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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 5 | Page : 388-399 |
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Partner of NOB1 homolog transcriptionally activated by E2F transcription factor 1 promotes the malignant progression and inhibits ferroptosis of pancreatic cancer
Qin Yang1, Bin Yang2, Min Chen1
1 Department of Laboratory Medicine, General Hospital of Central Theatre Command, Wuhan, Hubei, China
2 Department of Burn and Plastic Surgery, General Hospital of Central Theatre Command, Wuhan, Hubei, China
| Date of Submission | 05-May-2023 |
| Date of Decision | 18-Jun-2023 |
| Date of Acceptance | 25-Jun-2023 |
| Date of Web Publication | 26-Oct-2023 |
Correspondence Address:
Dr. Qin Yang
Department of Laboratory Medicine, General Hospital of Central Theatre Command, Wuluo Road 627, Wuhan 430070, Hubei
China
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00063
Pancreatic cancer (PC) is one of the deadliest malignancies. Partner of NOB1 homolog (PNO1) has been reported to be involved in tumorigenesis. However, the role of PNO1 in PC remains to be elucidated. The purpose of this study was to examine the effects of PNO1 on the progression of PC and the possible mechanism related to E2F transcription factor 1 (E2F1), a transcription factor predicted by the JASPAR database to bind to the PNO1 promoter region and promoted the proliferation of pancreatic ductal adenocarcinoma. First, PNO1 expression in PC tissues and its association with survival rate were analyzed by the Gene Expression Profiling Interactive Analysis database. Western blot and reverse transcription-quantitative polymerase chain reaction were used to evaluate PNO1 expression in several PC cell lines. After PNO1 silencing, cell proliferation, migration, and invasion were measured by colony formation assay, 5-ethynyl-2'-deoxyuridine staining, wound healing, and transwell assays. Then, the lipid reactive oxygen species in PANC-1 cells was estimated by using C11-BODIPY581/591 probe. The levels of glutathione, malondialdehyde, and iron were measured. The binding between PNO1 and E2F1 was confirmed by luciferase and chromatin immunoprecipitation (ChIP) assays. Subsequently, E2F1 was overexpressed in PANC-1 cells with PNO1 knockdown to perform the rescue experiments. Results revealed that PNO1 was highly expressed in PC tissues and PNO1 expression was positively correlated with overall survival rate and disease-free survival rate. Significantly elevated PNO1 expression was also observed in PC cell lines. PNO1 knockdown inhibited the proliferation, migration, and invasion of PANC-1 cells. Moreover, ferroptosis was promoted in PNO1-silenced PANC-1 cells. Results of luciferase and ChIP assays indicated that E2F1 could bind to PNO1 promoter region. Rescue experiments suggested that E2F1 overexpression reversed the impacts of PNO1 depletion on the malignant behaviors and ferroptosis in PANC-1 cells. Summing up, PNO1 transcriptionally activated by E2F1 promotes the malignant progression and inhibits the ferroptosis of PC.
Keywords: E2F transcription factor 1, ferroptosis, invasion, pancreatic cancer, PNO1
How to cite this article:
Yang Q, Yang B, Chen M. Partner of NOB1 homolog transcriptionally activated by E2F transcription factor 1 promotes the malignant progression and inhibits ferroptosis of pancreatic cancer. Chin J Physiol 2023;66:388-99 |
How to cite this URL:
Yang Q, Yang B, Chen M. Partner of NOB1 homolog transcriptionally activated by E2F transcription factor 1 promotes the malignant progression and inhibits ferroptosis of pancreatic cancer. Chin J Physiol [serial online] 2023 [cited 2023 Dec 4];66:388-99. Available from: https://www.cjphysiology.org/text.asp?2023/66/5/388/388469 |
| Introduction | |  |
Pancreatic cancer (PC), a devastating malignancy with a median survival time of 6 months after diagnosis and a 5-year overall survival rate of only 5%, is one of the leading causes of cancer-related deaths worldwide.[1],[2] Although there are many treatments available, such as chemotherapy and radiofrequency ablation, surgical excision is the most effective way for the treatment of PC. Most patients are in advanced stages by the time of diagnosis, which led to the limitation of the possibility of surgical treatment of the disease.[3] Understanding the biological process and molecular mechanism of PC progression may contribute to the effective treatment of this disease.
