR-486-3p and NPTX1 expression showed that as miR-486-3p expression increased, NPTX1 levels decreased (Figure 4F). Notably, the level of NPTX1 was markedly elevated in ACC cells in comparison to HEK293T cells (p <0.001), a result that was consistent with the GEPIA database (Figure 4G,H). In addition, the expression of NPTX1 mRNA was notably higher in ACC tissues than in normal adjacent tissues (p < 0.001, Figure 4I).
3.5 Overexpression of NPTX1 Partially Attenuated the Suppressive Impact of Overexpression miR-486-3p on the Biological Behaviors of ACC Cells |
To explore the influence of Overexpression NPTX1 on the malig- nant behaviors of ACC cells, we transfected OE-NPTX1 and detected its overexpression efficiency through RT-qPCR and Western blot. Following transfection with OE-NPTX1, the results indicated a notable rise in NPTX1 mRNA and protein level in ACC cells (p <0.01), which satisfied the requirements of subsequent ex- periments (Figure 5A,B). By clone formation assay, we found that overexpression NPTX1 attenuated the inhibitory impact of over- expression miR-486-3p on clone formation in ACC cells (p <0.001, Figure 5C). More than that, overexpression NPTX1 also weakened the suppressive impact of overexpression miR-486-3p on cell
A
B
GEPIA database
miRWalk
miRDB
TargetScan
12506
3
8
3980
39
0
GEPIA
NPTX1 3’UTR (MUT 5’ - 3’)
GGACUCUCUCAGGCAACGGCCAU
NPTX1 expression
0
772
455
0
56
miR-486-3p(3’-5’)
UAGGACAUGACUCGACGGGGC
.
43
1
31
NPTX1 3’UTR (WT 5’ - 3’)
GGACUCUCUCAGGCAUGCCCCAU
2
NPTX1
0
10
0
T
2
ACC (num(T)=77; num(N)=128)
C
D
E
Relative luciferase activity
SW-13
Relative luciferase activity
NCI-H295R
1.5
mimic NC
mimic
1.5
mimic NC
mimic
SW-13
NCI-H295R
Relative NPTX1 protein
1.5
mimic NC
mimic
ns
ns
1.0-
1.0
NPTX1
47KDa
GAPDH
36KDa
level
1.0
0.5
0.5
mimic-NC
0.5
mimic
mimic-NC
mimic
0.0
0.0
0.0
WT
MUT
WT
MUT
SW-13
NCI-H295R
F
G
Relative NPTX1 mRNA level
H
Relative NPTX1 protein level
4-
4.
6-
NPTX1 expression
Relative NPTX1 mRNA level
3-
3-
3.
NPTX1
47KDa
4.
2-
2-
2
1.
GAPDH
36KDa
2.
1-
R2=0.6624
1
P<0.001
HEK293T
SW-13
NCI-H295R
0-
0
0
0
0.0
0.2
0.4
0.6
0.8
1.0
HEK293T
SW-13
NCI-H295R
HEK293T
SW-13
NCI-H295R
Adjacent
ACC
miR-486-3p expression
migration ability (p <0.01, Figure 5D,E). Flow cytometry results indicated that overexpression NPTX1 impaired the promotion influence of overexpression miR-486-3p on apoptosis (p<0.01, Figure 5F). The above findings indicated that overexpression NPTX1 attenuated the suppressive impact of miR-486-3p on the biological behaviors of ACC cells, suggesting that miR-486-3p ex- erted its anticancer effect by targeting downregulation of NPTX1.
