Abstract
Nuclear pore membrane protein 121 (POM121) is a part of the nuclear pore complex, which regulates intracellular signaling and maintains normal cellular functions. However, the role of POM121 in gastric cancer (GC) remains unclear. Quantitative real-time polymerase chain reaction was performed to detect POM121 mRNA in 36 pairs of GC and adjacent non-tumor tissues. POM121 protein expression was determined by immunohistochemistry in 648 GC tissues and 121 normal gastric tissues. Connections between POM121 levels, clinicopathological parameters, and the prognosis of GC patients were explored. The influence of POM121 on proliferation, migration, and invasion was detected in vitro and vivo. The mechanism underlying the involvement of POM121 in GC progression was demonstrated via bioinformatics analysis and Western blot. Both the mRNA and protein levels of POM121 in GC tissues were higher than those in normal gastric tissues. High POM121 expression in GC was associated with deep invasion, advanced distant metastases and TNM stage, and positive HER2 expression. A negative connection was found between POM121 expression and the overall survival (OS) of GC patients. Downregulation of POM121 inhibited the proliferation, clone formation, migration, and invasion of GC cells, and overexpression of POM121 showed the opposite trend. POM121 promoted the phosphorylation of PI3K/AKT pathway and increased the expression of MYC. In conclusion, this study suggested that POM121 has the potential to act as an independent prognostic factor for GC patients.
Keywords: POM121, gastric cancer, proliferation, metastasis, PI3K/AKT, MYC
Introduction
Gastric cancer (GC) is one of the most common and fatal cancers globally, with most of the new cases diagnosed in developing countries [1]. The prevalence of GC is second only to lung carcinoma and becomes the second leading cause of cancer-related mortality in China [2]. The symptoms of early-stage GC are not prominent, thus a large proportion of GC patients have advanced-stage disease at the time of diagnosis. Despite the continuous advances in GC treatment methods, the prognosis of most GC patients is still not optimistic. Therefore, the identification of new prognostic biomarkers and therapeutic strategies is indispensable to curing this disease and improving quality of life for GC patients.
The nuclear pore complex (NPC) is the sole gateway embedded in the nuclear envelope and controls transport between the cytoplasm and the nucleus [3]. NPC is reported to have many transport-independent functions such as chromatin organization [4], RNA processing [5], DNA repair [6], transcriptional regulation [7], and cell cycle control [8]. NPC plays an important role in maintaining the homeostasis of cellular signaling and protein localization, the dysregulation of which is closely related to carcinogenesis [9]. The involvement of several nucleoporins (NUPs) has been demonstrated in tumorigenesis and progression. TPR is the first validated NUP associated with cancer in osteogenic sarcoma cells and is thought to be involved in the early stages of gastric carcinomas [10]. NUP98 fusion proteins are important in many myeloid malignancies, and NUP98 acts as a potential tumor suppressor by regulating the expression of genes targeted by p53 [11-13]. NUP62, NUP88, NUP214, and NUP358/RANBP2 are also connected with tumorigenesis [14]. POM121 is reported to promote lethal prostate cancer, but little is known about its role in GC [15].
The present study aimed to determine the role of POM121 in GC. The differences in POM121 expression in GC tissues and normal tissues were investigated. We also analyzed the relationships between the POM121 level and clinicopathological features of GC patients. Then the POM121 expression was downregulated, and the biological behavior of GC cells was detected. Finally, the variation of the PI3K/AKT/MYC pathway was demonstrated in the mechanisms of POM121 in GC progression. Our findings indicate that POM121 may be a potential prognostic biomarker and new therapeutic target for GC patients in the future.
Materials and methods
POM121 expression in database
The gene expressions of GC patients have downloaded from The Cancer Genome Atlas (TCGA) Data Portal website (https://portal.gdc.cancer.gov/projects/TCGA-STAD), including 27 GC tissues and 27 normal gastric tissues. The data were analyzed by R package ggplot2.
Human gastric tissue samples
All pathological specimens were collected from the Pathology Department of Inner Mongolia Autonomous Region People’s Hospital between February 2004 and November 2014. A total of 983 tissue samples were obtained from patients after surgery or gastric endoscopic biopsy, including 684 GC tissues, 121 paired para-carcinoma tissues, 37 chronic gastritis, 34 intestinal metaplasia, 46 low-grade intraepithelial neoplasia, and 61 high-grade intraepithelial neoplasia. None of the volunteers underwent any chemotherapy or radiotherapy before surgery or biopsy. Histopathological confirmation of tissues was performed by two pathologists blinded to each other. Clinicopathological data such as age, tumor size, grade, lymph node status, and TNM stage were retrieved from patient medical records. This study was performed following medical ethical standards and was approved by the Ethics Committee of Inner Mongolia Medical University. Written informed consent were obtained from all study participants.
