Upregulated patterns of DLGAP5 in pan-cancer
As described above, extensive studies have demonstrated that DLGAP5 is overexpressed in many cancers. The pan-cancer gene expression patterns were therefore performed to examine the differential expression of DLGAP5 across TCGA cancer types. The results were shown that the upregulated levels were observed in all tissues of cancer types compared with the corresponding adjacent tissues (Fig. 2a) Interestingly, the degree of upregulated in three types of kidney cancer (KICH, KIRC/ccRCC, KIRP) were not very high at P < 0.001 level, which exhibited a similar expression manner to that of kidney tumor cells (Fig. 1b). Considering the small number of normal samples in TCGA, and to extend this comparison, we combined the normal tissue data from GTEx database and TCGA tumor tissue data to analyze the expression of 33 tumors, and found also the upregulated of DLGAP5 in all tumors tested (*P < 0.05, **P < 0.01, ***P < 0.001), of which kidney cancers, including ccRCC, again showed the relatively low expressions (Fig. 2b).
Figure 2 DLGAP5 expression in Pan-cancer (a) The expression of DLGAP5 in different human tumors within TCGA.(b)DLGAP5 expression in 33 human tumors was analyzed by integrating normal tissue data from GTEx database and TCGA tumor tissue. (TCGA sample data and GTEx normal sample data of 33 tumors, Additional file 1:Table 1) (blue: normal; red: tumor; log2(TPM + 1)*P < 0.05 ,**P < 0.01, ***P < 0.001).
Prognostic analysis of DLGAP5 in pan-cancer
To investigate the potential prognostic value of DLGAP5 upregulated in ccRCC and other cancers, data of the gene expression profiles from TCGA 33 tumor samples was analyzed for the relationship between expression and prognosis (overall survival, OS) using univariate analysis, the survival forest map comparing 33 tumors is presented in (Fig. 3a), and part of K-M survival plots are presented in (Additional file 2: Figure S1). Among these, about half of patients with upregulated show a worse prognosis than those with low expression (P < 0.05), and KIRC/ccRCC, KIRP, as well as LGG show a worse prognosis at a very significant level (P < 1 × 10− 10). Considering that there may be factors other than tumor death during the follow-up period, a comparison of the disease-specific survival (DSS) and the progression-free interval (PFI) was also presented in Fig. 3b and 3c. (part of K-M survival plots as shown in Additional file 3: Figure S2 and Additional file 4: Figure S3), respectively. The similar patterns of prognostic values, however, were also observed in those contexts, showing that KIRC/ccRCC, KIRP, as well as LGG exhibit a worse prognosis at a very significant level (P < 1 × 10− 8).
Figure 3 The survival forest map for prognostic analysis of DLGAP5 expression in pan-cancer. (a)HR(95% CI) for OS.(b)HR༈95% CI) for DSS. (c)HR༈95% CI) for PFI (Using a single-factor survival analysis ,DSS, disease-specific survival; OS, overall survival; PFI, progression-free interval; *P < 0.05 ,**P < 0.01, ***P < 0.001).
Association of DLGAP5 expression with immune infiltration in pan-cancer
Tumor-infiltrating lymphocytes (TILs) are the independent predictors of overall survival rate and sentinel lymph node status [31, 32]. Therefore, we investigated whether DLGAP5 expression was correlated with immune infiltration levels in different types of cancer. Six main types of immune cells, including B cell, CD4 T cell, CD8 T cell, Dendritic cell, Macrophage, and Neutrophil, were selected, of which the abundance of cells was scored and analyzed by Spearman’s correlation. Among 33 types of cancers analyzed, three types, namely KIRC/ccRCC, LGG and LIHC showed a significant positive correlation between the gene expression and immune infiltration levels, and an extremely significant level was observed in KIRC/ccRCC from R = 0.288, P = 1.99 × 10− 11 for CD4 T cell to R = 0.513, P = 5.04 × 10− 37 for Dendritic cell. These results indicate that DLGAP5 plays a key role in immune infiltration in ccRCC, especially for Dendritic cell (Fig. 4a).