3.6 | ß-Elemene Regulates ACC Cell Proliferation, Migration and Apoptosis Through miR-486-3P/ NPTX1
Next, we explored whether ß-elemene influenced ACC cells via the miR-486-3p/NPTX1 axis. After transfection with inhibitor, RT-qPCR findings indicated a markedly decline in miR-486-3p expression in ACC cells (p<0.001), which satisfied the requirements of subsequent experiments (Figure 6A). Following ß-elemene treatment, RT-qPCR and Western blot assays revealed a notable decline in NPTX1 level in ACC cells, whereas
silencing miR-486-3p or overexpression NPTX1 both resulted in a significant increase in NPTX1 expression (p <0.001, Fig- ure 6B,C). ß-elemene treatment significantly reduced the clone formation rate and the number of migrating cells in ACC cells, whereas silencing miR-486-3p or overexpression of NPTX1 both attenuated the suppressive influence of ß-elemene treatment on the clone formation and migratory capability of ACC cells (p < 0.001, Figure 6D-F). Moreover, ß-elemene notably elevated the apoptosis rate of ACC cells, but silencing miR-486-3p or overexpression NPTX1 both attenuated this influence (p <0.001, Figure 6G). It is implied that ß-elemene may hinder ACC cell proliferation and migration while promote apoptosis through regulating the miR-486-3p/NPTX1 axis.
3.7 | ß-Elemene Inhibits Tumor Growth In Vivo By Upregulating miR-486-3p and Targeting NPTX1
Finally, to investigate the effect of ß-elemene on tumor growth in vivo, we constructed a subcutaneous
A
B
C
Relative NPTX1 mRNA level
Relative NPTX1 protein
mimic-NC+ OE-NC
mimic+ OE-NC
mimic+ OE-NPTX1
3
OE-NC
OE-NPTX1
SW-13
NCI-H295R
3
OE-NC
OE-NPTX1
SW-13
2.
NPTX1
47KDa
level
2
**
1.
GAPDH
36KDa
1
OE-NC
OE-NPTX1
OE-NC
OE-NPTX1
NCI-H295R
0
T
0
SW-13
NCI-H295R
SW-13
NCI-H295R
T
D
mimic-NC+ OE-NC
mimic+ OE-NC
mimic+
E
mimic-NC+OE-NC
OE-NPTX1
Colony formation rate (% of NC)
200
mimic+OE-NC
mimic-NC+OE-NC
mimic-NC+OE-NC
mimic+OE-NPTX1
SW-13
Migration cell numbers
200-
mimic+OE-NC
mimic+OE-NC
mimic+OE-NPTX1
2.0
mimic+OE-NPTX1
150
150
1.5
**
**
100
**
NCI-H295R
Migration rate (% of NC)
100
**
T
T
1.0
T
T
50
50
0.5
0
SW-13
NCI-H295R
0
SW-13
NCI-H295R
0.0
SW-13
NCI-H295R
F
mimic-NC+ OE-NC
mimic+ OE-NC
mimic+
OE-NPTX1
-
01
a2
1
a
.
137
01
2.89
3.27
02
4.50
-
mimic-NC+ mimic+ mimic+ mimic-NC+ mimic+ mimic+
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«
OE-NC
OE-NC OE-NPTX1 OE-NC
OE-NC OE-NPTX1
SW-13
«
mimic-NC+OE-NC
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1
50
mimic+OE-NC
0h
Apoptosis rate (%)
-
-
.
40-
mimic+OE-NPTX1
GA
0
₩
1.30
1
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17.4
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7.30
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Annexin V(APC) FL-4
transplantation tumor model in nude mice. Injection of SW- 13 cells transfected with miR-486-3p inhibitor markedly reduced miR-486-3p levels in nude mice, and was further reduced by overexpression of NPTX1, whereas ß-elemene treatment notably increased miR-486-3p level (p <0.001, Figure 7A). Not only that, ß-elemene treatment reduced the expression of NPTX1 protein in ACC tissues (p <0.001), confirming that ß-elemene also regulates the miR-486-3p/ NPTX1 axis in vivo (Figure 7B). Silencing miR-486-3p and overexpressing NPTX1 resulted in significantly higher weight and increased volume of tumors in nude mice, while ß-elemene treatment significantly reduced the weight and volume of tumors (p <0.05, Figure 7C-E). Additionally, silencing miR-486-3p and overexpressing NPTX1 resulted in an increase in Ki-67 expression in ACC tissues (Figure 7F) and a notable decline in TUNEL-positive cell rate (p <0.05, Figure 7G). Injection of ß-elemene, on the other hand, decreased Ki-67 expression and increased the rate of TUNEL-positive cells, suggesting that ß-elemene can inhibit ACC cell proliferation and promote apoptosis.
