3.1 Different expression of PSMD family in ovarian tissue
Differences in mRNA expression of PSMDs in ovarian cancer and normal ovarian tissues were analyzed using GEPIA database (Fig. 1). The mRNA expression levels of PSMD8 and PSMD14 in ovarian cancer tissues were significantly higher than those in normal ovarian tissues (Fig. 1h, n).
The relationship between PSMDs mRNA expression level and clinical stage in ovarian serous adenocarcinoma was further analyzed using the TISIBD database (Fig. 2). The results showed that PSMD3 expression significantly decreased with increase in FIGO stage (Fig. 2c, Spearman: rho = 0.128, P = 0.0264), while PSMD5 expression increased significantly with increase in FIGO stage (Fig. 2e, Spearman: rho = 0.123, P = 0.0329).
3.2 Gene variation in PSMDs in ovarian cancer
Genetic variations of PSMDs in 1691 cases retrieved from three studies (489 cases from TCGA, Nature 2011; 585 cases from TCGA, PanCancer Atlas; and 617 cases from TCGA, Firehose legacy) were analyzed using the cBioPortal database (Fig. 3). We found varying degrees of genetic variation among the 14 PSMD family members, among which PSMD2 displayed the highest incidence rate (26.37% in TCGA) of genetic variations (the incidence rates of amplification) (Fig. 3b), followed by PSMD4 whose incidence rate of amplification was 11.13% and PSMD8 whose incidence rates of amplification and deep deletion were 10.96% and 0.68%, respectively (in TCGA) (Fig. 3e, i).
3.3 PSMD8 showed the best prognostic value in patients with ovarian serous tumors
The correlation between PSMDs mRNA expression levels and PFS in ovarian cancer patients was analyzed using the Kaplan–Meier Plotter database (Fig. 4). Among them, PSMD2, PSMD3, PSMD4, PSMD5, PSMD8, PSMD11, PSMD12, and PSMD14 mRNA expression levels were associated with prognosis. These findings indicate the prognostic significance of PSMDs in the context of ovarian cancer.
Ovarian serous tumors are the most common pathological type of ovarian tumors. Therefore, we assessed the correlation of the expression of the above-mentioned 8 genes in ovarian serous tumors cancer with OS and PFS (Figs. 5 and 6). Among them, up-regulation of PSMD4, PSMD8, and PSMD14 mRNA expression was significantly associated with poor OS in patients with ovarian serous tumors (Fig. 5c,e,h). Up-regulation of PSMD2, PSMD3, PSMD5, and PSMD8 mRNA expression was significantly associated with poor PFS in patients with ovarian serous tumors (Fig. 6a, HR = 1.19, 95% CI: 1.02–1.39, P = 0.024; Fig. 6b, HR = 1.19, 95% CI: 1.03–1.38, P = 0.018; Fig. 6d, HR = 1.12, 95% CI: 1.05–1.34, P = 0.012; Fig. 6e, HR = 1.22, 95% CI: 1.04–1.44, P = 0.016), while down-regulation of PSMD12 and PSMD14 mRNA expression was associated with poor PFS (Fig. 6g, HR = 0.84, 95% CI: 0.71–0.98, P = 0.032; Fig. 6h, HR = 0.82, 95% CI: 0.71–0.95, P = 0.0085). On comprehensive comparison, up-regulation of PSMD8 mRNA expression was significantly associated with poor OS and PFS in patients with ovarian serous tumors.
PSMD8, which showed the greatest prognostic significance in patients with ovarian serous tumors, was selected for correlation analysis. We separately assessed the correlation between PSMD8 mRNA expression and PFS at different degrees of differentiation, FIGO stages, and TP53 mutation status (Fig. 7). There was no significant correlation between the up-regulation of PSMD8 mRNA expression and poor PFS in patients with moderately- and poorly-differentiated tumors (Fig. 7a-c); in patients with FIGO stage III-IV, the up-regulation of PSMD8 mRNA expression indicated poor PFS (Fig. 7d-e). Compared with wild type, PSMD8 mRNA upregulation in TP53 mutant patients was associated with significantly poor PFS (Fig. 7f). These findings indicated that PSMD8 can better reflect the prognosis of patients with ovarian serous tumors, and in patients with advanced FIGO stage and TP53 mutation, PSMD8 showed a more significant correlation with prognosis.
3.4 PSMDs gene interaction network construction and enrichment analysis
The gene interaction network map of the 14 genes of PSMDs was constructed using the GeneMANIA database, and the correlations were analyzed (Fig. 8a). The 14 nodes in the middle are members of PSMDs, and the surrounding 20 nodes are the 20 genes most related to the family in terms of physical interaction, interaction, co-localization, prediction, inheritance, and co-expression. The five most related genes are PSMC1, PSMC4, PSMC6, PSMC2, and PSMC3, which are members of the PSMCs family.