Furthermore, the tumor microenvironment (TME), including immune cells and stromal cells as the major non-tumour constituents of tumour samples, plays an important role in the occurrence and development of tumors, and has important reference values for tumor diagnosis and prognosis evaluation [33]. In addition, immune cells not only infiltrate the tumour cell region but have also been demonstrated to associate with stromal cells, in a cancer-type-specific manner [34]. We therefore assessed the correlations of DLGAP5 expression with immune and stromal scores, as well as combined stromal and immune scores (estimate score) in 33 TCGA tumor types from the Tumor Immune Estimation Resource (TIMER). As shown in Fig. 4b, both KIRC/ccRCC and THCA, showing positive correlation of the expression with three scores, and both STAD and LUSC, showing negative correlation of the expression with three scores, were found to exhibit the very significant differences (R = 0.356, P = 1.97 × 10− 17 for KIRC, R = 0.465, P = 0 for THCA, R = − 0.355, P = 1.96 × 10− 12 for STAD, and R = − 0.293, P = 2.64 × 10− 11 for LUSC;) among tumor types evaluated.
Figure 4 Association of DLGAP5 expression with immune infiltration in Pan-cancer. (a) Scores of six immune-infiltrating cells of 33 cancers were downloaded from TIMER database, and the correlation between DLGAP5 expression and scores of these immune cells was analyzed respectively. The expressions in three cancers (KIRC, LGG, and LIHC) were significantly associated with the Scores. (b) The Stromal Scores, Immune Scores, and ESTIMATE Scores for THCA, KIRC, STAC, and HUSC, with showing a significant correlation between the gene expression and Scores.
Upregulated DLGAP5 expression associated with clinical stage of ccRCC in TCGA dataset
To further investigate the effects of DLGAP5 expression on the clinical progression of ccRCC, publicly available CRC gene expression RNAseq datasets from TCGA database were analyzed for the relationship between its expression levels and clinical pathological characteristics. As shown in Fig. 5a-e, compared with the control group, DLGAP5 expression level in tumorous tissues was significantly (P < 0.05) positive correlated with clinical TNM stage and Fuhrman grade of patients with ccRCC. The highest level was found in the tissues of patients with stage T4, N1, M1 or G4, the most progressive clinicopathological stage. Thus, as shown in K-M curves (Fig. 5f-h), upregulation of DLGAP5 expression resulted in the low survival probability of ccRCC patients.
Figure 5 The association of DLGAP5 expression with clinical stage in TCGA dataset. (a) topography (T1, T2, T3 and T4), (b) lymph node metastasis (N0, N1), (c) distant metastasis (M0 and M1), (d) G stages (I, II, III and IV) and (e)pathological stage (I, II, III and IV) of patients with ccRCC. (f-h) Kaplan-Meier survival curves of patients with ccRCC based on DLGAP5 expression levels (f: DSS; g: OS; h: PFI). (DSS, disease-specific survival; OS, overall survival; PFI, progression-free interval; *P < 0.05. TCGA, The Cancer Genome Atlas; DLGAP5, DLG Associated Protein 5; ccRCC, clear cell renal cell carcinoma)
DLGAP5 expressions in ccRCC tissues and cell lines
Based on the above pan-cancer analysis, we further focused on the DLGAP5 expression in ccRCC tissues and the cell lines, Fig. 6a and b show the results of TCGA and GTEx samples, indicating that DLGAP5 expression was upregulated in cancer tissues, but not in adjacent normal tissues. The results from the western blotting released that the upregulated expression of DLGAP5 in the ccRCC cell lines 786-O and CAKI-1 was also occurred compared with the normal renal epithelial cell line HK-2 (both P < 0.05; Fig. 6c). By using immunohistochemistry, three pairs of representative results were presented in (Fig. 6d), positive staining of DLGAP5 protein measured by the gray pixels was higher in ccRCC tissue than normal, indicating that DLGAP5 protein expression was upregulated in ccRCC tissues compared with adjacent normal tissue.