4 Discussion |
Over the past few years, herbal extracts have garnered sig- nificant attention for their ability to hinder the malignant advancement of tumors, with their high safety levels, low side effects, multiple pathways, and multiple targets [24, 25]. Found in the medicinal plant Curcumae Rhizoma, B-elemene has a variety of targets, and its role in inhibiting the malig- nant progression of tumors has attracted much attention. It has been shown that ß-elemene reduces the survival and movement of non-small cell lung cancer cells, promotes their apoptosis, and enhances cell sensitivity to erlotinib [26]. According to Deng et al., ß-elemene caused a reduction in miR-1323 expression, resulting in the upregulation of Cbl-b, which in turn suppressed the metastasis of gastric cancer cells through the EGFR pathway [27]. The research by Cai et al. discovered that ß-elemene caused apoptosis in glio- blastoma cells by hindering the JAK2/Src-STAT3 pathway [28]. Yet, there is no information available on the impact of ß- elemene on ACC. In our research, ß-elemene reduced the
A
B
C
D
Relative miR-486-3p level
Relative NPTX1 mRNA level
Relative NPTX1 protein level
1.5
inhibitor-NC
inhibitor
1.5-
SW-13
NCI-H295R
…
1.5-
…
Colony formation rate (% of NC)
150-
…
…
…
NPTX1
47KDa
…
…
…
1.0-
1.0-
…
1.0-
100
…
GAPDH
ß-elemene+inhibitor-NC+
ß-elemene+inhibitor+OE-NC
36KDa
…
I
0.5
0.5
, inhibitor-NC+OE-NC
ß-elemene+inhibitor-NC+OE-NC
ß-elemene+inhibitor+OE-NC
inhibitor-NC+OE-NC
ß-elemene+inhibitor-NC+OE-NC
ß-elemene+inhibitor-NC+
0.5
50
L
0.0
SW-13
NCI-H295R
0.0
SW-13
NCI-H295R
0.0
OE-NPTX1
OE-NPTX1
SW-13
NCI-H295R
0
SW-13
NCI-H295R
inhibitor-NC+OE-NC
inhibitor-NC+OE-NC
inhibitor-NC+OE-NC
B-elemene+inhibitor-NC+OE-NC
B-elemene+inhibitor-NC+OE-NC
B-elemene+inhibitor-NC+OE-NC
ß-elemene+inhibitor+OE-NC
B-elemene+inhibitor+OE-NC
ß-elemene+inhibitor+OE-NC
B-elemene+inhibitor-NC+OE-NP
B-elemene+inhibitor-NC+OE-NPTX1
ß-elemene+inhibitor-NC+OE-NPTX1
inhibitor-NC +OE-NC
ß-elemene+ inhibitor-NC+
ß-elemene +inhibitor+
ß-elemene+ inhibitor-NC+ OE-NPTX1
E
ß-elemene+ inhibitor-NC+ OE-NC
ß-elemene +inhibitor+ OE-NC
ß-elemene+ inhibitor-NC+ OE-NPTX1
F
OE-NC
OE-NC
inhibitor-NC +OE-NC
Migration cell numbers
150-
…
1.5-
…
SW-13
NCI-H295R SW-13
…
Migration rate (% of NC)
…
100
…
1.0
…
…
I
50-
0.5-
1
NCI-H295R
0
SW-13
NCI-H295R
0.0
SW-13
NCI-H295R
inhibitor-NC+OE-NC
inhibitor-NC+OE-NC
B-elemene+inhibitor-NC+OE-NC
ß-elemene+inhibitor-NC+OE-NC
ß-elemene+inhibitor+OE-NC
ß-elemene+inhibitor+OE-NC
B-elemene+inhibitor-NC+OE-NPTX1
B-elemene+inhibitor-NC+OE-NPTX1
G
inhibitor-NC +OE-NC
ß-elemene+ inhibitor-NC+ OE-NC
ß-elemene +inhibitor+ OE-NC
ß-elemene+ inhibitor-NC+ OE-NPTX1
SW-13
NCI-H295R
200
-.