Quantitative real-time polymerase chain reaction (qRT-PCR)
A total of 36 GC tissues and 36 normal tissues were prepared to detect POM121 mRNA expression. Total RNA was extracted from frozen tissues using TRIzol Plus RNA Purification Kit (#12183555, Invitrogen, USA) following the manufacturer’s instructions. Reverse transcription was performed with SuperScript First-Strand Synthesis System for RT-PCR (#11904018, Invitrogen). Amplification of the cDNA with the Power SYBR Green PCR Master Mix (#4368706, Applied Biosystems, USA) was performed using a StepOne Real-Time PCR System (Applied Biosystems). The human POM121 primers used were as follows: forward 5’-AGTGGCAGTGGACATTCAGC-3’, and reverse 5’-CGTAAGC GCCTGTCAAGGA-3’. The human GAPDH primers were as follows: forward 5’-CTGGGCTACACTGAGCACC-3’, and reverse 5’-AAGTGGTCGTTGAGGGCAA TG-3’. GAPDH was utilized to normalize POM121 gene expression, and 2-ΔΔCt was calculated as the level of POM121. All analyses were performed independently three times.
Tissue microarray (TMA) construction and immunohistochemistry (IHC)
All tissue samples were embedded in paraffin after 4% formalin fixation. A TMA was constructed with paraffin-embedded blocks of donor tissues using a Quick Ray Master UATM-272A (UNITMA, Korea). Cores biopsies of 3 mm in diameter were acquired from donor blocks, and then were cut into 3 μm thicknesses. Sections of the tissue blocks were placed on glass slides and deparaffinized in gradient ethanol. TMA was heated in citrate buffer (0.01 M, pH 6.0) for antigen retrieval, and then incubated with 3% H2O2 to block endogenous peroxidase activity. Sections were incubated with a rabbit anti-POM121 polyclonal primary antibody (#ab190015, Abcam, UK) and were stained using the Lab Vision Ultra Vision ONE Detection System (Invitrogen, USA). The score of staining intensity was categorized into four levels: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The final score was set as 100 × the product of the staining intensity and the percentage of cells stained. Immunohistochemical staining for POM121 was estimated by two pathologists blinded to patient information. The cutoff value was selected as 115 by the X-tile-based TMA data analysis. Scores between 0 and 115 represented a low expression of POM121, and scores between 116 and 300 represented a high expression of POM121.
Stable cell lines
Human GC cell lines BGC823, MG803, SGC7901, and HGC27 (FuHeng Biology, Shanghai, China) were cultured in an incubator with 5% CO2 at 37°C. The lentiviral-based shRNA targeting POM121 and lentiviral vector were purchased from Genechem Co., Ltd., Shanghai, China. The two cells were infected with the lentivirus expressing shNC or shPOM21, respectively. Lentivirus-mediated POM121-cDNA and empty vector were transfected into SGC7901 and HGC27 cells, which were called NC and ovPOM121.
Western blot
Total protein was extracted from different stable cells using RIPA buffer (#89901, Thermo Scientific, USA). The protein samples were mixed with loading buffer (#P0015A, Beyotime, China) and then degenerated at 100°C for 10 min. The same amounts of proteins were separated by sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated with the primary antibodies to POM121 (#83525, CST, USA), P-PI3K (#17366, CST), PI3K (#4249, CST), P-AKT (#ab38449, Abcam, USA), AKT (#ab8805, Abcam), MYC (#ab9106, Abcam), and GAPDH (#AM4300, Invitrogen) at 4°C overnight. After three rinses with PBST buffer for 15 min each time, the membrane was incubated with horseradish-peroxidase-labeled secondary antibodies for 2 h at room temperature. Then, the membrane was rinsed with PBST and developed with High-sig ECL Western Blotting Substrate (#180-5001, Tanon, China).
