Apoptosis In Renal Cell Carcinoma Cells Research Articles

TGFBI promotes proliferation and epithelial–mesenchymal transition in renal cell carcinoma through PI3K/AKT/mTOR/HIF-1α pathway

BackgroundRenal cell carcinoma (RCC) is presently recognized as the most prevalent kidney tumor. However, the role and underlying mechanism of action of the conversion factor-inducible protein (TGFBI), an extracellular matrix protein, in RCC remain poorly understood.MethodsIn this study, we employed Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry techniques to assess the expression of TGFBI in RCC tissues or cells. Furthermore, we analyzed the proliferation and migration of RCC cells using CCK8, cloning, scratching, and migration assays. Additionally, we examined apoptosis and cell cycle progression through flow cytometry, analysis. Lastly, we employed gene set enrichment analysis (GSEA) to investigate the biological processes associated with TGFBI, which were subsequently validated.ResultsThe findings indicate that TGFBI exhibits significantly elevated expression levels in both renal cell carcinoma (RCC) tissues and cells. Furthermore, the knockdown of TGFBI in SiRNA transfected cells resulted in the inhibition of RCC cell proliferation, migration, invasiveness, apoptosis, and alteration of the cell cycle. Additionally, TGFBI was found to impede the epithelial-mesenchymal transition (EMT) process in RCC cells. Bioinformatics analysis suggests that TGFBI may exert its influence on various biological processes in RCC through the tumor immune microenvironment. Moreover, our study demonstrates that TGFBI promotes RCC progression by activating the PI3K/AKT/mTOR/HIF-1α.ConclusionsOur research indicates that TGFBI exhibits high expression in RCC and facilitate RCC progression and metastasis through various molecular mechanisms. Hence, TGFBI has the potential to be a novel therapeutic target for the diagnosis and treatment of RCC in the future.

Poria acid inhibit the growth and metastasis of renal cell carcinoma by inhibiting the PI3K/akt/NF-κb signaling pathway

BackgroundPoria acid (PAC) is a triterpene compound found in Poria cocos, a traditional Chinese medicine (TCM). The current study aims to explore the therapeutic effects and potential mechanisms of PAC on the migration and proliferation of human renal cell carcinoma (RCC) cells as well as tumor growth in animal model. MethodsCell viability and proliferative capacity of normal renal cells and RCC cells were investigated by MTT assay. In addition, 786-O cells were divided into four groups and treated with different concentrations of PAC (0, 20, 40, and 60 μM) for 48 h. Cell scratch test and cell invasion assay were performed to evaluate the effects of PAC on the invasion and migration of RCC cells, respectively. The effects of PAC on apoptosis of RCC cells and expression levels of PI3K/Akt/NF-kB signaling pathway-related biomarkers were investigated using TUNEL staining and Western blotting methods, respectively. Effects of PAC on the inhibitory activity of RCC tumor in mice were evaluated in a 786-O CDX model. ResultsThe study found that PAC inhibited the viability of RCC cells in a dose-dependent manner, as demonstrated by in vitro cell assays (p < 0.05). However, PAC showed no significant inhibitory effect on normal renal cells (p > 0.05). PAC also significantly inhibited the migration and invasion of RCC via EMT/MMP signaling pathways (p < 0.05). Immunofluorescence and immunoblotting results showed that PAC induced the apoptosis of RCC, which was accompanied by changes in the expression levels of apoptosis-related proteins (p < 0.05). Moreover, PAC significantly downregulated the PI3K/Akt/NF-kB signaling pathway in a concentration-dependent manner (p < 0.05). The effect of PAC on RCC apoptosis was dramatically reversed by 740Y–P (PI3K agonist) (p < 0.05) but significantly enhanced in the presence of LY294002 (PI3K inhibitor) (p < 0.05). The results of in vivo experiment also demonstrated that the antitumor activity of PAC was achieved by affecting the PI3K/Akt/NF-kB signaling pathway. ConclusionsPAC can effectively suppress the proliferation, invasion and migration of RCC cells, and exhibit anti-tumor effects in RCC model by inhibiting the PI3K/Akt/NF-kB signaling pathway.

Pan-Cancer Analysis Identifies BCLAF1 as a Potential Biomarker for Renal Cell Carcinoma.