Partner of NOB1 homolog (PNO1), also known as Dim2 or Rrp2, is a ribosome assembly factor and is critical in ribosome biogenesis.[4],[5] As a gene highly conserved from yeast to mammals, PNO1 has been reported to be involved in the occurrence and progression of cancers. For instance, PNO1 was significantly upregulated in tissues of breast cancer patients and associated with survival, and deletion of PNO1 inhibited tumorigenesis.[6] PNO1 was considered an oncogene of hepatocellular carcinoma (HCC) for its promotion of the tumor growth and metastasis of HCC cells.[7] It has also been shown that PNO1 could inhibit autophagy-mediated ferroptosis through reprogramming glutathione (GSH) metabolism in HCC.[8] PNO1 loss-of-function exhibited inhibitory effects on cell proliferation, migration, and invasion of esophageal cancer cells.[9] Chen et al. have demonstrated that PNO1 upregulation promotes glioma tumorigenesis.[10] The role of PNO1 in PC remains to be elucidated. Using the JASPAR database, we found that E2F transcription factor 1 (E2F1) might bind to the PNO1 promoter. E2F1 is the earliest and most widely studied member in the E2F family of transcription factors.[11] It is worthy of note that E2F1 can promote the proliferation of pancreatic ductal adenocarcinoma cells.[12] However, the role of PNO1 in PC and whether PNO1 could be transcriptionally activated by E2F1 to promote the malignant progression of PC has not been reported until now and remains largely unknown.
In the present study, the expression of PNO1 in PC tissues and cell lines was investigated. The further experiments evaluated the effects of PNO1 knockdown on the malignant progression and ferroptosis of PC cells and explore the relationship between PNO1 and E2F1 to reveal the mechanisms underlying PC.
| Materials and Methods | | |
Bioinformatic analysis
PNO1 expression in PC tissues and the relationship between PNO1 high expression and overall survival rate or disease-free survival rate in patients with PC were analyzed by Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/).
Cell culture
Several human PC cell lines (BxPC-3, SW1990, and PANC-1) and the pancreatic duct epithelial cell line HPDE6c7 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS) at 37°C in a humidified 95% air and 5% CO2 atmosphere.
Cell transfection
PANC-1 cells were harvested in the logarithmic growth phase and subsequently inoculated into 6-well plates at a density of 1 × 105 cells/mL. Short hairpin RNA specific to PNO1 (shRNA-PNO1#1/#2), the negative control (sh-NC), E2F1 overexpressed (Oe) plasmids (Oe-E2F1#1/#2) and the empty vector plasmid (Oe-negtive control, Oe-NC) were synthesized by GenePharma (Shanghai, China). Lipofectamine 2000 reagents (Invitrogen Co., Carlsbad, CA, USA) were used to transfect these plasmids into PANC-1 cells according to the instructions provided by the manufacturer. Forty-eight hours post-transfection, the transfection efficiency was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot.
Cell counting Kit-8 assay
Following transfection, CCK-8 (Solarbio, Beijing, China) was applied to detect the cell viability. PANC-1 cells were seeded into a 96-well plate at the density of 1 × 103 cells per well. After incubation for 24, 48, and 72 h, cells were exposed to 10 μL CCK-8 reagent at 37°C for 2 h. The absorbance reflecting the cell viability was detected by a microplate reader (Bio-Rad) at 450 nm.
Colony formation assay
The transfected PANC-1 cells were plated into a 6-well plate for 500 cells per well. After 14 days of culture in standard culture conditions, cells were fixed with 4% paraformaldehyde for 15 min for fixation and stained with 0.1% crystal violet solution. The visible colonies were photographed by a light microscope.
5-ethynyl-2'-deoxyuridine staining
An EdU kit (BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488; Beyotime Institute of Biotechnology) was used to detect cell proliferation. Following culture for 3 days in 24-well plates, PANC-1 cells were incubated with EdU, fixed with 4% paraformaldehyde, and stained with DAPI for 2, 0.5 h, and 10 min. At last, the proliferation of the cells was observed and imaged by an inverted fluorescence microscope.
