Expression levels of RNF180 in different types of cancer
The cancer-related database, GEPIA and Oncomine were used to analyze the expression of RNF180 mRNA in ccRCC. The results showed that RNF180 was usually overexpressed (green) in tumors compared with downregulated (red) in normal tissues based on GEPIA database (Fig. 1A). In addition, the expression profiles of RNF180 in various cancer types and corresponding normal controls were investigated in the Oncomine database. We found that RNF180 expression was significantly higher only in brain and CNS cancer. In addition, our results indicated that RNF180 mRNA expression levels in breast, colorectal, esophageal, gastric, head and neck, Liver Cancer, Lung cancer, pancreatic cancers were significantly under expressed compared to the corresponding normal tissues in some data sets (Fig. 1B). To further evaluate RNF180 expression in various cancer types, we investigated RNF180 expression using the RNA-seq data of multiple malignancies in TCGA by TIMER. The results indicated that the expression levels of RNF180 were significantly increased in GBM (glioblastoma multiforme) and PCPG (Pheochromocytoma and Paraganglioma) compared to adjacent control samples (Fig. 1C).
However, RNF180 expression was significantly lower in BLCA (Bladder Urothelial Carcinoma), BRCA (breast invasive carcinoma), CESC (Cervical squamous cell carcinoma and endocervical adenocarcinoma), COAD (Colon adenocarcinoma), ESCA (esophageal carcinoma), HNSC (Head and Neck squamous cell carcinoma), KICH (Kidney Chromophobe), KIRC (kidney renal clear cell carcinoma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma) , LUSC (lung squamous cell carcinoma), STAD (stomach adenocarcinoma), THCA (Thyroid carcinoma), UCEC (Uterine Corpus Endometrial Carcinoma) than in normal controls (Fig. 1b). Therefore, RNF180 showed the same expression trend in renal cancers in both the microarray and RNA-seq data.
RNF180 is a potential biomarker for ccRCC by TCGA
By reviewing the TCGA database, we found that RNF180 could serve as a potential biomarker for ccRCC. We first used the TCGA database to obtain the mRNA expression of RNF180 and its relationship with the prognosis of ccRCC. As shown in Figure 2A, the transcript level of RNF180 was significantly reduced in ccRCC compared with normal tissues (p < 0.001). Next, we further evaluated its relationship with clinicopathological features. RNF180 showed correlation with tumor grade, and its transcript level was well distinguished in G1 versus G3 and G4 (Figure 2B). We also observed that mRNA transcript levels of RNF180 were significantly correlated with the clinical stage of patients (Figure 2C). In terms of pathological samples, mRNA transcript levels of RNF180 were significantly distinguished in different TNM stages (Figure 2 D-F). In summary, the above evidences support our results that RNF180 may be a potential biomarker for patients with ccRCC.
Low expression of RNF180 predicts poor prognosis of ccRCC patients
The above results lead us to question whether the expression level of RNF180 could be a prognostic biomarker of ccRCC. To verify this hypothesis, TCGA database and GSE22541 dataset were used to analyze the overall survival between different expression level of RNF180 by Kaplan–Meier analysis (Figure 3 A-B). We found that low expression of RNF180 was correlated to low OS. To further validate the findings, IHC was performed to evaluate the RNF180 expression in clinical samples from 72 patients (Figure 3C). The patient characteristics and clinicopathological factors by RNF180 expression were displayed in Table 2. The results indicated that the low expression of RNF180 was significantly correlated to primary T stage (p=0.027), TNM stage (p=0.049) and Fuhrman grade (p=0.05). Despite age (p=0.380), gender (p=0.318) and tumor size (p=0.408) showed little relevant to expression of RNF180. IHC scores also supported the findings. The scores between tumor and adjacent normal tissues were significantly distinguished (p= 0.0424, Figure 3D), and such results could also be found between G1+G2 and G3+G4 (p=0.0491, Figure 3E) and between stage I+II and stage III+IV (p=0.0275, Figure 3F). Overall survival of patients with ccRCC was also reported by Kaplan–Meier curves (Figure 3G) and the results confirmed our previous hypothesis that low expression of RNF180 indicated low OS. We concluded that the expression level of RNF180 was a prognostic predictor for ccRCC patients
RNF180 is downregulated in ccRCCs clinical samples and cell lines
To further confirm the results of the bioinformatics analysis, we then evaluated the mRNA and protein expression of RNF180 in clinical samples and cell lines of ccRCCs. As shown in Figure 4A, in clinical samples of ccRCC, the results of Western Blot showed that protein levels of RNF180 were downregulated. The results of quantitative real-time PCR (qRT-PCR) corroborated the findings that the expression of RNF180 mRNA was relatively lower in tumors than in normal tissues (Figure 4B). Additionally, Western Blot and qRT-PCR were performed in ccRCCs cell lines. Similarly, the results in ccRCCs cell lines showed that both mRNA and protein levels of RNF180 were downregulated (Figure 4C-D). As expected, RNF180 expression was significantly decreased at both the cellular level and histological level in ccRCCs.
