UBE2L3 expression in human gastric cancer and its clinical significance

The UBE2L3 gene expression analysis of various tumors can be accessed from the GEPIA database (http://​gepia.​cancer-pku.​cn/​index.​html).

Human tissue specimens were obtained from GC patients who underwent surgical excision at the Second Hospital of Lanzhou University between December 2016 and June 2017. The informed consent was signed by all patients, and the study protocol received approval from the Ethics Committee of the Second Hospital of Lanzhou University. The study included 125 patients with primary gastric cancer, with 11 patients excluded due to less than 5 years of follow-up. The cohort consisted of 114 patients, comprising 87 men and 27 women, all of whom did not undergo chemotherapy or radiotherapy before the surgical procedure, and all completed a 5-year follow-up period. All 114 specimens underwent confirmation through pathological examination and TNM staging.

The six cell lines, namely GES-1, MKN45, MKN28, N87, AGS, and HGC27, were acquired from the Digestive Tumor Laboratory of Gansu Province. The six cell lines were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), and maintained in a humidified incubator at 37 °C with a 5% CO₂ atmosphere.

In the MKN45, MKN28, and AGS cell lines, the experimental group consisted of cells transfected with lentivirus obtained from GenePharma, China, while the negative control group comprised cells transfected with an empty lentiviral vector. Cell transfection was conducted using Invitrogen following the manufacturer’s protocol. Cells were cultured in six-well plates (Corning, USA) until reaching a cell density of 20–30%. Subsequently, the cells were harvested 18 h after transfection with lentivirus. After 72 h of continuous culture, the cells were subjected to puromycin screening for an additional 72 h. The transfection efficiency was assessed by observing green fluorescent protein expression under a microscope, and it was found that the fluorescence efficiency exceeded 80%, indicating a successful transfection.

In this study, the cell proliferation potential of GC cells was assessed using the Cell Counting Kit-8 (CCK-8) assay kit (Solarbio, China). The cells (MKN45, MKN28, AGS) were seeded at a density of 1000 cells per well in 96-well plates (Corning, USA). The medium in each well was removed at different time intervals (0, 24, 48, 72, and 96 h). Subsequently, 10 μL of the prepared CCK-8 reagent was introduced into each well and incubated for 1 h at 37 °C in a light-sheltered incubator. Following incubation, the absorbance value (OD) was assessed at a wavelength of 450 nm.

The cells were cultured at a density of 500 cells per well in 6-well plates. The cells were cultured without disturbance for a period of two weeks to facilitate the formation of proliferating colonies. Subsequently, the proliferating cells underwent two gentle washes with phosphate-buffered saline (PBS) to eliminate any non-adherent cells. Subsequently, the cells were treated with paraformaldehyde for 15 min and then subjected to staining with 0.1% crystal violet (Solarbio, China) for an additional 15 min. The number of colonies was determined using Image J software.

The cells that were treated with trypsin were subsequently placed in the upper chamber of the transwell system (Corning, USA) that was coated with Matrigel (BD Bioscience). The cell count was 1 × 10⁵ cells per upper chamber. The cells underwent two gentle washes with PBS following 18–72 h of incubation. Subsequently, the cells underwent fixation and staining using the identical procedure employed in the colony formation assay. The number of invasive cells was visualized and quantified.

Apoptosis in GC cells was evaluated using flow cytometry. To analyze apoptosis, the apoptosis rate of adherent cells was assessed using the AnnexinV-APC/7-ADD apoptosis kit (Multisciences, China). The GC cell samples were suspended again in a binding buffer. 5 μl of Annexin V-APC and 10 μl of 7-AAD were added to each tube, followed by a 5-min incubation at room temperature in the dark. At the conclusion of the incubation period, the samples underwent processing using flow cytometry (Canto BD).

Proteins were initially extracted using RIPA buffer containing a protease inhibitor cocktail (Beyotime), and the quantification of protein levels was determined using the BCA assay (Beyotime). Following the isolation of proteins, the entire protein content was separated using 10% SDS-PAGE and subsequently transferred to PVDF membranes for Western blot analysis. Subsequently, the membranes were blocked with 5% skim milk for 1 h, followed by sequential incubation with primary antibodies overnight and secondary antibodies for 1 h. To analyze and document the protein bands, the membrane underwent imaging using the enhancement chemiluminescence reagent (Biosharp, China) and a chemiluminescence imager (Tanon 4600, China). The antibodies used for Western blot analysis were as follows: anti-UBE2L3 (dilution 1:10,000, Abcam, USA) and anti-GAPDH (dilution 1:5000, ImmunoWay Biotechnology, USA).