Wound healing assay
Following transfection, PANC-1 cells were added to the each well of the 6-well plate, which was cultured to form a cell monolayer. A pipette tip was used to scratch the cells horizontally and then cultured in serum-free medium for 24 h at 37°C. Cells were photographed by an inverted microscope and the migration distance was analyzed by ImageJ software.
Transwell assay
Cell invasion assay was performed using transwell chambers which were coated with Matrigel (BD Biosciences, USA). Following transfection, PANC-1 cells were collected and 5 × 104 cells were added to the 200 μL serum-free medium in the upper chambers. The bottom chamber was supplemented with 800 μL DMEM containing 10% FBS as chemoattractant. After incubation for 24 h, the cells in the upper chamber were removed and the obtained cells were fixed with paraformaldehyde and stained with 0.1% crystal violet for 10 min. The observation of the invasive cells was performed by an inverted microscope under five randomly selected fields.
Detection of lipid reactive oxygen species
To detect the level of lipid reactive oxygen species (ROS), cells were stained with 10 μM C11-BODIPY581/591 probe (Invitrogen) for 30 min. Analysis of C11-BODIPY581/591 fluorescence was conducted using a confocal microscopy.
Measurement of glutathione, malondialdehyde, and iron levels
The contents of GSH, malondialdehyde (MDA), and iron in PANC-1 cells were measured using the GSH assay kit (A006-2-1; Nanjing Jiancheng Bioengineering Institute), MDA assay kit (A003-4-1; Nanjing Jiancheng Bioengineering Institute) and iron assay kit (ab83366; Abcam) following the manufacturer's protocols. The absorbance was detected by a microplate reader (Bio-Rad).
Luciferase reporter assay
The putative E2F1-binding site on the PNO1 promoter was identified through the JASPAR database.[13] Using Lipofectamine® 3000 (Thermo Fisher Scientific, Inc.), pGL3 vectors (Promega Corporation) containing the wild-type (WT) PNO1 promoter sequence or the corresponding mutant PNO1 promoter sequence (PNO1-MUT) were co-transfected with Oe-E2F1#1/#2 or Oe-NC into PANC-1 cells. 48 h after transfection, the luciferase activity was evaluated using Dual-Luciferase® Reporter Assay System (Promega Corp., USA). The luciferase activity was normalized to Renilla luciferase activity.
Chromatin immunoprecipitation assay
The ChIP assay was conducted with the help of a commercially available kit (Wanleibio, China) according to the kit instructions. PANC-1 cells were cross-linked with 1% formaldehyde solution for 10 min. Then, the cross-linking reaction was quenched with glycine. DNA fragments were extracted by sonication and immunoprecipitated overnight at 4°C with anti-E2F1 antibody or NC IgG. The immunoprecipitated DNA fragments were isolated and subsequently analyzed using PCR.
Reverse transcription-quantitative polymerase chain reaction
RNA was extracted using TRIzol kit (Invitrogen, USA) based on the manufacturer's instructions. RNA was then reverse-transcribed into cDNA using the RT kit (Toyobo, Osaka, Japan). Finally, qPCR was carried out by SYBR Real-time PCR kit (Takara, Japan) under the guidance of manufacturers. Relative gene expression levels were calculated using the 2−ΔΔCt method. GAPDH was used as endogenous control.
Western blot
Proteins were extracted using a RIPA lysis buffer (Beyotime, China), and the protein concentrations were measured using the BCA protein assay (Pierce, USA). Proteins were separated using 10% SDS-PAGE and were transferred to the PVDF membranes (Millipore, USA). Membranes were blocked with 5% skim milk and incubated with the indicated antibodies at 4°C overnight. After being washed three times with TBST, membranes were incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized using ECL Plus (Thermo Fisher Scientific) and the signals were quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). Blotting with GAPDH antibody was used as the loading control.