Figure 6 Up-regulated expression of DLGAP5 in ccRCC. (a) Data from TCGA database demonstrated a significant upregulation of DLGAP5 expression in ccRCC tumor samples (n = 531) compared with normal tubular tissue (n = 72). (b) Data from GTEx database and TCGA tumor tissue demonstrated a significant upregulation of DLGAP5 expression in ccRCC tumor samples (n = 531) compared with normal tubular tissue (n = 98). (c) Western blotting results revealed that DLGAP5 protein levels were higher in the ccRCC cell lines 786-O and CAKI-1 compared with the normal renal epithelial cells HK-2. (d) Representative images of DLGAP5 immunohistochemistry in paired tumor and normal tissue samples. (*P < 0.05; **P < 0.01 vs. control. DLGAP5, DLG Associated Protein 5; ccRCC, clear cell renal cell carcinoma; TCGA, The Cancer Genome Atlas.)
DLGAP5 Knockdown inhibits cell viability and proliferation in ccRCC cells.
To evaluate the contribution of DLGAP5 to ccRCC tumorigenesis, we designed two DLGAP-specific siRNAs (siRNA1, 2) for gene knockdown experiments. To test the efficacy of these siRNAs, they were transiently transfected into 786-O and CAKI-1cells. siRNA-NC was used as a negative control. We found that siRNA1 and siRNA2 significantly knocked down endogenous DLGAP5 expression compared with siRNA-NC (Fig. 7a). siRNA1 and siRNA2 was then used for cell growth analysis after DLGAP5 knockdown in various ccRCC cell lines, including 786-O and CAKI-1. Our data showed that the transient transfection of siRNA1 and siRNA2 suppressed the growth of the 786-O and CAKI-1 cells compared with siRNA-NC (Fig. 7b-d). These data further support the hypothesis that DLGAP5 promotes ccRCC cell viability and proliferation.
Figure 7 The knockdown of DLGAP5 levels inhibits the proliferation capability. (a) Reverse transcriptionqPCR and (b) western blotting were performed and revealed a significant decrease in the mRNA and protein levels of DLGP5 in siDLGAP51 and siDLGAP52-transfected human ccRCC, compared with those observed in siNCtransfected cells. The band intensities of DLGAP5 were quantified relative to ꞵ-actin and normalized to the siNC sample. (c) CCK8 results showed that DLGAP5 knockdown significantly inhibited the proliferation of 786O and CAKI1 cells. (d) Cell proliferation monitoring 786O and CAKI1 cells inhibited the proliferation. DLGAP5 knockdown significantly decreased the number of (e) 786O and CAKI-1 cell colonies compared with the control group. (*P < 0.05; **P < 0.01; ***P < 0.001; vs. control. DLGAP5, DLG Associated Protein 5; si, small interfering; NC, negative control.)
DLGAP5 knockdown inhibits cell migration and invasion in 786‑O and CAKI‑1 cells.
The up-regulated expression of DLGAP5 was closely related to the poor prognosis of ccRCC, indicating that its expression may promote the tumor development, The wound-healing and matrigel assays were therefore performed to assess the effects of DLGAP5 knockdown by RNAi on cell migration and invasion. The wound-healing assay revealed that cell migration was inhibited as a result of DLGAP5 knockdown in 786‑O and CAKI‑1 cells (Fig. 8a-b,). In comparison with the cells that were transfected with siRNA-NC, the cells of both cell lines that were transiently transfected siRNA2 also showed a significant inhibition of cell invasion through a Matrigel barrier when fibronectin was used as an attractant (Fig. 8c-d).
Figure 8 The knockdown of DLGAP5 levels significantly inhibits migration and invasion. (a) 786O and (b) CAKI1 cells Monolayer wound healing assay .(c) 786O and (d) CAKI1 cells Transwell assay demonstrated a significant decrease in the number of migration and invasion cells in siDLGAP51 and siDLGAP52-transfected human clear cell renal cell carcinoma compared with that observed in siNC-transfected cells. Scale bar, 100 µm. (*P < 0.05; **P < 0.01; ***P < 0.001; vs. control. DLGAP5, DLG Associated Protein 5; si, small interfering; NC, negative control.)