ME
A.
0h
SW-13
NE
Apoptosis rate (%)
30-
…
1
24h
20-
…
=
=
=
-
inhibitor-NC+
OF-NC
B-elemene+
inhibitor-NC+OE-NC
ß-elemene+
inhibitor+OE-NC
ß-elemene+
inhibitor-NC+
OE-NPTX1
OE-NC
inhibitor-NC+
ß-elemene+
inhibitor-NC+OE-NC
ß-elemene+
inhibitor+OE-NC
ß-elemene+
10
inhibitor-NC+
OE-NPTX1
NCI-H295R
1
-
HA
:
-
1
=
0
SW-13
NCI-H295R
inhibitor-NC+OE-NC
PI FL-2
ß-elemene+inhibitor-NC+OE-NC
=
:
136
=
ß-elemene+inhibitor+OE-NC
ß-elemene+inhibitor-NC+OE-NPTX1
Annexin V(APC) FL-4
viability of ACC cells, inhibited their clone formation, and induced apoptosis, confirming that ß-elemene has the capa- bility of hindering ACC malignant development.
The significance of miRNAs in the progression of malignant tumors, whether as oncogenes or tumor suppressors, is widely recognized. miR-486-3p is markedly reduced in multiple cancer types, and is considered to be a cancer suppressor gene [29-31]. Li et al. sequenced micro- RNAs from mouse adrenal tumors and demonstrated that miR-486-3p was notably diminished, while overexpression miR-486-3p caused a decline in the level of its target gene, FASN, which in turn hindered the proliferation of ACC cells [22]. Our study revealed a notable decrease in miR-486-3p level in ACC cells and tissues. Notably, ß-elemene treatment caused a markedly rise in miR-486-3p expression in ACC cells. Not only that, overexpression miR-486-3p hin- dered cell clone formation, decreased the amounts of mi- grating cells, and induced apoptosis. It indicated that an important mechanism for ß-elemene to exert cancer
inhibitory effects may be linked to the increased expression of miR-486-3p.
The primary way miRNAs identify a target gene is through base pairing and binding to the target gene’s 3’-UTR in a comple- mentary fashion to either degrade the target mRNA or block its translation, which may be the main mechanism by which miRNAs regulate cancer progression [32, 33]. We hypothesized that miR-486-3p may exert cancer suppressor gene effects by regulating downstream target gene expression. Using TargetS- can, miRWalk, miRDB, and GEPIA databases, we identified that NPTX1 is the downstream target gene of miR-486-3p, and further confirmed that miR-486-3p can indeed target negative regulate NPTX1.
NPTX1 is mainly found predominantly in central neurons, promotes neuronal growth, and is a secreted glycoprotein [34]. Various studies suggest a connection between NPTX1 and the progression of multiple cancer types [35-37]. Krsteski et al. discovered elevated level of NPTX1 in uterine smooth muscle
A
Relative miR-486-3p level
B
1.5
Relative NPTX1 protein
C
i-NC+O-NC+PBS
6-
i-NC+O-NC+PBS
Tumor volume (mm3)
1000-
i+O-NC+PBS
i+O-NPTX1+PBS
1.0-
NPTX1
47KDa
level
4
800
i+O-NPTX1+ß-elemene
*
600-
*
GAPDH
*
36KDa
*
0.5-
i+O-NC+PBS
i+O-NPTX1+PBS
i+O-NPTX1+ß-elemene
2
400
0.0
200
i-NC+O-NC+PBS
0
i-NC+O-NC+PBS
0
i+O-NC+PBS
i+O-NC+PBS
7
14
21
28
35
i+O-NPTX1+PBS
i+O-NPTX1+PBS
i+O-NPTX1+ß-elemene
i+O-NPTX1+ß-elemene
Time (d)
D
E
F
1000
i-NC+O-NC+PBS
Tumor weight (mg)
800
i+O-NC+PBS
600
Ki-67
i+O-NPTX1+PBS
400
200
i+O-NPTX1+ ß-elemene
i-NC+O-NC+PBS i+O-NC+PBS
i+O-NPTX1 +PBS
i+O-NPTX1
0
i-NC+O-NC+PBS
+ß-elemene
i+O-NC+PBS
0- 1 2 3 4 5 6 7 8 9 10 11
i+O-NPTX1+PBS
i+O-NPTX1+ß-elemene
G
TUNEL
TUNEL positive cells (%)
15
**
10
*
DAPI
5
0
Merge
i-NC+O-NC+PBS
i+O-NC+PBS
i+O-NPTX1+PBS
i+O-NPTX1+ß-elemene
i-NC+O-NC+PBS
i+O-NC+PBS
i+O-NPTX1+PBS
i+O-NPTX1+ß-elemene