Statistical analysis
All experiments were independently repeated in triplicate. The collected data were presented as mean ± standard deviation. GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA) was used for all statistical analyses. Comparisons among multiple groups were demonstrated by one-way analysis of variance with Tukey's post hoc test. Data with P < 0.05 were considered statistically significant.
| Results | | |
PNO1 expression was significantly elevated in pancreatic cancer tissues and cell lines
To figure out the role of PNO1 in PC, the GEPIA database was initially used to analyze PNO1 expression in PC tissues and the relationship between high expression of PNO1 and patient survival. Significantly elevated PNO1 expression was observed in the PC tissues when compared to the normal tissues [Figure 1]a. It was also found that highly expressed PNO1 predicted a lower overall survival rate and disease-free survival rate [Figure 1]b and [Figure 1]c. These results suggested that abnormally high expression of PNO1 was closely related to tumorigenesis of PC. Then, the in vitro experiments were performed to explore the effects of PNO1 on the malignant phenotypes of PC cells. Firstly, PNO1 expression in several PC cell lines (BxPC-3, SW1990, and PANC-1) and pancreatic duct epithelial cell line HPDE6c7 was detected by RT-qPCR and western blot to analyze whether there was abnormally high expression of PNO1 in PC cells. Results indicated that PNO1 was obviously upregulated in PC cell lines (BxPC-3, SW1990, and PANC-1) comparison to the pancreatic duct epithelial cell line HPDE6c7 [Figure 1]d and [Figure 1]e. The highest PNO1 expression was observed in PANC-1 cells, so PANC-1 cells were selected for subsequent experiments. These results revealed PNO1 may play a promoting role in PC. | Figure 1: PNO1 expression was significantly elevated in pancreatic cancer (PC) tissues and cell lines. (a) Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the PNO1 expression in tissues of patients with pancreatic adenocarcinoma (pancreatic adenocarcinoma; num (T) =179) and normal pancreatic tissues (num (N) =171). Tumor color is red and the mormal color is grey. We used log2 (TPM + 1) for log-scale. *P < 0.05. (b) The relationship between PNO1 upregulation and PC patient's overall survival rate was predicted by GEPIA database. (c) The relationship between PNO1 upregulation and disease-free survival rate was predicated by GEPIA database. (d) The mRNA expression of PNO1 expression in PC cell lines (BxPC-3, SW1990, and PANC-1) and the pancreatic duct epithelial cell line HPDE6c7 cells was measured using reverse transcription-quantitative polymerase chain reaction. (e) The protein expression of PNO1 expression in PC cell lines (BxPC-3, SW1990 and PANC-1) and the pancreatic duct epithelial cell line HPDE6c7 cells was measured using western blot assay. **P < 0.01, ***P < 0.001 versus HPDE6c7. All experiments were replicated three times. PNO1: Partner of NOB1 homolog.
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PNO1 silencing inhibited the proliferation, migration, invasion, and epithelial-to-mesenchymal transition of pancreatic cancer cells
Initially, shRNA-PNO1 was transfected into PANC-1 cells. To explore the effects of PNO1 silence on the proliferation, migration, and invasion of PANC-1 cells, CCK-8 assay, colony formation assay, EdU staining, wound healing assay, and transwell invasion assay were conducted. As exhibited in [Figure 2]a and [Figure 2]b, PNO1 was conspicuously downregulated in the transfected groups relative to the sh-NC group. The lower PNO1 level was observed in PANC-1 cells transfected with shRNA-PNO1#2, which was chosen to conduct the following experiments. PNO1 silencing remarkably decreased the viability and proliferation of PANC-1 cells, which was found by the results of CCK-8 assay, colony formation assay, and EdU staining [Figure 2]c, [Figure 2]d, [Figure 2]e. In addition, the capacities of migration and invasion of PANC-1 cells were alleviated in the PNO1 silenced group compared with the sh-NC group [Figure 2]f and [Figure 2]g. During EMT, loss of cell-cell adhesion occurred in epithelial cells and the cells acquired mesenchymal cell features and became more invasive and motile. At molecular levels, it was well known that the epithelial markers such as E-cadherin expression were reduced and mesenchymal markers such as vimentin and snail expression were elevated.[14],[15] The present study revealed that PNO1 knockdown led to upregulated expression of E-cadherin and downregulated expression of N-cadherin and Snail, suggesting the inhibition of EMT in PNO1-silenced group [Figure 2]h. The above data suggested that silencing of PNO1 inhibits the progression of PC. | Figure 2: PNO1 silencing inhibited the proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT) of pancreatic cancer cells. (a) The mRNA expression of PNO1 in PANC-1 cells transfected with shRNA-PNO1 was detected by reverse transcription-quantitative polymerase chain reaction. (b) The protein expression of PNO1 in PANC-1 cells transfected with shRNA-PNO1 was detected by western blot. (c) The viability of PNO1-silenced PANC-1 cells was evaluated by Cell counting Kit-8 assay. (d) The proliferation of PNO1-silenced PANC-1 cells was tested by colony formation assay. (e) The proliferation of PNO1-silenced PANC-1 cells was tested by 5-ethynyl-2'-deoxyuridine staining. (f) The migration of PNO1-silenced PANC-1 cells was detected using wound healing. (g) The invasion of PNO1-silenced PANC-1 cells was detected by transwell assay. (h) Western blot assay was adopted for the measurement of EMT-related proteins in PNO1-silenced PANC-1 cells. ***P < 0.001 versus sh-NC. All experiments were replicated three times. PNO1: Partner of NOB1 homolog. sh-NC: Short hairpin negative control.