The function and pathway enrichment analysis of PSMD8 co-expressed genes was carried out using the database. Among the signal pathways with strong correlation of PSMDs co-expressed genes, glutathione metabolism, yruvate metabolism, DNA replication, agrinine and proline metabolism were related to the occurrence and development of tumors (Fig. 8b). Functional analysis showed that PSMD8 co-expressed genes were mainly enriched in the following biological processes, including mitochodrial gene expression, mitochondrial transation, peptidase complex, ranslational termination, cellular protein complex disassembly, and mitochondrial membrane organization (Fig. 8c).
3.5 PSMD8 is involved in the regulation of immune molecules
Spearman's correlation analysis was performed to assess the correlation of PSMD8 expression with lymphocyte subsets and immunomodulators using the TISIDB database. Figure 9a and Fig. 9b showed the correlation between PSMD8 expression and tumor-infiltrating lymphocytes (TILs). The lymphocyte subsets displaying the greatest correlations included CD56dim (Spearman: ρ = 0.293, P = 1.88e − 07), Act_CD8 (Spearman: ρ = 0.209, P = 0.000231), Act_DC (Spearman: ρ = 0.189, P = 0.000877), and CD56bright (Spearman: ρ = 0.188, P = 0.000928). Immunomodulators were further classified into immunoinhibitors, immunostimulators, and major histocompatibility complex (MHC) molecules. Figure 9c and Fig. 9d showed the correlation of PSMD8 expression levels with immunoinhibitors. The immunoinhibitors displaying the greatest correlations included PVDL2 (Spearman: ρ = 0.254, P = 6.99e − 06), IDO1 (Spearman: ρ = 0.116, P = 0.042), IL10RB (Spearman: ρ = 0.114, P = 0.0463), and VTCN1 (Spearman: ρ = 0.113, P = 0.0479). Figure 9e and Fig. 9f showed the correlation between immunostimulators and PSMD8; the immunostimulators displaying the strongest correlation included PVR (Spearman:ρ = 0.194, P = 0.000657), TNFRSF4 (Spearman:ρ = 0.143, P = 0.0119), MICB (Spearman:ρ = 0.142, P = 0.0129), and CD48 (Spearman: ρ = 0.141, P = 0.0136). Figure 9g and Fig. 9h showed correlations between PSMD8 expression and MHC molecules. The MHC molecules displaying the strongest correlation included HLA-A (Spearman:ρ = 0.157, P = 0.00604), HLA-C (Spearman:ρ = 0.151, P = 0.00829), B2M (Spearman:ρ = 0.149, P = 0.00893), and TAP1 (Spearman:ρ = 0.133, P = 0.0202).
3.6 PSMD8 is highly expressed in different kinds of cancer tissues
The mRNA expression of PSMD8 in normal tissues and cancer tissues was analyzed using the human protein atlas website, and the results showed greater expression of PSMD8 in normal skeletal muscle, cardiac muscle, and tongue muscle (Fig. 10a). TCGA database showed that among malignant tumors, PSMD8 was more frequently expressed in ovarian cancer, testicular cancer, glioma, and melanoma (Fig. 10b). GEPIA website data analysis showed that PSMD8 is highly expressed in ovarian cancer, pancreatic cancer, gastric cancer, thymic cancer, glioblastoma multiforme, and diffuse large B-cell lymphoma, while it is lowly expressed in acute myeloid leukemia. In conclusion, PSMD8 has a higher abnormal expression in ovarian cancer (Fig. 10c).
3.7 Immunohistochemistry confirmed the high expression of PSMD8 in ovarian cancer tissue
PSMD8 is mainly located in the cytoplasm and its expression is indicated by brown staining (Fig. 11a-d). The positive rate in the malignant group was 96.19%, and the strong positive rate was 70.48%. In the borderline group, the positive rate was 41.67%, and the strong positive rate was 16.67%. In the benign group, the positive rate was 16.67% and the strong positive rate was 11.11%. In normal ovarian tissue, the positive rate was 6.25%, and the strong positive rate was 0.00%. The positive expression rate and strong positive rate of PSMD8 in the malignant group were significantly higher than that in borderline group, benign group, and normal group (P < 0.05 for all). The positive expression rate of PSMD8 in the borderline group was greater than that in the benign group and normal group (P < 0.05). The expression rate of PSMD8 in the benign group was higher than that in the normal group, but the difference was not statistically significant (P > 0.05) (Table 1, Fig. 11e).
Table 1
Expression of PSMD8 in different ovarian tissues
Groups
|
Cases
|
Low
|
High
|
Positive Rate (%)
|
High expression Rate (%)
|
-
|
+
|
++
|
+++
|
Malignant
|
80
|
13
|
20
|
24
|
23
|
83.75%*
|
58.75%*
|
Borderline
|
18
|
5
|
5
|
3
|
4
|
66.67%
|
38.89%
|
Benign
|
16
|
7
|
7
|
1
|
1
|
56.25%
|
12.50%
|
Normal
|
11
|
9
|
2
|
0
|
0
|
18.18%
|
0.00%
|
3.8 Relationship between PSMD8 expression and clinicopathological parameters of ovarian cancer
In order to compare the clinicopathological parameters and the expression of PSMD8 in ovarian tissue, we collected the pathological information of 80 patients with primary ovarian epithelial malignant tumor. The strong positive expression rate was significantly higher than that in FIGO I ~ II group (80.33% and 56.82%, P < 0.01), while there was no significant difference in other items (Table 2).