CCK-8 assay
The cells of each group were planted in a 96-well plate with 5 × 103 cells/well. The cell proliferation level was detected at 24 h, 48 h, 72 h, and 96 h, respectively. The cells in each well were incubated with a 100 μl mixture of RPMI 1640 medium and CCK-8 reagent (9:1) at 37°C in darkness for 2 h. The absorbance of each well in OD450 was measured with a microplate reader.
Clone formation assay
The cells of each group were planted in a 6-well plate with 600 cells/well. The plate was cultured in an incubator with 5% CO2 at 37°C for 2 w. Then the cells were rinsed with PBS buffer three times gently and soaked in methanol for 30 min. The cells were incubated with 0.5% crystal violet solution at room temperature for 30 min. Finally, the cell clones were rinsed by ddH2O.
Transwell assays
Migration assay
The cells were suspended in a serum-free medium and planted in the upper chamber. Place the Transwell chamber into the 24-well plate which contains medium with 20% FBS. The cells were cultured in an incubator for 24 h. The cells in the upper chamber were gently wiped with a swab. The chamber was fixated with 4% paraformaldehyde for 30 min and then stained with 0.2% crystal violet for 30 min. After rinsing and drying, cell clones were observed and counted.
Invasion assay
Matrigel basement membrane matrix (#356234, BD Biosciences, USA) was diluted with serum-free RPMI 1640 medium at a ratio of 1:3 at 4°C. 100 μl Matrigel mixture was spread on the surface of the upper chamber and dried at 37°C for 1 h. The following steps were the same as those of the migration assay.
Subcutaneous tumor model
A total of 12 five-week-old male BALB/C nude immunodeficient mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Shanghai, China). Cells of the BGC823 group were suspended with a density of 1 × 107/ml. Cell suspensions (100 µl) were injected into the left upper extremity armpit, and tumor size was measured with a vernier caliper every 6 days. The tumor volume was calculated as longest diameter × shortest diameter2/2.
Protein-protein interactions (PPI) and correlation analysis
To explore the possible mechanism of POM121, a putative protein–protein interaction network around POM121 was explored by STRING v11.5 (https://cn.string-db.org/). The visualization of PPI was performed in Cytoscape v3.9.1. The correlation between POM121 and MYC was calculated to use the Xiantao website (https://www.xiantao.love/) based on the RNAseq data of 375 GC patients downloaded from the TCGA database.
Statistical analysis
All statistical analyses were conducted on SPSS 19.0 statistical software package (SPSS, Inc., USA). The difference between the two groups was analyzed by paired test-test if the data came in pairs. If not, independent T test. Student’s t-test was used to compare differences in POM121 expression between GC and non-tumor tissues. The associations between the clinical characteristics of patients and POM121 expression were evaluated by the Chi-square test or Trend test. The Kaplan-Meier method was used to calculate overall survival, and the log-rank test was performed to analyze survival curves. Statistical significance was set at P<0.05.
Results
POM121 expression in GC tissues
The data of the TCGA database demonstrated that the gene expressions of POM121 in GC tissues were significantly higher than these in normal gastric tissues (P<0.001, Figure 1A). POM121 mRNA expression in 36 pairs of GC tissues and peritumoral normal tissues was examined, and the result confirmed that POM121 mRNA expression in GC tissues was significantly higher than that in matched normal gastric tissues (P<0.001, Figure 1B).
Figure 1.
POM121 expression in gastric tissues. A: POM121 expression in GC tissues and normal tissues in TCGA database. B: POM121 mRNA expression in 36 pairs of GC tissues and peritumoral normal tissues. C: POM121 protein expressions in gastric tissues. D: Representative images of POM121 protein in gastric tissues. a. Gastric papillary adenocarcinoma with high POM121 expression (staining score = 280). b. Low-differentiated adenocarcinoma with high POM121 expression (staining score = 260). c. Moderate-differentiated adenocarcinoma with low POM121 expression (staining score = 220). d. Well-differentiated adenocarcinoma with low POM121 expression (staining score = 190). e. Pericarcinomatous tissues with no POM121 expression (staining score = 0). (Original views ×4, bar = 500 μm; enlarged views ×40, bar = 50 μm). **P<0.01, ***P<0.001.