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PNO1 silencing induced the ferroptosis of pancreatic cancer cells
Ferroptosis is a recently discovered type of programmed cell death that is induced by iron-dependent lipid peroxidation and ROS.[16] A previous study revealed that lipid peroxidation is an important signaling event that triggers ferroptosis.[17] To investigate the changes in lipid peroxidation of PANC-1 cells, the lipid ROS and the lipid peroxidation end product MDA were detected using BODIPY 581/591 C11 kit and MDA assay kit. GSH plays a key role in maintaining the balance of oxidation and reduction, which exhibits corresponding changes when ferroptosis occurs.[18] Therefore, a GSH assay kit was used to evaluate GSH content in PANC-1 cells. As what is observable from [Figure 3]a, [Figure 3]b, [Figure 3]c, PNO1 loss-of-function significantly increased the level of lipid ROS and elevated the levels of MDA and iron when compared to the sh-NC group. Concurrently, GSH content was markedly reduced after transfection with sh-PNO1 [Figure 3]d. Solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1), acyl-CoA synthetase long-chain family (ACSL4) and transferrin receptor protein 1 (TFR1) were several key proteins involved in ferroptosis.[19] Results of western blot showed the decreased SLC7A11 and FTH1 expression as well as increased ACSL4 and TFR1 expression relative to the sh-NC group [Figure 3]e. Overall, these data suggested that PNO1 silencing promotes the ferroptosis of PC cells. | Figure 3: PNO1 silencing induced the ferroptosis of pancreatic cancer cells. (a) The generation of lipid reactive oxygen species in PNO1-silenced PANC-1 cells was detected using C11-BODIPY581/591 staining. (b) The level of GSH in PNO1-silenced PANC-1 cells was measured by the glutathione assay kit. (c) The level of MDA in PNO1-silenced PANC-1 cells was measured by the MDA assay kit. (d) The level iron in PNO1-silenced PANC-1 cells was measured by the iron assay kit. (e) The expression of ferroptosis-related proteins in PNO1-silenced PANC-1 cells was determined by western blot. ***P < 0.001 versus sh-NC. All experiments were replicated three times. PNO1: Partner of NOB1 homolog, sh-NC: Short hairpin negative control.
