Knockdown of SETD2 promotes erastin-induced ferroptosis in ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is associated with poor prognosis. The histone H3 lysine 36 methyltransferase SET-domain-containing 2 (SETD2) has been reported to be expressed at low levels and frequently mutated in ccRCC. Ferroptosis, a form of death distinct from apoptosis and necrosis, has been reported in recent years in renal cancer. However, the relationship between SETD2 and ferroptosis in renal cancer is not clear. Here, we demonstrated that SETD2 was expressed at low levels in ccRCC and was associated with poor prognosis. Moreover, we found that knockdown of SETD2 increased lipid peroxidation and Fe2+ levels in tumor cells, thereby increasing the sensitivity of erastin, a ferroptosis inducer. Mechanistically, histone H3 lysine 36 trimethylation (H3K36me3) which was catalyzed by SETD2, interacted with the promoter of ferrochelatase (FECH) to regulate its transcription and ferroptosis-related signaling pathways. In conclusion, the presesnt study revealed that knockdown of the epigenetic molecule, SETD2, significantly increases the sensitivity of ferroptosis inducers which promotes tumor cell death, thereby indicating that SETD2 may be a potential therapeutic target for ccRCC.

RBM4 inhibits the growth of clear cell renal cell carcinoma by enhancing the stability of p53 mRNA.

RBM4 has been reported as a tumor suppressor gene in cancers, including lung cancer, colon cancer and gastric cancer. However, the role of RBM4 in clear cell renal cell carcinoma (ccRCC) remains unclear. Therefore, the present study investigated the expression and biological function of RBM4 in ccRCC. Analysis of the differential expression of RBM4 and its relationship with clinicopathological features using ccRCC samples data from TCGA database deminstrated that RBM4 expression in tumor samples of ccRCC was lower than that in normal samples, and RBM4 expression was closely related to the survival time of patients. RBM4 overexpression (RBM4-oe) cell lines were constructed to investigate the effect of RBM4 on biological function using CCK-8, EdU, flow cytometry and wound-healing assays. In addition, the regulatory effect of RBM4 on signaling pathways was investigated by GSEA and WB assays. RBM4-oe significantly reduced the proliferation of ccRCC cells by controlling the p53 signaling pathway, inhibited cell cycle progression and promoted apoptosis. In addition, RBM4-oe suppressed the migration and invasion of cells by EMT. Mechanistically, RBM4-oe facilitated the activity of the p53 signaling pathway by enhancing the stability of p53 mRNA. Finally, RBM4-oe markedly inhibited the growth of tumors formed with 786-O cells in vivo. In summary, there findings suggeated that RBM4 inhibits the progression of ccRCC by promoting p53 signaling pathway activity by enhancing the stability of p53 mRNA, suggesting that RBM4 may be a potential target for the treatment of patients.

FBXL6 depletion restrains clear cell renal cell carcinoma progression.

BACKGROUND: F-box proteins play important roles in cell cycle and tumorigenesis. However, its prognostic value and molecular function in clear cell renal cell carcinoma (ccRCC) remain unclear. In this study, we established a survival model to evaluate the prognosis of patients with ccRCC using the F-box gene signature and investigated the function of FBXL6 in ccRCC. METHODS: Comprehensive bioinformatics analyses were used to identify differentially expressed F-box and hub genes associated with ccRCC carcinogenesis. Based on the F-box gene signature, we constructed a risk model and nomogram to predict the overall survival (OS) of patients with ccRCC and assist clinicians in decision-making. Finally, we verified the function and underlying molecular mechanisms of FBXL6 in ccRCC using CCK-8 and EdU assays, flow cytometry, and subcutaneous xenografts. RESULTS: A risk model based on FBXO39, FBXL6, FBXO1, and FBXL16 was developed. In addition, we drew a nomogram based on the risk score and clinical features to assess the prognosis of patients with ccRCC. Subsequently, we identified FBXL6 as an independent prognostic marker that was highly expressed in ccRCC cell lines. In vivo and in vitro assays revealed that the depletion of FBXL6 inhibited cell proliferation and induced apoptosis. We also demonstrated that SP1 regulated the expression of FBXL6. CONCLUSIONS: FBXL6 was first identified as a diagnostic and prognostic marker in patients with ccRCC. Loss of FBXL6 attenuates proliferation and induces apoptosis in ccRCC cells. SP1 was also found to regulate the expression of FBXL6.

A pyroptosis-associated signature plays a role in prognosis prediction in clear cell renal cell carcinoma.