To confirm the protein expression of POM121 in gastric tissues, we performed IHC analysis on TMAs consisting of 684 GC tissues, 121 adjacent non-tumor tissues, 37 chronic gastritis, 34 intestinal metaplasia, 46 low-grade intraepithelial neoplasia, and 61 high-grade intraepithelial neoplasia. IHC staining demonstrated that POM121 was mainly expressed in the nuclear envelope (Figure 1D). Our results showed only 12.4% (15/121) of normal gastric tissues presented high POM121 expression, while the occurrence rate of high POM121 expression was 63.0% (433/684) in GC tissues. High POM121 expression was also detected more often in chronic gastritis (24.32%, 9/37), low-grade intraepithelial neoplasia (26.09%, 12/46), and high-grade intraepithelial neoplasia (22.95%, 14/61) than in neighboring tumor tissues (Figure 1C).
Association between POM121 expression and clinicopathological characteristics
To ascertain if POM121 affects the clinical manifestations of GC patients, we analyzed the association between POM121 expression and pathological features. POM121 level was significantly linked to depth of invasion (χ2 = 17.831, P = 0.001), distant metastases (χ2 = 7.505, P = 0.006), TNM stage (χ2 = 18.412, P = 0.001), and HER2 expression (χ2 = 6.285, P = 0.012). However, POM121 expression was found to have no connection with sex, age, histological type, differentiation, lymph node metastases, preoperative CEA, preoperative CA199, and Helicobacter pylori. Our results indicated that gastric carcinomas with high POM121 expression tend to enter a more advanced clinical stage with distant metastases than those with low POM121 expression (Table 1).
Table 1.
POM121 protein expression level and GC patient clinicopathological characteristics
| Characteristics | n | POM121 expression (%) | Pearson χ2 | P-value | |
|---|---|---|---|---|---|
|
| |||||
| Low or no | High | ||||
| Total | 684 | 251 | 433 | ||
| Gender | 2.928 | 0.087 | |||
| Male | 495 | 172 (34.75) | 323 (65.25) | ||
| Female | 189 | 79 (41.80) | 110 (58.20) | ||
| Age | 0.443 | 0.506 | |||
| <60 | 256 | 98 (38.28) | 158 (61.72) | ||
| ≥60 | 428 | 153 (35.75) | 275 (64.25) | ||
| Histological type | 0.287 | 0.991 | |||
| Tubular | 485 | 180 (37.11) | 305 (62.89) | ||
| Mucinous | 89 | 32 (35.96) | 57 (64.04) | ||
| Mixed (tubular and mucinous) | 16 | 5 (31.25) | 11 (68.75) | ||
| Signet ring cell | 45 | 16 (35.56) | 29 (64.40) | ||
| Othersa | 49 | 18 (36.73) | 31 (63.27) | ||
| Differentiation | 1.281 | 0.527 | |||
| Well | 198 | 77 (38.89) | 121 (61.11) | ||
| Moderate | 261 | 89 (34.10) | 172 (65.90) | ||
| Poor | 225 | 85 (37.78) | 140 (62.22) | ||
| Depth of invasion | 17.831 | 0.001* | |||
| Tis | 23 | 14 (60.87) | 9 (39.13) | ||
| T1 | 78 | 36 (46.15) | 42 (53.85) | ||
| T2 | 138 | 57 (41.30) | 81 (58.70) | ||
| T3 | 405 | 137 (33.83) | 268 (66.17) | ||
| T4 | 40 | 7 (17.50) | 33 (82.50) | ||
| Lymph node metastasis | 5.826 | 0.120 | |||
| N0 | 258 | 93 (36.05) | 165 (63.95) | ||
| N1 | 193 | 72 (37.31) | 121 (62.69) | ||
| N2 | 117 | 52 (44.44) | 65 (55.56) | ||
| N3 | 116 | 34 (29.31) | 82 (70.69) | ||
| Distant metastasis | 7.505 | 0.006* | |||
| M0 | 573 | 223 (38.92) | 350 (61.08) | ||
| M1 | 111 | 28 (25.23) | 83 (74.77) | ||
| TNM stage | 18.412 | 0.001* | |||
| 0 | 29 | 16 (55.17) | 13 (44.83) | ||
| I | 110 | 49 (44.55) | 61 (55.45) | ||
| II | 297 | 111 (37.37) | 186 (62.63) | ||
| III | 183 | 64 (34.97) | 119 (65.03) | ||
| IV | 65 | 11 (16.92) | 54 (83.08) | ||
| Preoperative CEA, ng/ml | 4.347 | 0.114 | |||
| <5 | 272 | 87 (31.99) | 185 (68.01) | ||
| ≥5 | 373 | 149 (39.95) | 224 (60.05) | ||
| Unknownb | 39 | 15 (38.46) | 24 (61.54) | ||
| Preoperative CA199, ng/ml | 5.007 | 0.082 | |||
| <37 | 246 | 96 (39.02) | 150 (60.98) | ||
| ≥37 | 301 | 116 (38.54) | 185 (61.46) | ||
| Unknownb | 137 | 39 (28.47) | 98 (71.53) | ||
| Helicobacter pylori | 0.451 | 0.502 | |||
| Positive | 420 | 150 (35.71) | 270 (64.29) | ||
| Negative | 264 | 101 (38.26) | 163 (61.74) | ||
| Her-2 | 6.285 | 0.012* | |||
| Negative | 561 | 218 (38.83) | 343 (61.14) | ||
| Positive | 123 | 33 (26.83) | 90 (73.17) | ||
others include: papillary adenocarcinoma 12 cases; adeno-squamous carcinoma 10 cases; squamous cell carcinoma 9 cases; undifferentiated carcinoma 8 cases; neuroendocrine carcinoma 10 cases.