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PNO1 could be transcriptionally activated by E2F1 in pancreatic cancer cells
To figure out the relationship between PNO1 and E2F1 in PC cells, the putative E2F1-binding site on the PNO1 promoter was first identified through the JASPAR database. The binding sites of E2F1 and PNO1 promoters are presented in [Figure 4]a. PANC-1 cells exhibited higher E2F1 mRNA and protein expression than HPDE6c7 cells [Figure 4]b and [Figure 4]c. Then, E2F1 was overexpressed by transfection with Oe-E2F1#1 and Oe-E2F1#2, and significantly elevated E2F1 mRNA and protein expression was observed after transfection as a comparison to the empty vector group [Figure 4]d and [Figure 4]e. Subsequently, to validate the transcriptional activation effect of E2F1 on PNO1, we conducted the dual luciferase reporter assay and ChIP assay. We mutated the site in the PNO1 promoter region where E2F1 primarily binds and then examined the luciferase activity. E2F1 upregulation led to the enhanced luciferase activity in the PNO1-WT group, especially in the Oe-E2F1#2 transfected group [Figure 4]f. Results of ChIP assay indicated that DNA fragments of PNO1 promoter region were precipitated by E2F1 antibody, but not IgG control [Figure 4]g. The higher Oe-E2F1 expression was observed in the Oe-E2F1#2 group. To validate the regulatory effect of E2F1 on PNO1 expression, PNO1 expression was assessed after E2F1 overexpression. It was found that E2F1 gain-of-function conspicuously elevated PNO1 mRNA and protein expression relative to the Oe-NC group [Figure 4]h and [Figure 4]i. PANC-1 cells transfected with Oe-E2F1#2 exhibited higher PNO1 expression. Therefore, Oe-E2F1#2 plasmid was selected to perform the subsequent experiments. These observations revealed that PNO1 could be transcriptionally activated by E2F1 in PC cells. | Figure 4: PNO1 could be transcriptionally activated by E2F1 in pancreatic cancer cells. (a) The putative E2F1-binding site on the PNO1 promoter was identified through the JASPAR database. (b) The mRNA expression of E2F1 in PANC-1 cells and HPDE6c7 cells was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (c) The protein expression of E2F1 in PANC-1 cells and HPDE6c7 cells was detected using western blot assay. ***P < 0.001 versus HPDE6c7. (d) The mRNA expression of E2F1 in E2F1-overexpressed PANC-1 cells was tested by RT-qPCR. (e) The protein expression of E2F1 in E2F1-overexpressed PANC-1 cells was tested by western blot assay. ***P < 0.001 versus Oe-NC. (f) The interaction between E2F1 and PNO1 promoter was confirmed by dual-luciferase reporter assay. (g) The interaction between E2F1 and PNO1 promoter was confirmed by chromatin immunoprecipitation assay. ***P < 0.001 versus Oe-NC or IgG. (h) The mRNA expression of PNO1 in E2F1-overexpressed PANC-1 cells was detected using RT-qPCR. (i) The protein expression of PNO1 in E2F1-overexpressed PANC-1 cells was detected using western blot assay. ***P < 0.001 versus Oe-NC. All experiments were replicated for three times. PNO1: Partner of NOB1 homolog, E2F1: E2F transcription factor 1. Oe-NC: Overexpressed negtive control.
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E2F1 overexpression reversed the impacts of PNO1 knockdown on the proliferation, migration, invasion and EMT of pancreatic cancer cells
With the aim of exploring the molecular mechanism of PNO1 and E2F1 in PC, sh-PNO1, and Oe-E2F1 were transfected into PANC-1 cells to clarify whether PNO1 affected the progression of PC through transcriptional activation by E2F1. Firstly, it was observed that Oe-E2F1 overexpression significantly upregulated PNO1 expression when compared to the sh-PNO1 + Oe-NC group [Figure 5]a and [Figure 5]b. Additionally, sh-PNO1 transfection had no significant difference in E2F1 expression compared to the control group [Figure 5]c and [Figure 5]d. However, the further E2F1 overexpression elevated PNO1 level in comparison to the sh-PNO1 + Oe-NC group. As shown in [Figure 5]e, [Figure 5]f, [Figure 5]g, [Figure 5]h, E2F1 overexpression significantly increased the viability and proliferation capacity of PANC-1 cells when compared to the sh-PNO1 + Oe-NC group. As expected, cell migration and invasion were also promoted in PANC-1 cells with PNO1 knockdown and E2F1 overexpression [Figure 6]a and [Figure 6]b. Results obtained from western blot suggested that E2F1 upregulation partially counteracts the impacts of PNO1 knockdown on the EMT of PANC-1 cells, evidenced by the decreased E-cadherin expression and increased N-cadherin and Snail expression after Oe-E2F1 transfection [Figure 6]c. Through the above findings, we proved that PNO1 transcriptionally activated by E2F1 promoted the malignant progression of PC cells. | Figure 5: E2F1 overexpression reversed the impacts of PNO1 knockdown on the proliferation of pancreatic cancer cells. PNO1 expression in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was tested by (a) reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and (b) western blot. E2F1 expression in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was detected by (c) RT-qPCR and (d) western blot. (e) The viability of PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was evaluated by cell counting kit-8 assay. The proliferation of PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was tested by (f and g) colony formation assay and (h) 5-ethynyl-2'-deoxyuridine staining. ***P < 0.001 versus control; ##P < 0.01, ###P < 0.001 versus sh-PNO1 + Oe-NC. All experiments were replicated for three times. E2F1: E2F transcription factor 1, PNO1: Partner of NOB1 homolog.