B-cell lymphoma-2-associated transcription factor 1 (BCLAF1) is a versatile protein involved in the regulation of gene transcription and post-transcriptional processing. Although BCLAF1 exerts a broad tumor suppressor effect or tumor promoter effect in many cancer types, the specific roles concerning its expression levels, and its impact on tumorigenesis in Renal cell carcinoma (RCC) remain unclear. Here, we utilized the Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) datasets alongside R software and online tools to unravel the specific roles of BCLAF1 in 33 cancer types, including its expression levels, tumor immune and molecular subtypes, and its correlation with prognosis, diagnosis, DNA methylation, and immune microenvironment. Additionally, we carried out cell biology experiments to independently investigate the expression of BCLAF1 in RCC and its effects on tumor progression. BCLAF1 was differentially expressed in tumor tissues compared to normal tissues across various cancer types and was also differentially expressed in different immune and molecular subtypes. In RCC, patients with high BCLAF1 expression had a better prognosis and BCLAF1 was tightly correlated with the stage, gender, and histological grade of patients. Furthermore, BCLAF1 had higher DNA methylation levels and higher immune infiltration levels in tumor tissues. Additionally, cell functional experiments confirmed the low expression of BCLAF1 in RCC and that BCLAF1 significantly inhibited the proliferation, migration, and invasion, while inducing apoptosis and cell cycle arrest in RCC cells in vitro. Our study under-scored the potential of BCLAF1 as an important actor in tumorigenesis, especially concerning RCC where it may serve as an effective prognostic marker.

Disruption of Autophagic Flux and Treatment with the PDPK1 Inhibitor GSK2334470 Synergistically Inhibit Renal Cell Carcinoma Pathogenesis.

Background: Renal cell carcinoma (RCC) frequently exhibits activating PI3K-Akt-mTOR pathway mutations. 3-Phosphoinositide-dependent kinase 1 (PDPK1 or PDK1) has been established to play a pivotal role in modulating PI3K pathway signaling. mTOR is the main autophagy-initiating factor. However, limited advances have been made in understanding the relationship between PDPK1 and autophagy in RCC. Methods: GSK2334470 (GSK470), a novel and highly specific inhibitor of PDPK1, was selected to investigate the anticancer effects in two RCC cell lines. Cell growth was assessed by CCK-8 test and colony formation. Changes in the protein levels of key Akt/mTOR pathway components and apoptosis markers were assessed by Western blotting. Autophagy was assessed by using LC3B expression, transmission electron microscopy, and a tandem mRFP-EGFP-LC3 construct. The effect of PDPK1 and autophagy inhibitor chloroquine in RCC in vivo was examined in a mouse tumor-bearing model. Results: GSK470 significantly inhibited cell proliferation and induces apoptosis in A498 and 786-O RCC cells. GSK470 downregulates the phosphorylation of PDPK1, thereby inhibiting downstream phosphorylation of Akt1 at Thr308 and Ser473 and mTOR complex 1 (mTORC1) activity. Treatment with insulin-like growth factor-1 (IGF-1) partially restored GSK470-induced behaviors/activities. Interestingly, treatment of A498 and 786-O cells with GSK470 or siPDPK1 induced significant increases in the hallmarks of autophagy, including autophagosome accumulation, autophagic flux, and LC3B expression. Importantly, GSK470 and chloroquine synergistically inhibited the growth of RCC cells in vitro and in xenograft models, supporting the protective role of autophagy activation upon blockade of the PDPK1-Akt-mTOR signaling pathway. Conclusion: Our study provides new insight into PDPK1 inhibition combined with autophagy inhibition as a useful treatment strategy for RCC.

FOXK1 upregulation is correlated with tumor progression and tumor associated macrophages infiltration in renal cell carcinoma.

Renal cell carcinoma (RCC)is one of the most common urological cancers in adults. Forkhead box k1 (FOXK1)is a transcription factor involved in the progression of various malignant tumors. In this study, we aimed to investigate the expression and roles of FOXK1 in RCC development. Our findings revealed increased expression of FOXK1 in RCC tumor tissues and cell lines compared with normal controls. Functional assays demonstrated that knockdown of FOXK1 significantly inhibited proliferation, migration, invasion, and promoted apoptosis in RCC cells. Furthermore, FOXK1 knockdown suppressed epithelial-mesenchymal transition and Wnt signaling in RCC cells. Additionally, we observed a correlation between FOXK1 upregulation and tumor associated macrophages infiltration in RCC. These results suggest that FOXK1 acts as an oncogene in RCC and may serve as a potential therapeutic target for RCC treatment.

Circular RNA CSPP1 motivates renal cell carcinoma carcinogenesis and the Warburg effect by targeting RAC1 through microRNA-493-5p.