Two unknown groups had no exact records, and thus, were not used in the P-value calculation.
P<0.05.
Relationship between POM121 expression and prognosis for GC patients
The median survival time of the 684 patients we investigated was 27±5.78 months. Patients with high POM121 expression showed significantly shorter median survival time than patients with low POM121 expression (21±2.43 vs 36±5.69 months, P<0.001; Figure 2A). Distant metastases and TNM stages were also significantly connected with OS (Figure 2). Univariate analysis indicated that risk factors for OS of GC patients included POM121 expression (P<0.001), age (P = 0.008), differentiation (P<0.001), distant metastasis (P<0.001), TNM stage (P<0.001), and HER2 expression (P<0.001). Multivariate analysis suggested that high POM121 expression (HR, 1.502; 95% CI: 1.197-1.883; P<0.001) was significantly associated with poor OS, as well as differentiation (HR, 1.137; 95% CI: 1.006-1.287; P = 0.040), distant metastasis (HR, 2.539; 95% CI: 1.967-3.277; P<0.001), TNM stage (HR, 1.180; 95% CI: 1.089-1.279; P<0.001), and HER2 expression (HR, 0.329; 95% CI: 0.287-0.377; P<0.001) (Table 2). These results showed that POM121 may be an independent prognostic biomarker for GC patients.
Figure 2.
Survival curves of GC patients using the Kaplan-Meier method and the log-rank test. A: Overall survival curves for patients with low POM121 expression (blue line) and patients with high POM121 expression (green line). B: Overall survival curves by TNM stage, TNM 0, I and II (blue line), TNM III and IV (green line). C: Overall survival curves by distant metastases, M0 (blue line), M1 (green line).
Table 2.
Univariate and multivariate analyses of the prognostic factors for overall survival in GC
| Univariate analysis | Multivariate analysis | |||||
|---|---|---|---|---|---|---|
|
|
|
|||||
| HR | P-value | 95% CI | HR | P-value | 95% CI | |
| POM121 expression | ||||||
| High vs Low or no | 2.020 | <0.001* | 1.619 2.520 | 1.502 | <0.001* | 1.197 1.883 |
| Age (year) | ||||||
| <60 vs ≥60 | 1.319 | <0.001* | 1.073 1.621 | 1.171 | 0.143 | 0.948 1.447 |
| Gender | ||||||
| Male vs Female | 1.024 | 0.831 | 0.825 1.270 | |||
| Differentiation | ||||||
| Well vs Moderate vs Poor | 1.259 | <0.001* | 1.108 1.430 | 1.137 | 0.040* | 1.006 1.287 |
| Tumor disameter | ||||||
| Tis + T1 + T2 vs T3 + T4 | 1.074 | 0.226 | 0.957 1.205 | |||
| Lymph node metastasis | ||||||
| N0 vs N1 + N2 | 0.975 | 0.577 | 0.893 1.065 | |||
| Distant metastasis | ||||||
| M0 vs M1 | 5.253 | <0.001* | 4.164 6.627 | 2.539 | <0.001* | 1.967 3.277 |
| TNM stage | ||||||
| 0 + I + II vs III + IV | 1.477 | <0.001* | 1.376 1.586 | 1.180 | <0.001* | 1.089 1.279 |
| Her-2 | ||||||
| Negative vs Positive | 0.270 | <0.001* | 0.238 0.306 | 0.329 | <0.001* | 0.287 0.377 |
HR: Hazard Ratio; CI: Confidence Interval.