BACKGROUND: Approximately 90% of renal malignancies are RCCs (renal cell carcinomas), and the primary subtype in histology is ccRCC (clear cell RCC). In recent years, pyroptosis has been considered a kind of inflammation-related programmed cell death that participates in the invasion, metastasis, and proliferation of tumour cells, thereby influencing tumour prognosis. Nonetheless, the expression level of pyroptosis-associated genes in RCCs and their relationship with prognosis remain obscure. RESULTS: In our research, 44 regulators of pyroptosis that were differentially expressed between normal kidney and ccRCC tissues were identified. ccRCC cases were categorized into 2 subgroups according to prognostic-related DEGs (differentially expressed genes), and there was a significant difference in OS (overall survival) between them. The prognostic value of pyroptosis-associated genes was assessed as a signature based on a cohort from TCGA (The Cancer Genome Atlas). Following Cox regression with DEGs and LASSO (least absolute shrinkage and selection operator), a 6-gene signature was established, and all ccRCC cases in the cohort from TCGA were categorized into an LR (low-risk) or HR (high-risk) group (P < 0.001). In combination with clinical features, risk scores were considered a predictive factor of OS in ccRCC. KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) analyses suggest increased immunity and enrichment of genes related to immunity in the HR group. CONCLUSIONS: Our findings indicate that genes related to pyroptosis have an important role in tumour immunity and may be used to predict the prognosis of ccRCC.

Identification of a differentiation-related prognostic nomogram based on single-cell RNA sequencing in clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is a kidney cancer that is originated from the lined proximal convoluted tubule, and its major histological subtype is clear cell RCC (ccRCC). This study aimed to retrospectively analyze single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, to explore the correlation among the evolution of tumor microenvironment (TME), clinical outcomes, and potential immunotherapeutic responses in combination with bulk RNA-seq data from The Cancer Genome Atlas (TCGA) database, and to construct a differentiation-related genes (DRG)-based prognostic risk signature (PRS) and a nomogram to predict the prognosis of ccRCC patients. First, scRNA-seq data of ccRCC samples were systematically analyzed, and three subsets with distinct differentiation trajectories were identified. Then, ccRCC samples from TCGA database were divided into four DRG-based molecular subtypes, and it was revealed that the molecular subtypes were significantly correlated with prognosis, clinicopathological features, TME, and the expression levels of immune checkpoint genes (ICGs). A DRG-based PRS was constructed, and it was an independent prognostic factor, which could well predict the prognosis of ccRCC patients. Finally, we constructed a prognostic nomogram based on the PRS and clinicopathological characteristics, which exhibited a high accuracy and a robust predictive performance. This study highlighted the significance of trajectory differentiation of ccRCC cells and TME evolution in predicting clinical outcomes and potential immunotherapeutic responses of ccRCC patients, and the nomogram provided an intuitive and accurate method for predicting the prognosis of such patients.

RHBDD1 promotes proliferation, migration, invasion and EMT in renal cell carcinoma via the EGFR/AKT signaling pathway.

Renal cell carcinoma (RCC) is a common malignant tumor of the urinary system with a poor prognosis and high mortality rate. The increasing incidence of RCC poses a serious threat to human health. It is well­documented that rhomboid domain­containing protein 1 (RHBDD1) plays a vital role in cancer progression. The present study was designed to identify the biological functions of RHBDD1 in RCC and investigate the underlying regulatory mechanism, aiming to explore the novel molecular therapeutic targets for RCC. The protein and mRNA expression levels of RHBDD1 in normal renal tubule epithelium and human RCC cell lines were analyzed using western blotting and reverse transcription­quantitative PCR. Cell proliferation was determined using Cell Counting Kit­8 assays. Wound healing and Transwell assays were performed to determine cell migration and invasion, respectively. In addition, key proteins related to migration, invasion and epithelial­mesenchymal transition (EMT), such as matrix metalloproteinase (MMP)2, MMP9, MMP13, E­cadherin, N­cadherin, vimentin and Slug, were analyzed using western blotting. In addition, the EGFR/AKT signaling pathway was further studied using western blotting to determine the potential molecular mechanism. The results of the present study revealed that RHBDD1 expression levels were significantly upregulated in RCC cell lines. The knockdown of RHBDD1 inhibited cell proliferation, migration, invasion and EMT, while the overexpression of RHBDD1 promoted cell proliferation, migration, invasion and EMT in RCC. In addition, the knockdown of RHBDD1 suppressed the activation of the EGFR/AKT signaling pathway, while the overexpression of RHBDD1 activated the EGFR/AKT signaling pathway. Moreover, these stimulatory effects of RHBDD1 overexpression on RCC progression and the EGFR/AKT signaling pathway were partly reversed by gefitinib, an EGFR inhibitor. In conclusion, the findings of the present study suggested that RHBDD1 may be a crucial regulator of RCC by modulating the EGFR/AKT signaling pathway. The present study may provide a theoretical basis and potential targets for RCC treatment.

