Upregulation of BMP1 through ncRNAs correlates with adverse outcomes and immune infiltration in clear cell renal cell carcinoma

Renal cell carcinoma (RCC) remains a major type of cancer, with a significant increase in incidence over the past years [1]. In 2019, there were approximately 73,750 new cases of kidney cancer in the United States, resulting in approximately 14,830 deaths [1]. Clear cell renal cell carcinoma (ccRCC) accounts for approximately 80% of clinical cases of renal cell carcinoma in adults [2]. Localized ccRCC can be treated by surgery and has good prognosis, but approximately 20% of cases are at an advanced stage at the time of diagnosis, resulting in relatively poor prognosis [1, 3]. Despite current application of tyrosine kinase inhibitor (TKI) therapy and immunotherapy, the median survival time for advanced ccRCC is only approximately 3–4 years [4‐6]. Moreover, TKI is associated with increased risk of multiple cardiovascular events, and immunotherapy may increase the occurrence of such adverse reactions [7]. Therefore, it is very important to understand the mechanism of development and progression of ccRCC.

Bone morphogenic proteins (BMPs) participate in cartilage and bone formation. BMP1 is a metalloendopeptidase belonging to the astacin superfamily and is a splice variant of mammalian tolloid protein, whereas BMP2 to BMP16 belong to the transforming growth factor-β (TGF-β) superfamily [8]. BMP is commonly found in various tissues of the human body. BMPs participate in the genesis and development of tumors by inducing apoptosis and inhibiting proliferation through regulation of the epithelial–mesenchymal transition (EMT), the G2M checkpoint, angiogenesis, and the hypoxia pathway [9, 10]. BMP1 can activate the TGF-β signaling pathway, which initiates cleavage and release of the TGF-β complex from the extracellular matrix [11]. In recent years, it was found that BMP1 is highly expressed in some cancers and associated with cancer invasiveness in gastric cancer [12], lung cancer [13], osteosarcoma [14], colon cancer [15] and renal cancer [10]. However, expression and the mechanism of BMP1 in ccRCC and how they relate to prognosis are still unresolved. In addition, current understanding regarding the association between BMP1 and tumor immune infiltration in ccRCC remains unclear.

To address these questions, we first investigated expression of BMP1 and its prognostic significance in a variety of human cancers. Then, the mechanism of noncoding RNA (ncRNA)-associated regulation of BMP1 in ccRCC was examined. Finally, we explored the relationship of BMP1 expression with biomarkers of immune cells, immune cell infiltration, and immune checkpoints in ccRCC. Overall, our results reveal that high expression of BMP1 in ccRCC is associated with poor prognosis and tumor immune infiltration through ncRNAs.

In this study, we identified BMP1 as being upregulated in GBM, HNSC, KIRC, KIRP, and STAD. Additionally, we found that BMP1 can serve as a biomarker for poor prognosis in patients with KIRC. We also established the LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15, and CCDC18-AS1/hsa-miR-532-3p/BMP1 axis as the most potential upstream ncRNA-related pathway of BMP1 in ccRCC. Moreover, our findings revealed a significant positive correlation between BMP1 levels and tumor immune cell infiltration, biomarkers of immune cells, and immune checkpoint expression.

We first investigated expression of BMP1 across cancers using TCGA data, after which the GEPIA database was also used to confirm BMP1 expression. Survival analysis for BMP1 in cancer types of interest indicated that ccRCC patients with high expression of BMP1 had poor prognosis. The preliminary results of Xiao et al. [10] also demonstrated that overexpression of BMP1 is related to short survival time in ccRCC patients. The results of this report and our analysis demonstrate the carcinogenic role of BMP1 in ccRCC.

LncRNAs sponge miRNAs and regulate miRNA-targeted mRNAs at the posttranscriptional level in the cytoplasm [19, 20]. To explore the ceRNA mechanism of BMP1, we used 7 prediction programs to find possible miRNAs that might bind to BMP1, and 4 miRNAs were finally obtained. Survival analysis showed that ccRCC patients with high expression of these 4 miRNAs had better prognosis. Research has shown that miRNAs interact with the 3’-UTR of target genes to decrease the protein level by impeding the translation process or enhancing degradation of respective mRNAs [21]. For instance, hsa-miR-532-3p acts as a tumor suppressor and inhibits cell proliferation in lung cancer, breast cancer, ovarian cancer and renal cell carcinoma by targeting ETS1 [22, 23]. Hsa-miR-29c-3p modulates FOS expression to repress EMT and cell proliferation in age-related cataract tissues [24]. Combined with the results of survival analysis and differential expression of miRNAs in renal cancer and normal renal tissues, we selected hsa-miR-532-3p for further analysis.

Based on the ceRNA hypothesis [25], the potential lncRNAs that regulate hsa-miR-532-3p should be oncogenic in ccRCC. Next, upstream lncRNAs of the hsa-miR-532-3p/BMP1 axis were predicted, and 148 potential lncRNAs were identified. Combined with expression analysis, correlation analysis and survival analysis, six of the most likely upregulated lncRNAs, including LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15 and CCDC18-AS1, were obtained. It has been reported that most of these six lncRNAs play a role as oncogenes in a variety of malignant tumors, including ccRCC. For example, SNHG15 stimulates renal cell carcinoma proliferation and EMT by regulating the NF-κB signaling pathway [26]. Downregulation of SLC16A1-AS1 inhibits the proliferation, viability and migration of ccRCC [27]. PVT1 is significantly upregulated in ccRCC tissues, and high expression of PVT1 is associated with poor prognosis in ccRCC patients [28, 29]. In general, LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15 and the CCDC18-AS1/hsa-miR-532-3p/BMP1 axis were deemed to be potential regulatory pathways in ccRCC.

In recent years, the tumor microenvironment (TME) has gained much attention and is considered to be a key factor affecting treatment resistance, tumor development and prognosis [30‐32]. Our results demonstrate that BMP1 is significantly positively associated with various immune cells, including CD4⁺ T cells, B cells, CD8⁺ T cells, macrophages, neutrophils, and dendritic cells, in ccRCC. Furthermore, BMP1 is significantly positively associated with biomarkers of these infiltrated immune cells. These findings indicate that BMP1 might regulate development of ccRCC through tumor immune infiltration. In addition, the effectiveness of immunotherapy depends not only on adequate infiltration of immune cells into the tumor microenvironment but also on adequate expression of immune checkpoints [33]. Thus, we also investigated the relationship between BMP1 and immune checkpoints. Our results suggest that BMP1 correlates significantly positively with CTLA-4 and PD1 but significantly negatively with PD-L1 in ccRCC. These results indicate that targeting BMP1 might increase the efficacy of immunotherapy in ccRCC.

In general, we demonstrate that BMP1 is highly expressed in multiple types of human cancer (including ccRCC) and strongly correlates with unfavorable prognosis in ccRCC. We explored the upstream regulatory mechanism of BMP1 in ccRCC, namely, LINC00685, SLC16A1-AS1, PVT1, VPS9D1-AS1, SNHG15 and the CCDC18-AS1/hsa-miR-532-3p/BMP1 axis (Fig. 6). In addition, our results suggest that BMP1 might exert its oncogenic roles by increasing tumor immune cell infiltration and immune checkpoint expression.

Although our study has some advantages, it also has some limitations. First, the results were obtained from the TCGA database only, and verification in other databases is needed. Second, all the results were obtained by analyzing public datasets. In addition, further experiments should be performed in vivo and in vitro to verify these results.