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 | Figure 6: E2F1 overexpression reversed the impacts of PNO1 knockdown on the migration, invasion and epithelial-to-mesenchymal transition (EMT) of pancreatic cancer cells. (a) The migration of PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was detected by means of wound healing assay. (b) The invasion of PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was detected by means of transwell assay. (c) Western blot assay was used to evaluate the expression of EMT-related proteins in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1. ***P < 0.001 versus control; ##P < 0.01, ###P < 0.001 versus sh-PNO1 + Oe-NC. All experiments were replicated for three times. E2F1: E2F transcription factor 1, PNO1: Partner of NOB1 homolog.
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E2F1 overexpression alleviated the induction of PNO1 knockdown on the ferroptosis of pancreatic cancer cells
The impacts of E2F1 overexpression on ferroptosis in PNO1-silenced PANC-1 cells were discussed through rescue experiments. As exhibited in [Figure 7]a, the significantly increased level of lipid ROS in PNO1 silenced group was abrogated by the further E2F1 overexpression in PANC-1 cells. Remarkable increased GSH levels and decreased MDA and iron levels were also found in the sh-PNO1 + Oe-E2F1 group when compared to the sh-PNO1 + Oe-NC group [Figure 7]b, [Figure 7]c, [Figure 7]d. Moreover, the results of the western blot showed that E2F1 overexpression alleviated the impact of PNO1 depletion on the expression of ferroptosis-related proteins, as reflected by the upregulated expression of SLC7A11 and FTH1, as well as the downregulated expression of ACSL4 and TFR1 [Figure 7]e. Together, these findings confirmed that PNO1 transcriptionally activated by E2F1 suppressed the ferroptosis of PC cells. | Figure 7: E2F1 overexpression alleviated the induction of PNO1 knockdown on the ferroptosis of pancreatic cancer cells. (a) The lipid reactive oxygen species in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was detected using C11-BODIPY581/591 staining. (b) The level of GSH in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was measured by GSH assay kit. (c) The level of MDA in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was measured by MDA assay kit. (d) The level ofiron in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was measured by iron assay kit. (e) The expression of ferroptosis-related proteins in PANC-1 cells co-transfected with sh-PNO1 and Oe-E2F1 was determined by western blot. ***P < 0.001 versus control; ##P < 0.01, ###P < 0.001 versus sh-PNO1 + Oe-NC. All experiments were replicated for three times. E2F1: E2F transcription factor 1, PNO1: Partner of NOB1 homolog, GSH: Glutathione.
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| Discussion | | |
PC is one of the deadliest gastrointestinal cancers with a poor prognosis due to nonspecific early symptoms and early metastasis.[20] As cancer with high mortality, the pathogenetic mechanism of which remains vague. Previous reports have affirmed that alterations in the oncogenes and tumor suppressor genes contribute to the progression and metastasis of PC.[21],[22] Meanwhile, the progress of molecular genetics has greatly accelerated the identification of molecular markers of PC, which is conducive to the diagnosis and treatment of PC. Therefore, the exploration of potential genes related to the carcinogenesis and development of PC is requisite for the treatment of this disease.