Overexpression of RNF180 inhibits proliferation, migration and invasion of ccRCCs in vitro
Altered expression levels of RNF180 in ccRCC may have a potential impact on the progression of ccRCC. To confirm this hypothesis, we constructed 786-O and A498 cell lines overexpressing RNF180 by transfection with overexpression plasmids and performed Western-Blot assays and qRT-PCR to verify this (Figure 5A). Next, CCK-8 assays were performed to assess the proliferation of the cells. The results showed that overexpression of RNF180 significantly reduced the proliferation rate of ccRCCs cells (Figure 5B). Similarly, the results of the EdU assays suggested that overexpression of RNF180 inhibited the proliferation of ccRCCs cells (Figure 5C). Migration and invasion are also hallmarks of tumor progression. We performed transwell assays to assess the migratory and invasive abilities of the cells. Our results showed that migration and invasion of ccRCCs cells were inhibited after RNF180 overexpression (Figure 5D). In summary, overexpression of RNF180 inhibited the proliferation, migration and invasion of ccRCCs cells in vitro.
Effects of RNF180 on tumor growth in vivo
To confirm the effect of RNF180 protein expression on tumor growth in vivo, we established a tumor transplantation model in nude mice by inoculating 4 × 106 786-O cells into nude mice and transfecting them with RNF180 expression vectors or the corresponding negative control vectors. After 8 weeks, the transplanted tumor area was smaller in the RNF180 overexpression group than in the control group (Figure 6A). Similarly, IHC staining showed that RNF180 was highly expressed in xenografts consisting of flag-RNF180 treated cells. The overexpressed RNF180 group had lower Ki67 levels than the control group (Figure 6B). In addition, the mean tumor volume in the group with increased RNF180 expression was significantly smaller than that in the group with decreased RNF180 expression (Figure 6C). Similar results were found for the tumor mass measurement (Figure 6D).
According to the biological effects of RNF180 mentioned above, the role of RNF180 overexpression on metastasis in vivo needs to be further proved. Cells with RNF180 overexpression or empty vector transfection were subcutaneously injected into nude mice to inhibit cell liver metastasis. Compared with the control group, less nodules of liver metastasis were observed in mice injected with RNF180 overexpression cells (Figure 6E). Histopathological studies confirmed that liver disease was caused by ccRCC cell invasion and subsequent tumor growth (Figure 6F). Therefore, we believe that RNF180 overexpression can inhibit the growth and metastasis of renal carcinoma.
These results were consistent with the results of the CCK8 and EdU assays described above. Therefore, we concluded that RNF180 was closely associated with the progression of renal cancer.
Overexpression of RNF180 significantly reduces EMT in ccRCC cells.
To investigate how RNF180 is involved in tumorigenesis and development in ccRCCs, a gene set enrichment analysis (GSEA) was performed to explore the biological pathways regulated by RNF180 based on the TCGA database. The results showed that RNF180 was highly correlated with the signatures of MAPK signalling, TGF-β signalling and WNT-β signalling (Figure 7A-C). EMT represents cancer progression and metastasis. TGF-β signaling, WNT-β signaling and MAPK signaling are involved in the EMT process by regulating many transcription factors, such as Vimentin, Snail1 [18,21,27]. Altered expression levels of E-cadherin and N-cadherin are also the biomarkers of EMT process . Therefore, we hypothesized that RNF180 influences the EMT process. To test this hypothesis, we assessed the alteration of several EMT markers in RNF180 overexpressing cells. The results indicated that in RNF180 overexpression plasmid-transfected 786-O and A498 cells, E-cadherin expression was increased at the protein and mRNA levels, while Vimentin, Snail1 and N-cadherin expression was decreased (Figure 7D-F). Accordingly, it can be concluded that overexpression of RNF180 is able to inhibit EMT of ccRCCs in vitro.
Correlations of RNF180 expression with immune infiltration level in ccRCC
Somatic copy number alterations (sCNA) of RNF180 have significant correlations with infiltrating levels of neutrophils (Figure 8A). Immune cells in the TME can affect patient survival, and the above findings support a prognostic role of RNF180 in ccRCC. Hence, it would be meaningful to explore the association between immune infiltration and RNF180 expression. We determined whether RNF180 expression was correlated with the immune infiltration level by calculating the coefficient of RNF180 expression and immune infiltration level in ccRCC in TIMER. The results indicated that RNF180 expression was also significantly correlated with the infiltration levels of CD8+ T cells, CD4+ T cells, macrophages and neutrophils in ccRCC, while showed no significant correlation of infiltration levels of B cells and dendritic cells (Figure 8B).
Immune infiltration is associated with survival in ccRCC patients with low RNF180 expression
By mining the EPIC database, we were able to explore the correlation between immune infiltration and survival of ccRCC patients. In general, immune infiltration had a significant effect on survival of ccRCC patients in patients with low RNF180 expression, while no significant effect was seen in patients with high RNF180 expression. Specifically, we found that among lymphocytes, high infiltration of B cells (Figure 9A), CD8+ T (Figure 9B) cells and CD4+ T (Figure 9C) cells significantly increased the overall survival time of patients, while high infiltration of NK cells (Figure 9D) decreased the overall survival time of patients. In contrast, among other components of the tumor microenvironment (TME), we found that high levels of cancer-associated fibroblasts (CAF) decreased the overall survival (OS) of patients (Figure 9E), while high levels of vascular endothelial cells significantly increased the OS of patients (Figure 9F). With the above findings, it is obvious to indicate that the type and level of immune infiltration has a significant impact on the prognosis of patients with ccRCC with low expression of RNF180.