Circular RNAs (circRNAs) take on regulatory roles in renal cell carcinoma (RCC). The research's goal was to figure out circ-CSPP1's role and molecular mechanism in RCC. The results clarified that circ-CSPP1 expression was enhanced in RCC. Down-regulating circ-CSPP1 refrained the proliferation, migration, invasion, and Warburg effect (aerobic glycolysis), but accelerated apoptosis of RCC cells. The luciferase activity assay exhibited that circ-CSPP1 could perform as an endogenous sponge for miR-493-5p. Elevating miR-493-5p repressed RCC progression. The bioinformatics website starBase confirmed that ras-related C3 botulinum toxin substrate 1 (RAC1) was a target gene of miR-493-5p. Circ-CSPP1 up-regulated RAC1 by sponging miR-493-5p, and elevating RAC1 could turn around the effect of down-regulating circ-CSPP1 on RCC cells. Taken together, circ-CSPP1 is identified as a novel RCC-promoting RNA that could serve as a latent therapeutic target for RCC therapy.

S100 Calcium-Binding Protein A8 Functions as a Tumor-Promoting Factor in Renal Cell Carcinoma via Activating NF-κB Signaling Pathway

Background Renal cell carcinoma (RCC), arising from the renal tubular epithelium, is one of the most common types of genitourinary malignancies. Based on the Gene Expression Omnibus (GEO) database (GSE100666), S100 calcium-binding protein A8 (S100A8) was highly expressed in RCC tissues. S100A8, an inflammatory regulatory factor, has emerged as an important mediator associated with the occurrence and development of cancer. Materials and Methods The Gene Expression Omnibus (GEO) database was used to identify the key genes and investigate the main signaling pathways in RCC. Human RCC samples and corresponding adjacent normal tissues were collected in our hospital. The expression of S100A8 in human RCC samples was detected using western blotting and immunohistochemical analysis. S100A8 overexpression or knockdown was mediated by using Lipofectamine 3000 in human renal cell carcinoma cell line 786-O and ACHN cells. Basic experiments, including MTT and cell apoptosis assays, were utilized for investigating the function of S100A8 in RCC. Furthermore, the levels of inflammation were also evaluated in 786-O and ACHN cells. Results In the current study, we found that downregulation of S100A8 inhibited proliferation and promoted apoptosis in 786-O and ACHN RCC cells. Of note, S100A8 silencing downregulated the phosphorylation of NF-κB p65, thereby decreasing the levels of TNF-α, cleaved caspase1, and MMP9. By contrast, S100A8 upregulation could increase these expressions. Conclusion Overall, S100A8 knockdown restrained RCC malignant biological properties, which was associated with the deactivation of the NF-κB signaling pathway. This present study demonstrates new insights that S100A8 may be a potential therapeutic target in RCC.

Melatonin acts synergistically with pazopanib against renal cell carcinoma cells through p38 mitogen-activated protein kinase-mediated mitochondrial and autophagic apoptosis.

Mounting evidence indicates that melatonin has possible activity against different tumors. Pazopanib is an anticancer drug used to treat renal cell carcinoma (RCC). This study tested the anticancer activity of melatonin combined with pazopanib on RCC cells and explored the underlying mechanistic pathways of its action. The 786-O and A-498 human RCC cell lines were used as cell models. Cell viability and tumorigenesis were detected with the MTT and colony formation assays, respectively. Apoptosis and autophagy were assessed using TUNEL, annexin V/propidium iodide, and acridine orange staining with flow cytometry. The expression of cellular signaling proteins was investigated with western blotting. The in vivo growth of tumors derived from RCC cells was evaluated using a xenograft mouse model. Together, melatonin and pazopanib reduced cell viability and colony formation and promoted the apoptosis of RCC cells. Furthermore, the combination of melatonin and pazopanib triggered more mitochondrial, caspase-mediated, and LC3-II-mediated autophagic apoptosis than melatonin or pazopanib alone. The combination also induced higher activation of the p38 mitogen-activated protein kinase (p38MAPK) in the promotion of autophagy and apoptosis by RCC cells than melatonin or pazopanib alone. Finally, tumor xenograft experiments confirmed that melatonin and pazopanib cooperatively inhibited RCC growth in vivo and predicted a possible interaction between melatonin/pazopanib and LC3-II. The combination of melatonin and pazopanib inhibits the growth of RCC cells by inducing p38MAPK-mediated mitochondrial and autophagic apoptosis. Therefore, melatonin might be a potential adjuvant that could act synergistically with pazopanib for RCC treatment.