Cancer cells rely on enhanced ribosome biogenesis, which is increased in response to elevated protein synthesis and to sustain unrestricted growth.[23] Accumulating research has demonstrated the abnormal expression of PNO1, a ribosome assembly factor critical for ribosome biogenesis, in multiple human cancers.[24] PNO1 participates in almost all aspects of cancer biology.[24] For instance, significantly elevated PNO1 expression was observed in lung adenocarcinoma compared to normal lung tissues, and PNO1 loss-of-function suppressed the cell viability and colony formation of lung cancer cells.[25] In urinary bladder carcinoma, PNO1 is closely associated with promoting proliferation, migration, metastasis, and caulogenesis, and silencing PNO1 has also been confirmed to alleviate the tumorigenic ability of urinary bladder carcinoma in mice.[26] By depressing PNO1 expression, early B-cell factor 1 restrains tumor growth in vivo and in vitro in colorectal cancer.[27] It is worth noting that PNO1 knockdown inhibits the proliferation, migration, and invasion of esophageal cancer cells.[9] In line with previous findings, we also found in this study that PNO1 was highly expressed in PC tissues and cells, and PNO1 depletion inhibited the proliferation, migration, and invasion of PC cells. In addition, EMT is undoubtedly one of the critical mechanisms responsible for tumor metastasis including in PC, during which epithelial cells acquire mesenchymal characteristics.[27],[28],[29] EMT is a cellular process which is often defined by the loss of the epithelial marker E-cadherin and the gain of the mesenchymal marker N-cadherin and snail.[30] In particular, PNO1 silencing inhibited the EMT process of lung cancer cells.[25] In the present study, the knockdown of PNO1 led to the upregulated E-cadherin expression and downregulated N-cadherin and snail expression in PANC-1 cells, suggesting the inhibitory effect of PNO1 deletion on EMT.
Ferroptosis is a recently discovered type of programmed cell death that is induced by iron-dependent lipid peroxidation and ROS.[31] The essence of ferroptosis is the metabolic disorder of intracellular lipid oxidation. Under the catalysis of iron ions, abnormal metabolism leads to the generation of a large number of lipids, disrupting the intracellular redox balance, damaging biological macromolecules, and ultimately triggering cell death.[32] Cancer cells exhibit increased iron dependence and enhanced sensitivity to ferroptosis compared to normal cells.[19] In recent years, ferroptosis has been found in pathological processes related to tumor formation and a growing body of evidence suggests that PC is susceptible to ferroptosis through elevating iron uptake and reducing iron efflux.[33],[34] FTH1 is an important member in the process of iron metabolism and is responsible for the storage of Fe2+.[35] ACSL4 plays an essential role in ferroptosis execution by regulation of lipid composition.[36] Moreover, TFR1 is considered a specific marker of ferroptosis as it mediates the transfer of iron-containing ferritin from the extracellular to the intracellular compartment.[37] Importantly, SLC7A11 is one of the two core components of system Xc-, which plays a key role in regulating ferroptosis by serving as the rate-limiting precursor for GSH.[38] Emerging evidence supports that PNO1 inhibits autophagy-mediated ferroptosis through the reprogramming of GSH metabolism in HCC.[8] The regulatory effect of PNO1 on ferroptosis in PC has been investigated in our study. We demonstrated that PNO1 silencing induced ferroptosis in PANC-1 cells, as evidenced by increased lipid ROS and MDA levels, reduced GSH content, downregulated expression of SLC7A11 and FTH1, as well as upregulated expression of ACSL4 and TFR1.
To explore the mechanisms of PNO1 in PC, the JASPAR database was used to find the transcription factor that could regulate PNO1 expression, and it was noticed that E2F1 might bind to the PNO1 promoter. It is well known that E2F1 is the earliest and most widely studied member of the transcription factor E2F family.[11] E2F1 participates in the control of the cell proliferation, differentiation, and metabolic activity.[39],[40] Numerous studies have suggested that increased E2F1 expression leads to abnormal cell growth, thereby affecting the occurrence and development of malignant tumors.[41],[42] E2F1 has been reported to accelerate the proliferation and metastasis of clear cell renal cell carcinoma, cervical cancer, gastric cancer, and non-small cell lung cancer.[43],[44],[45],[46] It is worthy of note that E2F1 also promotes the proliferation, migration, and invasion of PC cells.[47] Much work thus far has shown the inhibitory effect of E2F1 in the regulation of ferroptosis.[48],[49] Results of our present study further explore the mechanisms of E2F1 in PC, and we demonstrated that PNO1 could be transcriptionally activated by E2F1 in PC cells, and E2F1 overexpression reversed the impacts of PNO1 knockdown on the malignant progression and inhibited ferroptosis of PC cells.
| Conclusion | | |
Taken together, our study reveals that PNO1, transcriptionally activated by E2F1, promotes malignant progression and inhibits ferroptosis in PC. This finding provides new insights into the mechanism underlying PC and offers a promising therapeutic strategy for PNO1-targeted treatment in PC.
Data availability statement
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
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