The total RNA was isolated using AG RNAex Pro Reagent (Accurate Biology, China), followed by RNA conversion to cDNA, and subsequently subjected to quantitative real-time PCR using a CFX96 Touch qRT-PCR system (Bio-Rad). GAPDH was employed as an internal control for mRNA analysis. The values of the target genes were determined using the 2^−ΔΔCt method. The primer sequences have been documented in Table 1.

In this investigation, two cohorts of stably transfected MKN45 cells (1 × 10⁶ cells) were subcutaneously administered to female Balb/c nude mice aged 5–6 weeks old, obtained from Chengdu Yaokang Biotechnology Co., LTD. Tumor dimensions were assessed at 3-day intervals over a period of 21 days. Subsequently, the mice were euthanized following inhalation of ether anesthesia, and the xenograft tumors were removed, weighed, and then stored in a refrigerator at − 80 ℃ after resection for future experiments.

The immunohistochemical method was used to detect the expression level of UBE2L3 protein in human tissues and tumor tissues of nude mice. Tissue microarrays were created using gastric cancer (GC) tissues and adjacent normal tissues from 125 GC patients. Each sample was represented by two cores with a diameter of 1mm. Subsequently, the tumor tissue samples in nude mice were fixed, embedded in paraffin, and sectioned into 5 µm slices. Subsequently, the sections underwent antigen retrieval, followed by overnight incubation at 4 °C with anti-UBE2L3 antibody (1:500, Abcam, USA) and anti-Ki67 antibody (1:500, Servicebio, China). Following incubation with secondary antibodies, the sections underwent staining with 3,3ʹ-diaminobenzidine, and subsequent incubation with hematoxylin. Two pathologists assessed UBE2L3 immunostaining based on the percentage of stained cells and the intensity of staining in each section. The scoring system was determined by the proportion of stained cells in the positive area (0 = 0–5%, 1 = 6–25%, 2 = 26–50%, 3 = 51–75%, 4 = 76–100%) and the intensity of staining (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining). The immunohistochemical score is calculated by multiplying the score of the staining area by the score of the staining intensity. The samples were subsequently categorized as negative (-, 0 points), weakly positive (+ , 1–4 points), moderately positive (+ + , 5–8 points), and strongly positive (+ + +, 9–12 points).

The GEPIA public database was utilized for the collection of UBE2L3 mRNA expression data in various tumor tissues, with the aim of further investigating the UBE2L3 mRNA expression in GC tissues. The various cancer tissues examined in this study comprised pancreatic cancer (n = 179) and normal pancreatic tissue (n = 171), gastric cancer (n = 408) and normal gastric tissue (n = 211), cholangiocarcinoma (n = 36) and normal bile duct tissue (n = 9), and normal pancreatic tissue (n = 10). Thymoma (n = 118) and normal thymic tissue (n = 339) were compared, as well as diffuse large B-cell tumor (n = 47) and normal tissue (n = 337). The data presented in this study provide insight into the high expression of UBE2L3 in various human cancers, suggesting that UBE2L3 may be involved in the pathogenesis of malignant tumors (Fig. 1A).