Abstract 3866: Targeting AXL can effectively inhibit c-Met-induced therapeutic resistance in renal cancer

Abstract Advanced stage renal cell carcinoma (RCC) often becomes resistant to the currently available therapeutic options. Receptor Tyrosine Kinase (RTK), c-Met, is overexpressed and hyperactivated in renal cancer; and it promotes tumor-promoting pathways following binding with its ligand, Hepatocyte Growth Factor (HGF). Cabozantinib (Cabo), an inhibitor of c-Met/few other RTKs (like, AXL and KDR), is being used for the treatment of RCC. However, acquired therapeutic resistance is a major hurdle in its clinical use. Mechanism(s) of drug resistance against c-Met inhibition is largely unknown. Although Cabo (XL184) can effectively inhibit c-Met/RTK phosphorylation, very interestingly, we found that, prolonged treatment of Cabozantinib (24 hours, in vitro) increased the expression of total c-Met and AXL in renal cancer cells in a dose-dependent manner. AXL is also overexpressed in RCC and has a novel cross-talk with c-Met. We found that silencing AXL (either by gene knock-down approach or using a specific inhibitor (TP-0903)) markedly down-regulates Cabo-mediated total c-Met expression and enhances apoptosis of human RCC cells. We have also utilized murine model(s) of renal cancer growth (both xenograft and syngeneic), and found that Cabo treatment increases the levels of c-Met and AXL in tumor tissues, and the inhibition of AXL using TP-0903 can markedly inhibit the expression levels of total c-Met and AXL. We observed that HGF-mediated c-Met phosphorylation is short-lived when AXL is silenced. We also found that a lower concentration of Cabo can mediate cancer cell survival; and either the knock-down of AXL or using AXL inhibitor in combination with Cabo can reverse these effects. We have studied the downstream signaling pathways (Akt, ERK1/2 and STAT3) of c-Met, and found that AXL inhibition is crucial to down-regulate c-Met-induced signaling molecules following Cabo treatment. Our observations indicate that Cabo-mediated increase in total c-Met in RCC cells is not regulated at the transcriptional level; however, Cabo can interfere with the receptor degradation pathway leading to increased accumulation of c-Met. We have also studied the levels of c-Met and AXL in renal tumor tissues (archived specimens) from renal cancer patients before and after Cabozantinib treatment. Interestingly, we found increased levels of total c-Met and AXL in tumor tissues following Cabo treatment. Together our data suggest that AXL plays a critical role in mediating therapeutic resistance against c-Met inhibitor; and a combination of Cabo and an AXL/other RTK inhibitor can effectively block the resistance. Citation Format: Akash Sabarwal, Laxminarayan Rawat, Johannes Wedel, Yuzuru Sasamoto, Sabina Signoretti, Toni K. Choueiri, Murugabaskar Balan, Soumitro Pal. Targeting AXL can effectively inhibit c-Met-induced therapeutic resistance in renal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3866.

Phospholipase C delta 1 inhibits WNT/β‐catenin and EGFR-FAK-ERK signaling and is disrupted by promoter CpG methylation in renal cell carcinoma

BackgroundPLCD1, located at 3p22, encodes an enzyme that mediates cellular metabolism and homeostasis, intracellular signal transduction and movement. PLCD1 plays a pivotal role in tumor suppression of several types of cancers; however, its expression and underlying molecular mechanisms in renal cell carcinoma (RCC) pathogenesis remain elusive.MethodsRT-PCR and Western blot were used to detect PLCD1 expression in RCC cell lines and normal tissues. Bisulfite treatment, MSP and BGS were utilized to explore the CpG methylation status of PLCD1 promoter. Online databases were analyzed for the association between PLCD1 expression/methylation and patient survival. In vitro experiments including CCK8, colony formation, wound-healing, transwell migration and invasion, immunofluorescence and flow cytometry assays were performed to evaluate tumor cell behavior. Luciferase assay and Western blot were used to examine effect of PLCD1 on WNT/β‐catenin and EGFR‐FAK-ERK signaling.ResultsWe found that PLCD1 was widely expressed in multiple adult normal tissues including kidney, but frequently downregulated or silenced in RCC due to its promoter CpG methylation. Restoration of PLCD1 expression inhibited the viability, migration and induced G2/M cell cycle arrest and apoptosis in RCC cells. PLCD1 restoration led to the inhibition of signaling activation of WNT/β-catenin and EGFR-FAK-ERK pathways, and the EMT program of RCC cells.ConclusionsOur results demonstrate that PLCD1 is a potent tumor suppressor frequently inactivated by promoter methylation in RCC and exerts its tumor suppressive functions via suppressing WNT/β‐catenin and EGFR‐FAK-ERK signaling. These findings establish PLCD1 as a promising prognostic biomarker and treatment target for RCC.