tumors by RNA sequencing [38]. The results of Yamaguchi et al. also showed that NPTX1 was highly expressed in pancreatic ductal adenocarcinoma cells in humans and mice, and that upregulation of its expression resulted in liver metastasis [39]. It was discovered that there was a marked elevation in NPTX1 expression in ACC cells and tissues in contrast to normal, a result that was consistent with the GEPIA database. Addition- ally, overexpression miR-486-3p led to a notable decline in NPTX1 protein expression in ACC cells, and miR-486-3p was negatively correlated with the expression of NPTX1, suggesting that miR-486-3p targets to negatively regulate NPTX1. Not only that, overexpression NPTX1 weakened the suppressive impact of overexpression miR-486-3p on the malignant progression of ACC cells, indicating that miR-486-3p can affect ACC and exert antitumor effects through reverse regulation of NPTX1 expression.
Finally, to investigate whether ß-elemene exerts antitumor ef- fects via the miR-486-3p/NPTX1 axis, we transfected both inhibitor and OE-NPTX1 in ACC cells. We found that ß- elemene treatment reduced NPTX1 expression in ACC cells. Not only that, silencing miR-486-3p or overexpression NPTX1 both attenuated the inhibitory impacts of ß-elemene treatment on proliferation and migration, and reduced the promotion of apoptosis by ß-elemene. To explore the safety and efficacy of ß- elemene in vivo, we constructed a subcutaneous transplantation tumor model in nude mice. The results of in vivo experiments similarly showed that silencing miR-486-3p and overexpressing NPTX1 promoted tumor growth, whereas injection of ß- elemene inhibited the proliferation of ACC cells in vivo and promoted apoptosis. This further confirmed that ß-elemene hinderd the malignant biological progression of ACC via the miR-486-3p/NPTX1 axis.
|
5 Conclusion
In summary, ß-elemene downregulated NPTX1 levels by increasing miR-486-3p expression, reduced ACC cell viability, hindered cell proliferation and migration, and induced apo- ptosis, thereby inhibiting the malignant progression of ACC. This research elucidated the potential action mechanism of ß- elemene in inhibiting the malignant progression of ACC by in vitro cellular and in vivo experiments, which offers a new reference for the clinical use of ß-elemene. However, there are still some shortcomings in this research, the sample size of this experiment was small, and the use of ß-elemene as a potent anti-ACC agent and its application in the clinic will require a great deal of in-depth research.
Author Contributions
Yan Lin: Developed and planned the study, performed experiments, and interpreted results. Edited and refined the manuscript with a focus on critical intellectual contributions. Tailin Guo, Lishuang Che, Jieqiong Dong, Ting Yu, Chaiming Zeng: Participated in collecting, assessing, and interpreting the data. Made significant contributions to data interpretation and manuscript preparation. Ziyu Wu: Provided substantial intellectual input during the drafting and revision of the manuscript.
Acknowledgments
The authors have nothing to report.
Ethics Statement
The study was approved by the Ethics Committee of Fuzhou Univer- sity Affiliated Provincial Hospital (2022-197). The animal experiment protocol received approval from the Experimental Animal Ethics Committee of Fuzhou University Affiliated Provincial Hospital (2022-201).
Consent
The manuscript has not been published before, and it is not being reviewed by any other journal. The authors have all approved the content of the paper.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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