Apoptosis In Renal Cell Carcinoma Cells Research Articles

Role of Sam68 in Sunitinib induced renal cell carcinoma apoptosis.

Sunitinib is one of the first‐line targeted drugs for metastatic renal cell carcinoma (RCC) with dual effects of antiangiogensis and proapoptosis. Sam68 (Src‐associated in mitosis, 68 KDa), is found being involved in cell apoptosis. This article reveals that Sam68 impacts the sensitivity to sunitinib by mediating the apoptosis of RCC cells. Immunohistochemical staining indicated that the Sam68 expression levels in sunitinib sensitive tumor tissues were markedly higher than those in sunitinib resistant tumor tissues. Sunitinib induced RCC cell apoptosis in a concentration‐dependent manner and inhibited the expression of total and phosphorylated Sam68 (p‐Sam68). Downregulation of Sam68 expression inhibited RCC cell apoptosis induced by sunitinib. While upregulation of Sam68 expression could enhance apoptosis induced by sunitinib. Xenograft models showed that tumors in the Sam68‐knockdown group did not shrink as much as those in the control group after treatment with sunitinib for 4 weeks. Together, our results suggest that Sam68 expression is associated with the sensitivity of ccRCC patients to sunitinib. Sam68 may promote cell apoptosis induced by sunitinib, and the Sam68 expression level may be a biomarker for predicting sunitinib sensitivity in ccRCC patients.

LncRNA GIHCG promoted the proliferation and migration of renal cell carcinoma through regulating miR-499a-5p/XIAP axis

BackgroundOur previous study demonstrated that lncRNA GIHCG is upregulated in renal cell carcinoma (RCC) and that knockdown of lncRNA GIHCG suppresses the proliferation and migration of RCC cells. However, the mechanism of lncRNA GIHCG in RCC needs further exploration. MethodsThe proliferation, cell cycle, migration, and apoptosis of RCC cells were tested using CCK-8, flow cytometry, wound healing and Annexin-V/-FITC/PI flow cytometry assays, respectively. Dual-luciferase reporter and RNA pull-down or RNA immunoprecipitation assays (RIPs) were performed to analyze the interactions among lncRNA GIHCG, miR-499a-5p and XIAP. A tumour xenograft study was conducted to verify the function of lncRNA GIHCG in RCC development in vivo. ResultsKnockdown of lncRNA GIHCG inhibited cell proliferation and migration and induced G0/G1 arrest while promoting apoptosis. Overexpression of lncRNA GIHCG led to the opposite results. LncRNA GIHCG sponged miR-499a-5p and downregulated its expression in RCC cells. MiR-499a-5p overexpression suppressed RCC cell growth. MiR-499a-5p targeted XIAP and inhibited its expression. LncRNA GIHCG knockdown reduced the growth of tumour xenografts in vivo and the expression of XIAP while increasing miR-499a-5p levels. ConclusionLncRNA GIHCG accelerated the development of RCC by targeting miR-499a-5p and increasing XIAP levels.

Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation.

Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9, γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.

Circular RNA circMTO1 suppressed the progression of renal cell carcinoma progression by sponging miR-211 and miR-204.

Despite considerable improvements in renal cell carcinoma (RCC) diagnostic and therapeutic strategy, the clinical prognosis of patients is far from satisfactory due to its recurrence and metastasis. Here, we attempted to explore the role of circMTO1 in RCC progression, and the underlying mechanism was further elucidated. We first detected the expression of circMTO1 in 90 pairs of RCC tissues and adjacent normal tissues using qRT-PCR. Besides, circMTO1, miR-211, miR-204 and KLF6 expression levels in RCC cells were also measured using qRT-PCR. MTT assay, cell migration, flow cytometry analysis, qRT-PCR and western blotting analysis were applied to evaluating the effect of circMTO1 in RCC cells. The bioinformatics analysis and the rescue experiment were devoted to the underlying mechanism. The results demonstrated CircMTO1 expression was significantly down-regulated in RCC tissues and cell lines. Besides, CircMTO1 inhibited cell proliferation, migration and invasion, induced apoptosis in RCC cells. In addition, CircMTO1 serves as a sponge for miR-211 and miR-204, KLF6 is a direct target of miR-211 and miR-204. Furthermore, circMTO1 and KLF6 overexpression rescued the suppression of miR-211/204 in RCC cell proliferation. In short, circMTO1 repressed RCC progression by regulating KLF6 via sponging miR-211 and miR-204, which may provide new idea of diagnosis and treatment in renal cell carcinoma.