IGLL5 is correlated with tumor-infiltrating immune cells in clear cell renal cell carcinoma.

Renal cell carcinomas (RCCs) account for about 90% of renal tumors, and their major histological subtype is ccRCC (clear cell RCC). Increasing evidence has indicated that the tumor microenvironment plays a significant role in the occurrence and development of ccRCC. In this study, we used ESTIMATE and CIBERSORT computational methods to calculate the proportion of immune and stromal components and the rate of TICs (tumor-infiltrating immune cells) in 539 ccRCC samples from The Cancer Genome Atlas database. By examining the intersection of the differentially expressed genes obtained by the protein-protein interaction network and Cox regression analysis, we identified only one overlapping gene: IGLL5 (immunoglobulin lambda-like polypeptide 5). We report that IGLL5 expression is correlated with TICs. Furthermore, our immunoinfiltration analyses revealed that three types of TIC are positively correlated with IGLL5 expression. IGLL5 may have potential as a prognostic biomarker of ccRCC.

Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma.

Nucleobindin 2 (NUCB-2) is a multifunctional protein that contains several functional domains and is associated with a wide variety of biological processes, such as food intake and energy homeostasis. NUCB-2 has been demonstrated to be associated with worse malignant outcomes and cell migration in breast and prostate cancer. However, to the best of our knowledge, its clinical and biological significance in renal cell carcinoma remains unknown. In the present study, tissue specimens from 68 patients with renal cell carcinoma and 10 normal controls were collected for NUCB-2 mRNA and protein assays. The NUCB-2 level in the patients with renal cell cancer was significantly increased compared with the normal control patients. NUCB-2-knockout in the renal cancer cell line SK-RC-52 inhibited migration and invasion. In addition, the expression levels of molecules associated with epithelial-mesenchymal transition (EMT), including E-cadherin, ß-catenin, Slug and Twist, were affected by NUCB-2 suppression and the zinc finger E-box binding to homeobox 1 (ZEB1)-dependent pathway. The AMP-dependent protein kinase (AMPK)/target of rapamycin complex (mTORC) 1 signaling pathway participates in the regulation of NUCB-2-mediated metastasis and EMT. Suppression of NUCB-2 also inhibited tumor nodule formation in a murine renal cell carcinoma tumor model. In summary, NUCB-2 increased migration, invasion and EMT in renal cell carcinoma cells through the AMPK/TORC1/ZEB1 pathway in vitro and in vivo.

BMP8A promotes survival and drug resistance via Nrf2/TRIM24 signaling pathway in clear cell renal cell carcinoma.

There is increasing evidence that bone morphogenetic proteins (BMP) are involved in the proliferation and drug tolerance of kidney cancer. However, the molecular mechanism of BMP8A in renal cell proliferation and drug tolerance is not clear. Here we showed that BMP8A was highly expressed in renal cell carcinoma, which suggests a poor prognosis of ccRCC. Promotion of cell proliferation and inhibition of apoptosis were detected by CCK-8 assay, Trypan Blue staining, flow cytometry and bioluminescence. BMP8A promoted resistance of As2 O3 by regulating Nrf2 and Wnt pathways in vitro and in vivo. Mechanistically, BMP8A enhanced phosphorylation of Nrf2, which, in turn, inhibited Keap1-mediated Nrf2 ubiquitination and, ultimately, promoted nuclear translocation and transcriptional activity of Nrf2. Nrf2 regulates the transcription of TRIM24 detected by ChIP-qPCR. BMP8A was highly expressed in ccRCC, which suggests a poor prognosis. BMP8A was expected to be an independent prognostic molecule for ccRCC. On the one hand, activated Nrf2 regulated reactive oxygen balance, and on the other hand, by regulating the transcription level of TRIM24, it was involved in the regulation of the Wnt pathway to promote the proliferation, invasion and metastasis of ccRCC and the resistance of As2 O3 . Taken together, our findings describe a regulatory axis where BMP8A promotes Nrf2 phosphorylation and activates TRIM24 to promote survival and drug resistance in ccRCC.