The Effects of circ_000558/miR-1225-5p/ARL4C on Regulating the Proliferation of Renal Cell Carcinoma Cells.

Renal cell carcinoma (RCC) is one of the top ten tumors over the world. RCC is not sensitive to radiotherapy and chemotherapy. Therefore, it is necessary to find new targets for the treatment. CircRNAs are a special type of noncoding RNAs, which play important roles in many types of cancer. In this study, we found circ_000558 was upregulated in RCC cells, and it elevated the proliferation ability of RCC cells. The relationship between miR-1225-5p and circ_000558 or ARL4C was predicted via circBank and circular RNA interactome and confirmed by dual-luciferase reporter assay. Then, the effects of circ_000558/miR-1225-5p/ARL4C on RCC cell proliferation and apoptosis were assessed by CCK-8 assay. The results revealed that the knockdown of ARL4C significantly reduced RCC cell proliferation and overexpression of circ_000558 could significantly induce RCC cell proliferation after miR-1225-5p treatment further promoted the inhibitory ability of ARL4C knockdown. Overall, our study suggested that circ_000558/miR-1225-5p/ARL4C network was related to the RCC cell proliferation. This finding could provide new targets for the treatment and prognosis of RCC.

Speckle-type POZ protein could play a potential inhibitory role in human renal cell carcinoma

BackgroundSpeckle-type POZ protein(SPOP), a substrate adaptor of Cul3 ubiquitin ligase, plays crucial roles in solid neoplasms by promoting the ubiquitination and degradation of substrates. Limited studies have shown that SPOP is overexpressed in human renal cell carcinoma (RCC) tissue. However, the exact role of SPOP in RCC remains unclear and needs to be further elucidated. The present study showed that SPOP was expressed at different levels in different RCC cell lines. The purpose of this study was to explore the roles of SPOP in the biological features of RCC cells and the expression levels of SPOP in human tissue microarray (TMA) and kidney tissues.MethodsHere, SPOP was overexpressed by lentiviral vector transfection in ACHN and Caki-1 cells, and SPOP was knocked down in Caki-2 cells with similar transfection methods. The transfection efficiency was evaluated by quantitative PCR and western blotting analyses. The role of SPOP in the proliferation, migration, invasion and apoptosis of cell lines was determined by the MTT, wound-healing, transwell and flow cytometry assays. Moreover, the cells were treated with different drug concentrations in proliferation and apoptosis assays to investigate the effect of sunitinib and IFN-α2b on the proliferation and apoptosis of SPOP-overexpressing cells and SPOP-knockdown RCC cells. Finally, immunohistochemical staining of SPOP was performed in kidney tissues and TMAs, which included RCC tissues and corresponding adjacent normal tissues.ResultsOverexpression of SPOP inhibited cell proliferation, migration and invasion and increased cell apoptosis. Interestingly, sunitinib and IFN-α2b at several concentrations increased the proliferation inhibitory rate and total apoptosis rate of cells overexpressing SPOP. The findings of the present study showed that the SPOP protein was significantly expressed at low levels in most clear cell RCC (ccRCC) tissues and at relatively high levels in the majority of adjacent normal tissues and kidney tissues. Kaplan–Meier survival analysis showed that there was no statistically significant difference in cumulative survival based on the data of different SPOP expression levels in TMA and patients.ConclusionsIn contrast to previous studies, our findings demonstrated that overexpression of SPOP might suppress the progression of RCC cells, which was supported by cell experiments and immunohistochemical staining. SPOP could be a potential tumour inhibitor in RCC.

Circ_0000069 contributes to the growth, metastasis and glutamine metabolism in renal cell carcinoma (RCC) via regulating miR-125a-5p-dependent SLC1A5 expression