Table 2
Relationships between the expression of PSMD8 and clinicopathological parameters of 80 ovarian cancer patients
Groups
|
cases
|
PSMD8
|
Positive rate(%)
|
P-value
|
High expression rate(%)
|
P-value
|
Age at diagnosis
|
|
|
|
|
|
< 55
|
42
|
37/42(88.10%)
|
P > 0.05
|
27/42(64.29%)
|
P > 0.05
|
≥ 55
|
38
|
30/38(78.95%)
|
20/38(72.63%)
|
Pathological type
|
|
|
|
|
|
Serous
|
58
|
50/58(86.21%)
|
P > 0.05
|
33/58(56.90%)
|
P > 0.05
|
Mucious
|
3
|
2/3(66.67%)
|
1/3(33.33%)
|
Endometrioid
|
10
|
7/10(70.00%)
|
6/10(60.00%)
|
Clear cell carcinoma
|
9
|
8/9(88.89%)
|
7/9(77.78%)
|
FIGO stage
|
|
|
|
|
|
I-II
|
26
|
15/26(57.69%)
|
P < 0.05
|
7/26(26.92%)
|
P < 0.001
|
III-IV
|
54
|
52/54(96.30%)
|
40/54(74.07%)
|
Differentiation
|
|
|
|
|
|
Well-moderate
|
30
|
23/30(76.67%)
|
P > 0.05
|
14/30(46.67%)
|
P > 0.05
|
Poor
|
50
|
44/50(88.00%)
|
33/50(66.00%)
|
Lymphatic metastasis
|
|
|
|
|
|
No
|
40
|
32/40(80.00%)
|
P > 0.05
|
18/40(47.50%)
|
P > 0.05
|
Yes
|
25
|
22/25(92.00%)
|
17/25(68.00%)
|
Unknown#
|
15
|
13/15(86.67%)
|
12/15(80.0%)
|
#15 patients without lymphadenectomy
3.9 Prognostic significance of PSMD8 expression in ovarian cancer patients
On follow-up of patients, it was found that 13 deaths occurred in the PSMD8 low expression group (n = 30), as compared to 23 deaths in the PSMD8 high expression group (n = 50). Kaplan-Meier survival analysis showed that the survival rate of patients in the PSMD8 high expression group was significantly shorter than that in the PSMD8 low expression group, and the survival rate of patients in FIGO stage III ~ IV was significantly lower than that in FIGO stage I ~ II (P < 0.05) (Fig. 11f,g).
We performed univariate and multivariate Cox regression analysis to assess the influence of PSMD8 expression, age, pathological type, degree of differentiation, FIGO stage, and lymph node metastasis on postoperative survival time of patients. The results indicated that PSMD8 expression and FIGO stage were prognostic risk factors for epithelial ovarian malignancies (Table 3).
Table 3
Univariate and Multivariate Cox Analysis of Different Clinicopathological Parameters with Ovarian Cancer
Variable
|
Categories
|
Univariate analysis
|
Multivariate analysis
|
HR
|
95% CI of HR
|
P
|
HR
|
95% CI of HR
|
P
|
Age at diagnosis
|
< 55
|
0.875
|
0.450–1.701
|
0.694
|
|
|
|
|
≥ 55
|
|
|
|
|
|
|
FIGO stage
|
I-II
|
2.562
|
1.153–5.695
|
0.021*
|
1.992
|
0.863 − 4.597
|
0.106
|
|
III-IV
|
|
|
|
|
|
|
Differentiation
|
Well-moderate
|
1.099
|
0.555–2.176
|
0.786
|
|
|
|
|
Poor
|
|
|
|
|
|
|
Lymphnode metastasis
|
No
|
1.864
|
0.858–4.051
|
0.116
|
|
|
|
|
Yes
|
|
|
|
|
|
|
PSMD8
|
Low
|
2.645
|
1.106–6.325
|
0.029*
|
2.424
|
1.034–5.681
|
0.042*
|
|
High
|
|
|
|
|
|
|
3.10 PSMD8 promotes the invasion, migration and proliferation of ovarian cancer cells
After differential expression of PSMD8, the effects on the invasion, migration, and proliferation of ovarian cancer cells were detected by transwell assay, cell scratch assay, and MTT assay. The results showed that: OVCAR3-PSMD8-H and A2780-PSMD8-H cells had significantly stronger invasion, migration, and proliferation abilities than the control group OVCAR3-PSMD8-MOCK, OVCAR3 and A2780-PSMD8-MOCK, A2780. The ability of OVCAR3-PSMD8-L1/L2 and A2780-PSMD8-L1/L2 cells were significantly weaker than that of the control group OVCAR3-PSMD8-MOCK and A2780-PSMD8-MOCK in invasion, migration, proliferation (P < 0.05 for both) (Fig. 12, 13). The results indicated that PSMD8 promoted the invasion, migration, and proliferation ability of ovarian cancer cells.