Objective:To explore the effects of ubiquitin-conjugating enzyme E2L3 (UBE2L3) on the proliferation and apoptosis of head and neck squamous cell carcinoma (HNSCC) cell line UMSCC-1 in vitro and its potential mechanisms, and to investigate the relationship between UBE2L3 and immune cell infiltration in the HNSCC tumor microenvironment.Methods:The plasmid carrying UBE2L3 gene sequence was transfected into UMSCC-1 cells by using liposome transfection technology (UBE2L3 overexpression group), and UMSCC-1 cells transfected with the empty plasmid was treated as the control group. The quantitative polymerase chain reaction (qPCR) was used to detect the expression level of UBE2L3 mRNA after transfection, and the transfection efficiency was evaluated. The proliferation levels of the 2 groups of cells were detected by using CCK-8 assay and the cell proliferation rate was calculated. The apoptosis rates of the 2 groups of cells after etoposide induced apoptosis for 24 h were detected by using flow cytometry. The expression levels of Cyclin D1, apoptosis-related proteins bcl-2, and Bax in the 2 groups of cells were detected by using Western blotting. The samples of 504 HNSCC tissues in the Cancer Genome Atlas (TCGA) were analyzed by using the single-sample gene set enrichment analysis (ssGSEA) database. The median expression level of UBE2L3 was used to distinguish between high and low expression of UBE2L3 transcriptional level. The differences in immune cell enrichment score between high and low expression samples were compared, and the relationship between UBE2L3 expression level and immune cell number was analyzed.Results:qPCR results showed that the relative expression level of UBE2L3 mRNA in UMSCC-1 cells carrying UBE2L3 gene sequence plasmid was higher than that in UMSCC-1 cells transfected with the empty plasmid (9.32±1.15 vs. 1.00±0.02), and the difference was statistically significant ( t = 7.23, P < 0.001), indicating successful transfection. The CCK-8 assay showed that the cell proliferation rate of UMSCC-1 cells in the UBE2L3 overexpression group for 24 h and 48 h of culture was higher than that in the control group [24 h: (184.0±7.9)% vs. (100.0±5.6)%; 48 h: (165.5±5.3)% vs. (100.0±5.3)%], and the differences were statistically significant (both P < 0.001). Flow cytometry showed that the apoptosis rate of UMSCC-1 cells in the UBE2L3 overexpression group was lower than that in the control group [(9.9±1.3)% vs. (15.6±1.3)%], and the difference was statistically significant ( t = 2.78, P = 0.005). Western blotting showed that the relative expression levels of Cyclin D1 and bcl-2 proteins in UMSCC-1 cells of the UBE2L3 overexpression group were higher than those in the control group, while the relative expression level of Bax protein was lower than that in the control group, and the differences were statistically significant (all P < 0.05). The ssGSEA analysis showed that the enrichment scores of B cells, CD8 + T cells, neutrophils, T cells, T helper 17 (Th17) cells, and regulatory T cells (Treg) in 252 HNSCC tissues samples with low UBE2L3 transcriptional expression level were higher than in 252 samples with high UBE2L3 transcriptional expression level in the TCGA database, while the enrichment scores of natural killer (NK) cells and T helper 2 (Th2) cells in patients with low UBE2L3 expression were lower than those in those with high UBE2L3 expression, and the differences were statistically significant (all P < 0.05). Correlation analysis showed that the expression level of UBE2L3 in HNSCC tissues was positively correlated with the number of Th2 cells ( R = 0.182) and NK cells ( R = 0.172), and negatively correlated with the number of Treg cells ( R = -0.095), dendritic cells ( R = -0.099), Th17 cells ( R = -0.129), CD8 + T cells ( R = -0.146), mast cells ( R = -0.161), T cells ( R = -0.171), and B cells ( R = -0.188), and the differences were statistically significant (all P > 0.05). Conclusions:UBE2L3 can promote the proliferation and inhibit the apoptosis of HNSCC cells probably by regulating the cell cycle and apoptosis signaling pathways through Cyclin D1, bcl-2, and Bax. UBE2L3 may be associated with immune cell infiltration in the HNSCC microenvironment.