BackgroundCircular RNAs (circRNAs) have emerged as critical mediators in various cancers, including renal cell carcinoma (RCC). In the present research, the functions of circ_0000069 in RCC were explored. MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) assay, western blot assay and immunohistochemistry (IHC) assay were performed for the expression of circ_0000069, microRNA-125a-5p (miR-125a-5p) and solute carrier family 1 member 5 (SLC1A5). Cell Counting Kit-8 (CCK-8) assay and 5′-ethynyl-2′-deoxyuridine (EdU) assay were performed for cell proliferation. Flow cytometry assay was manipulated for cell apoptosis. Transwell assay and wound-healing assay were utilized for cell invasion and migration. Glutamine metabolism level was evaluated by examining glutamine consumption, α-ketoglutarate production and glutamate production. Dual-luciferase reporter assay was used to analyze the relationships of circ_0000069, miR-125a-5p and SLC1A5. Murine xenograft model assay was conducted to analyze the function of circ_0000069 in vivo. ResultsCirc_0000069 level was abnormally upregulated in RCC tissues and cells. Knockdown of circ_0000069 inhibited the proliferation, invasion, migration and glutamine metabolism and promoted the apoptosis in RCC cells in vitro and restrained tumor growth in vivo. Circ_0000069 served as the sponge for miR-125a-5p. MiR-125a-5p inhibition ameliorated the effects of circ_0000069 knockdown on RCC cell malignant behaviors. SLC1A5 was identified as the target gene of miR-125a-5p. Moreover, miR-125a-5p overexpression repressed the progression of RCC cells, while SLC1A5 elevation abrogated the effect. ConclusionCirc_0000069 knockdown inhibited the carcinogenesis of RCC by regulating miR-125a-5p/SLC1A5 axis.

Ginsenoside Rg3 inhibits renal cell carcinoma cell migration, invasion, colony formation, and tube formation and enhances apoptosis through promoting the DNA demethylation and histone acetylation.

This study explored the effect and mechanism of Rg3 on renal cell carcinoma (RCC) progression. RCC cells were treated with different concentrations of Rg3, 5-Aza-dc (a methyltransferase inhibitor) or TSA (a deacetylase inhibitor). Rg3-induced cytotoxicity, migration, invasion, colony formation, tube formation and apoptosis of RCC cells were evaluated by CCK-8, wound healing, Transwell, colony formation, tube formation and flow cytometry assays, respectively. Methylation and expressions of p53, p21 and p16, and expressions of methylation-related genes and histone deacetylases and histone acetylation-related genes (H3 (acetyl K14), H3 (acetyl K9), H4 (acetyl K12), H4 (acetyl K5) and H4 (acetyl K16)) were analysed by qRT-PCR and western blot. Rg3 dose-dependently decreased the viability, inhibited migration, invasion, colony formation and tube formation, and enhanced apoptosis of RCC cells. Rg3 enhanced the demethylation levels and expressions of p53, p21 and p16 as well as the expressions of histone acetylation-related genes, but repressed the expressions of methylation-related genes and histone deacetylases. Rg3 had the same effect as 5-Aza-dc and TSA did on the above-mentioned cellular changes. Rg3 restrains RCC cell migration, invasion, colony formation and tube formation, yet enhances apoptosis through promoting demethylation of p53, p21 and p16, and histone acetylation.

Circular RNA circSDHC (hsa_circ_0015004) regulates tumor growth and angiogenesis via regulating centrosomal protein 55 expression in renal cell carcinoma.

Renal cell carcinoma (RCC) is the main aggressive subtype of kidney cancer. Circular RNAs have been shown to exert critical roles in RCC. However, little is known about the regulatory mechanism of hsa_circ_0015004 (circSDHC) in RCC. 35 patients with RCC were recruited in the research. Expression changes of circSDHC were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The effects of circSDHC inhibition on cell proliferation, apoptosis, angiogenesis, migration, and invasion were analyzed. The regulation mechanism of circSDHC was surveyed by bioinformatics analysis. The effect of circSDHC on tumorigenesis was validated by xenograft assay. We observed an observable elevation in circSDHC expression in RCC tissues and cell lines. Functionally, circSDHC silencing decreased xenograft tumor growth and induced RCC cell apoptosis, repressed RCC cell proliferation, angiogenesis, migration, and invasion in vitro. Mechanically, circSDHC modulated centrosomal protein 55 (CEP55) expression by functioning as a miR-130a-3p sponge. Also, miR-130a-3p silencing offset circSDHC knockdown-mediated impacts on malignant phenotypes and angiogenesis of RCC cells. Furthermore, exogenetic expression of CEP55 counteracted miR-130a-3p overexpression-mediated effects on malignant phenotypes and angiogenesis of RCC cells. Silencing of circSDHC restrained cell malignant phenotypes and angiogenesis via reducing CEP55 expression by releasing miR-130a-3p in RCC, providing a new mechanism for understanding the progression of RCC.

AKG induces cell apoptosis by inducing reactive oxygen species-mediated endoplasmic reticulum stress and by suppressing PI3K/AKT/mTOR-mediated autophagy in renal cell carcinoma.