P<0.05.
Decreased POM121 expression hindered the progression of GC
To explore the effect of POM121 on GC development, POM121 expression was knockdown in GC cell lines BGC823 and MGC803 (Figure 3A). CCK-8 assay showed that POM121 downregulation inhibited the proliferation of GC cells (Figure 3B). The result of the clone formation assay further confirmed the adverse impact of POM121 silence on cell viability (Figure 3C). Transwell assay demonstrated that the knockdown of POM121 hampered migration (Figure 3D) and invasion (Figure 3E) of GC cells. Finally, a tumor xenograft model was constructed to determine the influence of POM121 on GC development in vivo. The results displayed that shPOM121 significantly decreased the volume and weight of the subcutaneous tumor (Figure 3F-H). These results suggested that the knockdown of POM121 restrained malignant biological properties in GC.
Figure 3.
POM121 plays an oncogenic role in GC cells. A: POM121 expression of BGC823 and MGC803 cells after shPOM121 transfection. B: The proliferation of GC cells after POM121 knockdown. C: The clone formation of GC cells after POM121 silence. D: The migration of GC cells after downregulation of POM121. E: The invasion of GC cells after downregulation of POM121. F: The volumes of tumors in the xenograft mouse model. G: The growth curves of tumors in the xenograft mouse model. H: The weight of tumors in the xenograft mouse model.
Overexpression of POM121 expression promoted the progression of GC
Furthermore, we overexpressed POM121 in GC cell lines SGC7901 and HGC27 (Figure 4A). CCK-8 assay showed that high POM121 expression promoted the proliferation of GC cells (Figure 4B). The clone formation assay confirmed the promoting impact of POM121 overexpression on cell viability (Figure 4C). Transwell assay demonstrated that increased POM121 level accelerated migration (Figure 4D) and invasion (Figure 4E) of GC cells. These results suggested that overexpression of POM121 expedited malignant biological properties in GC.
Figure 4.
POM121 plays an oncogenic role in GC cells. A: POM121 expression of SGC7901 and HGC27 cells after ovPOM121 transfection. B: The proliferation of GC cells after POM121 overexpression. C: The clone formation of GC cells after POM121 overexpression. D: The migration of GC cells after upregulation of POM121. E: The invasion of GC cells after upregulation of POM121.
Silencing POM121 inhibited PI3K/AKT/MYC pathway
We constructed a protein interaction network to determine the putative mechanism as to how POM121 functions in GC. CCNB1, MYC, CDK1, and some other proteins were speculated to be linked to POM121 (Figure 5A). Pearson’s correlation analysis revealed a positive correlation between POM121 and MYC (r = 0.396, P<0.001, Figure 5B). Western Blot also confirmed that silencing POM121 decreased the expression of MYC (Figure 5C). Furthermore, phosphorylation of the PI3K/AKT pathway was inhibited after the downregulation of POM121 (Figure 5C). Overexpression of POM121 promoted the levels of MYC and phosphorylation of the PI3K/AKT pathway (Figure 5D). Furthermore, PI3K inhibitor wortmannin could reverse the promoting effect of high POM121 expression on cell proliferation (Figure 5E), migration, and invasion (Figure 5F). The results described above approved that POM121 promoted GC progression via PI3K/AKT/MYC pathway.
Figure 5.
POM121 promotes GC progression through PI3K/AKT/MYC pathways. A: PPI network around POM121. B: Pearson correlation analysis between POM121 and MYC. C: Expression and phosphorylation of PI3K/AKT/MYC pathways in shPOM121 cells compared with the control. D: Expression and phosphorylation of PI3K/AKT/MYC pathways in ovPOM121 cells compared with the control. E: The proliferation of GC cells after wortmannin treatment. F: The migration and invasion of GC cells after wortmannin treatment.
Discussion
There are three transmembrane NUPs embedded in the mammalian nuclear membrane (POM121, NUP210, andNDC1), and POM121 is the least conserved and is only expressed in vertebrates [16,17]. POM121 is essential in NPC and nuclear envelope assembly, empowering POM121 to have a vital role in nuclear transport [18]. Overexpression of POM121 has been detected in some tumors, including laryngeal cancer [19], colorectal cancer [20], oral squamous cell carcinoma [21], and lung cancer [22,23]. In the present study, we investigated POM121 mRNA and protein expression. The qRT-PCR confirmed the overexpression of POM121 mRNA in gastric carcinoma, and GC tissues were more often detected with high POM121 protein expression (63.30%) than normal gastric tissues (12.40%). We also detected POM121 expression in chronic gastritis, intestinal metaplasia, and intraepithelial neoplasia, and found that less POM121 was expressed in abnormal gastric tissues than in GC tissues.