Objective:To explore the effects of ubiquitin-conjugating enzyme E2L3 (UBE2L3) on the proliferation and apoptosis of head and neck squamous cell carcinoma (HNSCC) cell line UMSCC-1 in vitro and its potential mechanisms, and to investigate the relationship between UBE2L3 and immune cell infiltration in the HNSCC tumor microenvironment.Methods:The plasmid carrying UBE2L3 gene sequence was transfected into UMSCC-1 cells by using liposome transfection technology (UBE2L3 overexpression group), and UMSCC-1 cells transfected with the empty plasmid was treated as the control group. The quantitative polymerase chain reaction (qPCR) was used to detect the expression level of UBE2L3 mRNA after transfection, and the transfection efficiency was evaluated. The proliferation levels of the 2 groups of cells were detected by using CCK-8 assay and the cell proliferation rate was calculated. The apoptosis rates of the 2 groups of cells after etoposide induced apoptosis for 24 h were detected by using flow cytometry. The expression levels of Cyclin D1, apoptosis-related proteins bcl-2, and Bax in the 2 groups of cells were detected by using Western blotting. The samples of 504 HNSCC tissues in the Cancer Genome Atlas (TCGA) were analyzed by using the single-sample gene set enrichment analysis (ssGSEA) database. The median expression level of UBE2L3 was used to distinguish between high and low expression of UBE2L3 transcriptional level. The differences in immune cell enrichment score between high and low expression samples were compared, and the relationship between UBE2L3 expression level and immune cell number was analyzed.Results:qPCR results showed that the relative expression level of UBE2L3 mRNA in UMSCC-1 cells carrying UBE2L3 gene sequence plasmid was higher than that in UMSCC-1 cells transfected with the empty plasmid (9.32±1.15 vs. 1.00±0.02), and the difference was statistically significant ( t = 7.23, P < 0.001), indicating successful transfection. The CCK-8 assay showed that the cell proliferation rate of UMSCC-1 cells in the UBE2L3 overexpression group for 24 h and 48 h of culture was higher than that in the control group [24 h: (184.0±7.9)% vs. (100.0±5.6)%; 48 h: (165.5±5.3)% vs. (100.0±5.3)%], and the differences were statistically significant (both P < 0.001). Flow cytometry showed that the apoptosis rate of UMSCC-1 cells in the UBE2L3 overexpression group was lower than that in the control group [(9.9±1.3)% vs. (15.6±1.3)%], and the difference was statistically significant ( t = 2.78, P = 0.005). Western blotting showed that the relative expression levels of Cyclin D1 and bcl-2 proteins in UMSCC-1 cells of the UBE2L3 overexpression group were higher than those in the control group, while the relative expression level of Bax protein was lower than that in the control group, and the differences were statistically significant (all P < 0.05). The ssGSEA analysis showed that the enrichment scores of B cells, CD8 + T cells, neutrophils, T cells, T helper 17 (Th17) cells, and regulatory T cells (Treg) in 252 HNSCC tissues samples with low UBE2L3 transcriptional expression level were higher than in 252 samples with high UBE2L3 transcriptional expression level in the TCGA database, while the enrichment scores of natural killer (NK) cells and T helper 2 (Th2) cells in patients with low UBE2L3 expression were lower than those in those with high UBE2L3 expression, and the differences were statistically significant (all P < 0.05). Correlation analysis showed that the expression level of UBE2L3 in HNSCC tissues was positively correlated with the number of Th2 cells ( R = 0.182) and NK cells ( R = 0.172), and negatively correlated with the number of Treg cells ( R = -0.095), dendritic cells ( R = -0.099), Th17 cells ( R = -0.129), CD8 + T cells ( R = -0.146), mast cells ( R = -0.161), T cells ( R = -0.171), and B cells ( R = -0.188), and the differences were statistically significant (all P > 0.05). Conclusions:UBE2L3 can promote the proliferation and inhibit the apoptosis of HNSCC cells probably by regulating the cell cycle and apoptosis signaling pathways through Cyclin D1, bcl-2, and Bax. UBE2L3 may be associated with immune cell infiltration in the HNSCC microenvironment.

Objective To discuss the value of serum thymidine kinase 1 and DNA ploidy for the diagnosis of patients with acute myeloid lecukemia.Methods Determined TK1 and DI in 20 healthy people,6 patients with benign proliferate in hematological system and 66 patients with acute myeloid lecukemia by chemiluminescence detection technique and flow cytometry.Nonparametric comparisons among three group were done by rank sum test.ROC curve was used to determine the AUC and the diagnosis value serum thymidine kinase 1 and DNA.Results As showed by peripheral blood results,the TK1 (x2 =36.877,P＜0.001) and DI (x2=4.040,P＜0.05) had statistically difference among healthy people group,patients with benign proliferate in hematological system group and AML group.The normal control group compared with the AML group,TK1 (Z=-6.073,P＜0.001)and DI (Z=-2.012,P =0.044) had statistically difference;The normal control group compared with the benign proliferate patients,TK1 (Z=-1.234,P =0.169) and DI (Z=-1.084,P =0.278) had no statistically difference.The benign proliferate patients and that with AML patients,TK1 (Z=2.177,P=0.036) had statistically difference,but DI (Z=-1.801,P=0.061) had no statistically difference.The TK1 and DI area under the ROC curve were 0.950 (P＜0.001) and 0.638 (P=0.063),best cut-off were 1.73 pmol/L and 0.98,sensitivity were 0.95,0.78,and specifity were 0.88,0.39.Conclusion Serum TK1 and DI is a important diagnostic marker of early for AML patients,TK1 have a better diagnostic performance than DI significantly.