To clarify the potential clinical relevance of UBE2L3 in GC, we initially assessed the differential expression of UBE2L3 in GC tissues compared with adjacent normal tissues using immunohistochemical (IHC) staining on 125 specimens from 125 GC patients. As depicted in Fig. 1B, IHC data indicated that UBE2L3 is primarily expressed in the cytoplasm and nucleus of GC cells. The expression level of UBE2L3 in GC tissues was significantly higher than that in adjacent tissues (see Fig. 1C). Utilizing the median IHC score of UBE2L3, we segregated 114 GC patients into two categories: those with low UBE2L3 expression and those with high UBE2L3 expression. Table 2 illustrates the correlation between UBE2L3 expression and clinical pathological characteristics. The analysis of Table 2 reveals a strong correlation between the high expression of UBE2L3 in GC and the degree of differentiation (× 2 = 6.153, P = 0.0131) as well as TNM classification (× 2 = 6.216, P = 0.0447). However, no significant differences were observed in relation to age, gender, histological type, tumor size, and Lauren classification (refer to Table 2). As depicted in Fig. 1D, individuals exhibiting high UBE2L3 expression demonstrate a lower overall survival rate compared to those with low UBE2L3 expression. The CT data of the patients was collected over a 5-year follow-up period. In Fig. 2A, it was observed that Patient A did not develop metastases during the follow-up period, while Patient B was diagnosed with liver and lung metastases 24 months into the follow-up. A total of 23 patients were identified with metastases at various sites and at different times following surgery during the follow-up period. The specific patient data is depicted in Fig. 2B. Based on the IHC findings, the patients were categorized into two groups: those with metastasis and those without metastasis, and further divided into UBE2L3 high-expression group and UBE2L3 low-expression group. The findings indicated a significantly higher expression level of UBE2L3 in gastric cancer tissues compared to adjacent tissues in patients with and without metastasis, as illustrated in Fig. 2C and D. In summary, the heightened expression of UBE2L3 indicates an unfavorable prognosis in patients with GC.

The expression of UBE2L3 in GC was assessed by conducting Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) on five human GC cell lines and GES-1. The results indicate that UBE2L3 exhibited high expression in MKN45 and MKN28, and low expression in AGS (Fig. 3A, C). Subsequently, three UBE2L3 shRNAs were employed to suppress the expression of UBE2L3 in MKN28 and MKN45, while UBE2L3 was overexpressed in AGS to ascertain its function in GC. The transfection efficiency was assessed by observing the presence of green fluorescent proteins in MKN28, MKN45, and AGS cells. The experimental results indicated that the transfection efficiency exceeded 80% in all three cell lines (Fig. 3D, E). Furthermore, the transfection efficiency was assessed through Western blot analysis, revealing that two UBE2L3 knockdown cell lines and one UBE2L3 overexpressing cell line successfully attained the anticipated UBE2L3 protein levels (Fig. 3B). The aforementioned data suggests that the establishment of UBE2L3 knockdown and overexpression cell models has been successful, rendering them suitable for subsequent experiments.

Based on the discovery that UBE2L3 levels are linked to the prognosis of GC, we employed the following experimental approach to validate the functional role of UBE2L3 in the malignant behavior of GC in vitro. The study revealed that UBE2L3 promotes the proliferation rate of GC cells, as demonstrated by the CCK-8 assay results presented in Fig. 4A. Furthermore, the cells’ proliferation ability was verified through the colony formation assay. A reduced number of colonies were observed in the UBE2L3 knockdown group, while the overexpression group exhibited the opposite outcome (Fig. 4B). Subsequently, the Transwell assay was used to evaluate the invasive capacity of GC cells. Likewise, the suppression of UBE2L3 hindered cellular passage through the matrigel, whereas the up-regulation of UBE2L3 facilitated increased cellular invasion through the matrigel (Fig. 4C). Finally, the results of the apoptosis assay revealed a significant increase in the apoptosis rate of the sh-UBE2L3 group, and a significant decrease in the UBE2L3 overexpression group (Fig. 4D). In conclusion, the aforementioned findings unequivocally demonstrate the significant involvement of UBE2L3 in GC.

To validate the findings of the in vitro experiments, cell-derived xenograft (CDX) models were established in nude mice through the injection of MKN45 sh-NC and MKN45 sh-UBE2L3-3 cells. The growth of subcutaneous tumors in nude mice was monitored every 3 days, and the changes in tumor volume were measured and recorded (Fig. 5B). The results demonstrate that the tumor volume of the sh-NC group was greater than that of the sh-UBE2L3-3 group (Fig. 5A). Following the euthanasia of the nude mice, the tumor tissues were extracted, weighed, and the dimensions of each tumor were recorded. The findings indicated that the tumors in the sh-UBE2L3-3 group exhibited reduced size and weight compared to those in the sh-NC group (Fig. 5C). Finally, the findings from the hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) analysis (Fig. 5D), conducted on tumor tissues, revealed that the levels of UBE2L3 and Ki67 expression were higher in the sh-NC group compared to the sh-UBE2L3-3 group. The collective data presented above suggest that the suppression of UBE2L3 hinders the growth of gastric cancer in an in vivo setting.