POM121 expression in GC tissues and the matched clinical information of 684 patients was analyzed. The results confirmed that GC patients with high POM121 levels were associated with deeper invasion, advanced distant metastases, TNM stage, and positive HER2 expression, suggesting POM121 expression may influence the progression, invasion, and metastases of GC. Except for differentiation, distant metastasis, TNM stage, and HER2 expression, POM121 expression was also demonstrated as a relative factor for GC overall survival by multivariate Cox regression and Kaplan-Meier survival analyses. This evidence indicated that POM121 plays an important role in GC progression and invasion and has the potential to predict the prognosis of GC patients.
Soluble POM121, a variant that lacks the membrane-anchoring domain, was identified as a transcriptional regulator of gene promoters [24]. Kihlmark et al. reported POM121 may participate in the propagation of nuclear apoptosis [25]. The involvement of POM121 in transcriptional regulation and apoptosis suggests its potential role in tumorigenesis and progression. Rodriguez-Bravo et al. demonstrated the contribution of POM121 to tumorigenesis, cell proliferation, aggressiveness, and drug resistance in prostate cancer [15]. In lung cancer, decreased POM121 was confirmed to inhibit cell proliferation, clone formation, migration, and invasion [22]. We silenced and upregulated the POM121 expression in GC cells and detected the follow-up effects on cell biological functions. The results showed that downregulation of POM121 significantly hindered the proliferation, migration, and invasion of GC cells in vitro and vivo, while overexpression of POM121 expedited malignant biological properties in GC, suggesting the tumor-promoting role of POM121 in GC progression.
MYC, also named c-MYC, is an important proto-oncogene involved in cell regulation, proliferation, differentiation, and metabolism [26]. MYC is usually unexpressed in normal tissues, but can induce malignant transformation of normal cells when it is upregulated [27]. MYC is overexpressed in a variety of tumor tissues and shows significant influence on tumorigenesis and development [28,29]. We constructed a PPI network around POM121, and found the underlying connection between POM121 and MYC. In addition, Pearson correlation indicated that POM121 was significant associated with MYC in GC tissues. We confirmed that silencing POM121 inhibited the expression of MYC via Western Blotting. PI3K/AKT signaling pathway plays a crucial role in the proliferation, apoptosis, angiopoiesis and metastasis of tumor [30-32]. MYC is also a downstream protein of PI3K/AKT pathway [33,34]. We detected the expression and phosphorylation ofPI3K/AKT pathway after POM121 knockdown and overexpression, and found POM121 significantly promoted the phosphorylation level of the PI3K/AKT pathway. These results suggested that POM121 regulated MYC expression through PI3K/AKT pathway, thus affecting GC progression.
Conclusions
This study shows that POM121 is overexpressed in GC, and GC patients with high POM121 expression have a poor prognosis. POM121 promotes proliferation, clone formation, migration, and invasion of GC cells through PI3K/AKT/MYC pathway. Therefore, POM121 might serve as a new prognostic factor and therapeutic target for GC patients.
Acknowledgements
We thank all patients enrolled in the study. We also thank H. Nikki March, PhD, from LiwenBianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was supported by grants from National Natural Science Foundation of China (82060438), Natural Science Foundation of Inner Mongolia Autonomous Region (2020MS08002, 2020MS08060, 2020MS08149, 2022MS08031), Inner Mongolia Autonomous Region Science and Technology Plan Project (2020GG0273, 2022YFSH0018), Bayannur city Science and Technology Plan Project (BYZ2019-1-2, K202025), Academician workstation (YSZ2018-2, 2020PT0010), Inner Mongolia Medical University Joint Project (YKD2021LH072), Inner Mongolia Autonomous Region Health Science and Technology Plan Project (202202402, 202201625, 202202405).
This study was performed in accordance with medical ethical standards and was approved by the Ethics Committee of Inner Mongolia Medical University. Written informed consents were obtained from all study participants. The animal study was reviewed and approved by the Ethics Committee of Inner Mongolia Medical University.
Disclosure of conflict of interest
None.
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