CcRCC was believed to raise from the proximal tubular epithelium with aggressive behavior that was unparallel with its mild appearance. After surgery, the median survival time of patients with metastasis was only 13 months, and the 5-year survival rate was merely 12.3%[18]. As one of the main genetic events of ccRCC pathogenesis, the mutant VHL gene pathway has become the target of treatment towards ccRCC[4]. Hopefully, with the help of bio-informatic data management and further verification, potential key genes could be identified and act as new targets for ccRCC patients with metastasis.
In our study, bioinformatic analysis from the 104 co-expressed DEGs showed concrete pathways that might be related to ccRCC metastasis. GO analysis showed DEGs mainly distributed in extracellular space or exosome and were enriched in pathways with extracellular matrix organization, cell adhesion, integrin binding and extracellular matrix structural constituent, etc. KEGG pathway analysis revealed these DEGs enriched in focal adhesion, ECM-receptor interaction and PI3K-Akt signaling pathway. Exosomes, by carrying proteins, ceramide, mRNA or micro-RNA, can induce metastasis through participation in EMT, mesenchymal-epithelial reverting transition (MErT), and neoangiogenesis[19]. For example, in ccRCC cell lines, exosomes containing miR-19b-3p can initiate EMT and accelerate tumor metastasis by reducing PTEN expression[20]. EMT is essential in tumor metastasis through processes such as disorienting cell polarity, disconnection with the basement membrane, acquiring plasticity, invasiveness and anti-apoptosis[21]. Studies have shown that the PI3K-Akt signaling pathway might induce the EMT process directly or through cross-talk with the Wnt/b-catenin signaling pathway, TGF-b, or NF-kB and initiate distant metastasis[22]. In ccRCC, the phosphorylation of AKT, which was a key kinase of the PI3K signaling pathway was reported to be related to EMT[23]. Our results also showed DEGs enriched in the PI3K signaling pathway, suggesting that PI3K signaling pathway-mediated EMT was likely to participate in the metastasis of ccRCC. To sum up, our analysis results have indicated various processes that might be associated with ccRCC metastases.
During the effort of establishing key genes, we first obtained 17 hub genes from the PPI network analysis. Using survival analysis, PCOLCE, P4HB, COL6A2 and COL6A3 were found related to ccRCC patient survival, which were then further tested with qRT-PCR and IHC staining. We found that these 4 genes presented higher expression in the metastatic counterpart of ccRCC both in cell lines and in the paired ccRCC tissues. Among them, P4HB and COL6A3 have been reported to be associated with ccRCC, whereas PCOLCE and COL6A2 were found to be associated with ccRCC for the first time. By making primary exploration, we thought these 4 genes might play an important role during the metastases of ccRCC.
To our knowledge, this was the first time that PCOLCE was found to be related to ccRCC. PCOLCE is a secreted glycoprotein that participates in the maturation of procollagen and ECM reconstruction by enhancing the activity of bone morphogenetic protein-1 (BMP-1)[24–26]. PCOLCE was overexpressed in osteosarcoma, which could promote lung metastasis via twist family bHLH transcription factor 1 (TWIST1). Reducing PCOLCE expression could prevent the migration, invasion and lung metastasis of osteosarcoma cells[27]. BMP-1 was reported to be up-regulated in gastric cancer associated with poor survival and distant metastasis[28]. Importantly, BMP-1 is elevated in ccRCC and promoted proliferation, migration, and invasion in ccRCC cell lines[29]. Our study showed PCOLCE, as an active enhancer of BMP-1, was higher in metastatic ccRCC, which raised the hypothesis that PCOLCE might be involved in ccRCC metastasis through regulation on BMP-1. Therefore, PCOLCE might act as a potential research target on ccRCC metastasis and more data was needed for further research.
P4HB, a beta subunit of Prolyl 4-hydroxylase, might help cancer cells survive from apoptosis induced by endoplasmic reticulum (ER) stress[30, 31]. P4HB promoted proliferation, invasion, migration and angiogenesis in glioma through mitogen-activated protein kinase (MAPK) signaling pathway[32]. In gastric cancer, P4HB was proved to play an important role on regulating invasion and migration in the hypoxia-inducible factor 1a (HIF1a) network[33]. Previous studies showed that overexpression of P4HB in ccRCC was associated with poor outcome[34]. In our study, we further found that P4HB was specifically elevated in metastatic ccRCC. Hence, we considered P4HB might play a key role in modulating ccRCC metastasis.
Also, COL6A2 was found to be related to ccRCC progression for the first time. COL6A1-3 were three subunits of collagen VI[35, 36]. Strong evidence had demonstrated COL6A1-3’s role in promoting the progression of tumors[37]. COL6A2 was dysregulated and associated with poor prognosis by involving in TGFβ1 pathway in serous ovarian cancer[38, 39]. COL6A3 may take part in the tumorigenesis and progression of cholangiocarcinoma through E2F1/LMCD1-AS1/miR-345-5p/COL6A3 axis and might act as a prognostic factor for pancreatic cancer[40]. Up-regulation of COL6A1 in ccRCC was associated with poor prognosis of patients and promoted tumor growth in vivo[41]. COL6A3 was reported to be overexpressed in metastatic renal cell carcinoma (RCC) and associated with poor survival[42]. To date, there is no report mentioning the relation between COL6A2 and ccRCC. As COL6A2 and COL6A3 were identified as hub genes in our results, an in-depth research is worth trying to clarify their mechanism on ccRCC metastasis.
With meaningful findings in our study as to establishing 4 possible key genes related to ccRCC metastasis, there are many limitations. Firstly, we only conducted 22 paired ccRCC samples and 2 cell lines to verify the bio-informatic findings, the size of which needs to be enlarged to obtain more reliable results. Secondly, the mechanism of four key genes in regulating ccRCC cells was still unclear. Further exploration should be conducted to clarify the function and potential mechanism of the four key genes both in vitro and in vivo.