Alpha-ketoglutarate (AKG) or 2-oxoglutarate is a key substance in the tricarboxylic acid cycle (TCA) and has been known to play an important role in cancerogenesis and tumor progression. Renal cell carcinoma (RCC) is the most common type of kidney cancer, and it has a high mortality rate. Autophagy is a phenomenon of self-digestion, and its significance in tumor genesis and progression remains debatable. However, the mechanisms underlying how AKG regulates autophagy in RCC remain unknown. Thus, the purpose of this study was to assess the therapeutic efficacy of AKG and its molecular mechanisms. RCC cell lines 786O and ACHN were treated with varying doses of AKG for 24 h. CCK-8, Transwell, and scratch wound healing assays were utilized to evaluate the role of AKG in RCC cells. Autophagy protein and PI3K/AKT/mTOR pathway protein levels were analyzed by Western blot. AKG inhibited the proliferation of RCC cells 786O and ACHN in a dose-dependent manner according to the CCK-8 assay. In addition, flow cytometry and Western blot analysis revealed that AKG dose-dependently triggered apoptosis and autophagy in RCC cells. By promoting cell apoptosis and autophagy, AKG dramatically suppressed tumor growth. Mechanistically, AKG induces autophagy by promoting ROS generation and inhibiting the PI3K/AKT/mTOR pathway. The anti-tumor effect of AKG promotes autophagy in renal cancer cells via mediating ROS-PI3K/Akt/mTOR, and may be used as a potential anticancer drug for kidney cancer.

ZNF677 suppresses renal cell carcinoma progression through N6-methyladenosine and transcriptional repression of CDKN3.

BackgroundStudies on biological functions of N6‐methyladenosine (m6A) modification in mRNA have sprung up in recent years. Previous studies have reported m6A can determine mRNA fate and play a pivotal role in tumour development and progression. The zinc finger protein 677 (ZNF677) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence‐specific DNA.MethodsThe expression of ZNF677 and its clinicopathological impact were evaluated in renal cell carcinoma (RCC) patients. The m6A level of ZNF677 was determined by m6A methylated RNA immunoprecipitation‐sequencing (MeRIP‐seq) and MeRIP‐qPCR in RCC tissues and adjacent normal tissues. RNA immunoprecipitation‐qPCR (RIP‐qPCR) and luciferase assays were performed to identify the targeted effect of IGF2BP2 and YTHDF1 on ZNF677. RCC cells and subcutaneous models uncovered the role of ZNF677 methylated by CRISPR/dCas13b‐METTL3 in tumour growth. ZNF677‐binding sites in the CDKN3 promoter were investigated by chromatin immunoprecipitation (ChIP) and luciferase assays.ResultsZNF677 is frequently downregulated in RCC tissues and its low expression is associated with unfavourable prognosis and decreased m6A modification level. Further, we find the m6A‐modified coding sequence (CDS) of ZNF677 positively regulates its translation and mRNA stability via binding with YTHDF1 and IGF2BP2, respectively. Targeted specific methylation of ZNF677 m6A by CRISPR/dCas13b‐METLL3 system can significantly increase the m6A and expression level of ZNF677, and dramatically inhibit cell proliferation and induce cell apoptosis of RCC cells. In addition, ZNF677 exerted its tumour suppressor functions in RCC cells through transcriptional repression of CDKN3 via binding to its promoter. In vitro and clinical data confirm the negative roles of ZNF677/CDKN3 in tumour growth and progression of RCC.ConclusionZNF677 functions as a tumour suppressor and is frequently silenced via m6A modification in RCC, which may highlight m6A methylation‐based approach for RCC diagnosis and therapy.

Long non-coding RNA CYTOR modulates cancer progression through miR-136-5p/MAT2B axis in renal cell carcinoma

BackgroundTo explore the role of long noncoding RNAs (lncRNAs) cytoskeleton regulator RNA (CYTOR) in renal cell carcinoma (RCC). MethodsThe levels of CYTOR in RCC tissues and cell lines were detected by RT-qPCR. 786-O and Caki-1 cells were transfected with CYTOR-shRNA or pcDNA-CYTOR respectively, or co-transfected with CYTOR-shRNA and miR-136-5p inhibitor, or co-transfected with miR-136-5p mimic and pcDNA-MAT2B. MTT assay, Transwell assay and flow cytometry were used to evaluate cell proliferation, invasion and apoptosis. The relationship between lncRNA CYTOR and miRNA-136-5p was detected by dual luciferase reporter gene and RNA pull down assays, and the targeted relationship between miRNA-136-5p and MAT2B was verified by dual luciferase reporter gene assay. The interaction between MAT2B and BAG3 protein was verified by co-IP experiment. The role of lncRNA CYTOR in vivo was also examined. ResultsLncRNA CYTOR was up-regulated in RCC tissues and cell lines, and miR-136-5p was down-regulated in renal carcinoma cell lines and tissues. Downregulation of CYTOR inhibited cell proliferation and invasion and promoted apoptosis. miR-136-5p was sponged by lncRNA CYTOR, which negatively regulated the development of RCC. MAT2B was a target gene of miR-136-5p. MAT2B protein interacted directly with BAG3 protein to affect the proliferation, invasion and apoptosis of RCC cells. In vivo experiments showed that the expression level of miR-136-5p was increased, and MAT2B expression was decreased after CYTOR knockdown, thereby inhibiting the development of RCC. ConclusionsLncRNA CYTOR promoted the progression of RCC by targeting miR-136-5p to regulate the target gene MAT2B, which interacted with BAG3 protein.

