Expression of HAPLN3 in ccRCC
The TCGA database results revealed that the expression level of HAPLN3 mRNA in ccRCC samples was significantly higher than in normal tissues (Figure 1A, B). Subsequently, high HAPLN3 mRNA expression in ccRCC tissues was found after analyzing GSE40435 in the GEO database (Figure 1C).
To further examine HAPLN3 protein expression, we retrieved IHC data from the HPA database. In normal kidney tissue, the intensity of HAPLN3 staining was obviously weaker than in ccRCC tissue (Figure 1D).
Relationship between HAPLN3 expression and clinical characteristics
To explore the link between HAPLN3 expression and clinical characteristics, we downloaded 537 ccRCC samples with HAPLN3 gene expression and required clinical data from the TCGA database for Wilcox test analysis. The results showed that tumor grade (Grade) (P<0.001), clinical-stage (Stage) (P<0.001), tumor invasion depth (T) (P<0.001), lymph node invasion (N) (P=0.004), distant metastases (M) (P<0.001) were all significantly correlated with HAPLN3 gene expression (Figure 2A-E). Furthermore, we assessed the overall survival of HAPLN3 mRNA expression in ccRCC patients using Kaplan-Meier survival analysis. In patients with ccRCC, elevated levels of HAPLN3 mRNA were obviously associated with worse survival (P=0.004, Figure 2F). To sum up, this data suggests that high levels of HAPLN3 may be responsible for accelerating the malignant progression of ccRCC.
GSEA identifies signaling pathways associated with HAPLN3
To screen for potential signaling pathways that are differentially activated in ccRCC, we compared high and low HAPLN3 gene expression datasets by GSEA. GSEA indicated considerable disparities in the MSigDB collection's richness (c.2.cp.kegg.v7.0.symbols.gmt). According to the criteria of NOM P < 0.05 and FDR < 0.05, the differently enriched pathways in the high expression phenotype of the HAPLN3 gene were screened (Table 1). The JAK-STAT signaling pathway, MAPK signaling pathway, VEGF signaling pathway, Hedgehog signaling pathway, NOTCH signaling pathway, and Pathways in cancer were significantly enriched in the HAPLN3 high expression phenotype, according to GSEA results (Figure 3).
Table 1. Gene sets enriched in ccRCC samples with high expression of HAPLN3
Gene set name
|
ES
|
NES
|
NOM p-val
|
FDR q-val
|
KEGG_JAK_STAT_SIGNALING_PATHWAY
|
0.60
|
2.57
|
0.000
|
0.000
|
KEGG_MAPK_SINGALING_PATHWAY
|
0.49
|
2.21
|
0.000
|
0.002
|
KEGG_VEGF-SINGSLING_PATHWAY
|
0.52
|
2.19
|
0.000
|
0.002
|
KEGG_HEDGEHOG_SINGALING_PATHWAY
|
0.56
|
2.07
|
0.000
|
0.006
|
KEGG_NOTCH_SINGALING_PATHWAY
|
0.63
|
2.05
|
0.002
|
0.007
|
KEGG_PATHWAYS_IN_CANCER
|
0.48
|
2.02
|
0.006
|
0.009
|
The prognostic analysis of HAPLN3 in ccRCC
Using Cox regression models, we analyzed multivariate hazard ratios for different variables from ccRCC patients in the TCGA-ccRCC dataset. Age, grade, stage, T classification (P<0.001), TNM stage, and HAPLN3 overexpression (P<0.001) were all significantly linked to the poor prognosis of ccRCC patients in a univariate analysis. Multivariate analysis further revealed that age, grade, and HAPLN3 overexpression (P=0.006) could be considered independent prognostic variables for ccRCC (Figure.4B). This data suggests that HAPLN3 could serve as an independent prognostic marker in patients with ccRCC.
Correlation between HAPLN3 expression and immune markers
The relationship between HAPLN3 and six key immune cells, including neutrophils, B lymphocytes, CD4+T lymphocytes, CD8+T lymphocytes, macrophages, and dendritic cells, was analyzed by the TIMER database. In ccRCC, HAPLN3 expression was significantly associated with CD8+T lymphocytes, CD4+T lymphocytes, macrophages, neutrophils, and dendritic cells (all P<0.05). Among them, CD8+T lymphocytes, neutrophils, and dendritic cells were significantly positively correlated with HAPLN3 expression (Figure.5). These results suggest HAPLN3 may influence the occurrence and progression of ccRCC through immune cells.
The role of HAPLN3 in immune escape of ccRCC
CTLA-4, PD-1, and PD-L1 are the three main immune checkpoints associated with immune escape in cancer 15. To investigate the role of HAPLN3 in the occurrence and progression of ccRCC, we explored the correlation between HAPLN3 and the CTLA-4, PD-1, PD-L1 corresponding markers CTLA4, PDCD1, CD274 through the TIMER database. As illustrated in Figure.6, the level of HAPLN3 expression was significantly positively correlated with CTLA4 and PDCD1 in ccRCC after adjustment for purity. Based on these findings, HAPLN3 may affect the development of ccRCC by influencing tumor immune escape.
Validation of the expression of HAPLN3 in ccRCC in vitro
To validate the expression results of HAPLN3 in ccRCC, we used RT-qPCR and Western blotting. In ccRCC tissues, the protein expression level of HAPLN3 was significantly higher than in the corresponding adjacent tissues (Figure 7A). Moreover, HAPLN3 expression in the ccRCC cell line was significantly higher than in the normal cortex/proximal tubule epithelial cell line (HK2) (Figure 7B). To further evaluate the biological function of HAPLN3 in ccRCC, we employed small interfering RNA technology to knock down HAPLN3 expression in 786-O and A498 cells. According to the results, the level of HAPLN3 expression was significantly down-regulated after transfection (Figure 7C).
Knockdown of HAPLN3 inhibits the proliferation, migration, and invasion of ccRCC cells
To determine the effect of HAPLN3 on ccRCC cell proliferation, we used CCK-8 and EdU cell proliferation experiments for verification (experimental grouping: the negative control group, i.e., siNC, and experimental group, i.e., siHAPLN3 group). The CCK-8 experiment revealed that when HAPLN3 was knocked down (siHAPLN3 group), the proliferation ability of 786-O and A498 cells was significantly reduced compared with siNC (Figure 8A). The EdU experiment results further demonstrated that the siHAPLN3 group significantly inhibited the proliferation ability of ccRCC cell lines 786-O and A498 compared to the siNC group (Figure 8B). In addition, HAPLN3 knockdown significantly reduced the ability of 786-O and A498 cells to migrate and invade (Figure.9A, B). This data suggests that HAPLN3 may function as an oncogene in ccRCC, promoting tumor progression.
HAPLN3 regulates tumor growth via ERK1/2 signaling
Based on GSEA results, the group with high HAPLN3 expression was primarily involved in proliferation and metastasis pathways. Further defining the underlying molecular mechanisms of HAPLN3 regulation of ccRCC cell growth, by using Western blot assay, we discovered that knocking down HAPLN3 in ccRCCs reduces the expression of the p-ERK1/2 protein (Figure 10A). In addition, after knocking down the expression of HAPLN3, the expression of apoptosis-related proteins, Bax, was up-regulated, and the expressions of Bcl-2 and PARP were down-regulated. Also, the expression of E-cadherin was significantly up-regulated, and the expressions of N-cadherin and Vimentin were reduced (Figure 10B, C). Therefore, we speculate that HAPLN3 promotes ccRCC proliferation and metastasis via the ERK1/2 pathway.