Circ_0000274 contributes to renal cell carcinoma progression by regulating miR-338-3p/NUCB2 axis and JAK1/STAT3 pathway

BackgroundKidney transplant recipients (KTRs) are at increased risk of developing renal cell carcinoma (RCC). Accumulating evidence has demonstrated that circular RNAs (circRNAs) are essential players in tumor advancement. However, the functions of circ_0000274 in renal cell carcinoma (RCC) are barely explored. MethodsThe primary RCC cell lines 786-O and A498 were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was employed for the RNA levels of circ_0000274, microRNA-338-3p (miR-338-3p) and nucleobindin 2 (NUCB2). RNase R assay was conducted to analyze the feature of circ_0000274.Cell Counting Kit-8 (CCK-8) assay, colony formation assay, transwell assay, tube formation assay and flow cytometry analysis were conducted for cell viability, colony formation, metastasis, angiogenesis and apoptosis, respectively. Western blot assay was utilized for protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to analyze the associations of circ_0000274 RNA, miR-338-3p RNA and NUCB2 protein. Murine xenograft model was established to explore the function of circ_0000274 RNA in vivo. Immunohistochemistry (IHC) assay was used to analyze NUCB2 protein level in xenograft tumors. ResultsCompared to normal tissues and cells, circ_0000274 RNA level was elevated in RCC tissues and cells. Knockdown of circ_0000274 RNA suppressed cell viability, colony formation, metastasis and tube formation and promoted apoptosis in RCC cells in vitro. Circ_0000274 RNA sponged miR-338-3p RNA to positively regulate NUCB2 protein in RCC cells. Inhibition of miR-338-3p reversed the impacts of circ_0000274 knockdown on RCC cell malignant behaviors. MiR-338-3p RNA overexpression repressed the malignant phenotypes of RCC cells, while NUCB2 protein elevation could abrogate the effect. Moreover, circ_0000274 RNA knockdown blocked tumorigenesis in vivo. Besides, circ_0000274 RNA knockdown inactivated the JAK1/STAT3 protein signaling pathway. ConclusionCirc_0000274 RNA functioned as an oncogene in RCC development by regulating miR-338-3p RNA/NUCB2 protein axis and activating the JAK1/STAT3 protein signaling pathway.

Anlotinib combined with the PD-L1 blockade exerts the potent anti-tumor immunity in renal cancer treatment

Anlotinib have shown certain therapeutic effects of renal cell carcinoma (RCC), but drug resistance during treatment leads to the fact that the therapeutic effect is unsatisfactory. Herein, we investigated the tumor immune microenvironment about resistance mechanisms when application of Anlotinib and further improved its therapeutic effect. Our results showed that Anlotinib suppressed cell proliferation and promoted cell apoptosis in RCC cells. Meanwhile, the significantly up-regulated expression of PD-L1 was observed in Anlotinib-treated RCC cells by the concentration and time-dependent manner. Further study showed that Anlotinib-induced PD-L1 expression was regulated by autocrine IL-6 mediated JAK2/STAT3 signaling pathways. Interestingly, Anlotinib combined with PD-L1 blockade increased the infiltration of IFN-γ+CD8+ T cells and natural killer (NK) cells, also decreased the quantity of Treg cells and MDSCs in vivo. Likewise, the therapy above showed significantly synergistic therapeutic effect as demonstrated by reduced tumor volume and weight. These results indicated that the drug resistance might be attributed to the Anlotinib induced-PD-L1 mediated immunosuppression in renal cancer treatment. Anlotinib combined anti-PDL-1 treatment exerts the potential anti-tumor effect by promoting the induction and activation of immune killer cells. The therapeutic strategy of Anlotinib combined anti-PDL-1 could be a potential and promising approach for the therapy of renal cancer or other malignant tumors.