Circ_0039569 Competes with MARCKS for miR-133b Binding Sites to Promote the Progression of Renal Cell Carcinoma

Background: The dysregulation of circular RNAs (circRNAs) has been shown to be correlated with the aggressiveness of renal cell carcinoma (RCC). Hence, this study investigated the role and mechanism of circ_0039569 in RCC progression. Methods: The levels of circ_0039569, miR-133b, and MARCKS (myristoylated alanine-rich protein kinase C substrate) were detected using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by using 5-ethynyl-2′-deoxyuridine assay, colony-formation assay, Transwell assay, flow cytometry, and Western blot. The direct interactions between miR-133b and circ_0039569 or MARCKS were verified by using dual-luciferase reporter and pull-down assays. Xenograft mice models were established to conduct in vivo analysis. Results: Circ_0039569 was highly expressed in RCC tissues and cells. Functionally, silencing of circ_0039569 suppressed cell proliferation, migration, and invasion, but induced cell apoptosis in RCC cells in vitro. Moreover, mice subcutaneous xenograft assay suggested that circ_0039569 knockdown impeded tumor growth in vivo. Mechanistically, circ_0039569 acted as a sponge for miR-133b to regulate the expression of its target MARCKS. Importantly, miR-133b inhibition or MARCKS knockdown attenuated the anticancer effects of circ_0039569 knockdown on RCC growth. Conclusion: Diminished circ_0039569 restrains the growth and propagation of RCC cells via miR-133b/MARCKS axis, indicating an underlying effective therapeutic target for RCC patients.

CX3CL1 in the red bone marrow promotes renal cell carcinoma to metastasize to the spine by involving the Src-related pathway.

Spinal metastasis (SM) frequently occurs in renal cell carcinoma (RCC) patients. Our preliminary work showed that CX3CL1 plays a positive role in SM. The objective of the present study was to verify whether CX3CL1 activates the downstream pathway by binding to CX3CR1 in RCC cells, ultimately promoting RCC to metastasize to the spine. The expression of CX3CL1 and CX3CR1 in tissue samples was detected by immunohistochemistry and western blotting. ELISA was used to quantify the concentration of CX3CL1 in the serum. The expression level of CX3CR1 in RCC cell lines was also detected. The CellTiter-Glo assay and flow cytometry were used to analyze cell viability and apoptosis of RCC cells. Transwell and wound healing assay were used to analyze the effect of CX3CL1 on the invasion and migration ability of RCC cells. Specific inhibitors were used to interfere with key molecules in the signaling pathway to further explore the signal transduction in RCC cells after CX3CL1 stimulation. The expression of CX3CR1 in SM from RCC was higher than that in limb bone metastases. Among the five RCC cell lines, 786O cells expressed the highest level of CX3CR1. CX3CL1 neither inhibited the proliferation of 786O cells nor promoted the apoptosis of 786O cells. However, it promoted the migration and invasion of RCC cells. After CX3CL1 stimulation, Src and Focal adhesion kinase (FAK) phosphorylation levels increased in RCC cells. Bosutinib and PF-00562271 inhibited Src/FAK phosphorylation and cell motility and invasion triggered by CX3CL1 stimulation. CX3CL1 in the red bone marrow of spinal cancellous bone enhances migration and invasion abilities of RCC cells, thereby promoting RCC metastasize to the spine. The migration and invasion of RCC cells activated by CX3CL1 are at least partially dependent on Src/FAK activation.

C1QBP regulates apoptosis of renal cell carcinoma via modulating xanthine dehydrogenase (XDH) mediated ROS generation.

Background: Complement component 1 Q subcomponent binding protein (C1QBP) plays a vital role in the progression and metabolism of cancer. Studies have shown that xanthine dehydrogenase (XDH)-derived reactive oxygen species (ROS) accelerates tumor growth, and also induces mutations or produces cytotoxic effects concurrently. However, the role of C1QBP in metabolism, oxidative stress, and apoptosis of renal cell carcinoma (RCC) cells have not yet been explored.Methods: Metabolomics assay was applied to investigate the role of C1QBP in RCC metabolism. C1QBP knockdown and overexpression cells were established via lentiviral infection and subjected to apoptosis and ROS assay in vitro. RNA stability assay was applied to characterize the mechanism of C1QBP regulating XDH transcription. In vivo, orthotopic tumor xenografts assay was performed to investigate the role of C1QBP in RCC progression.Results: Metabolomics investigation revealed that C1QBP dramatically diminished the hypoxanthine content in RCC cells. C1QBP promoted the mRNA and protein expression of hypoxanthine catabolic enzyme XDH. Meanwhile, C1QBP may affect XDH transcription by regulating the mRNA level of XDH transcriptional stimulators IL-6, TNF-α, and IFN-γ. Moreover, the expression of C1QBP and XDH was lower in RCC tumors compared with the tumor-associated normal tissues, and their down-regulation was associated with higher Fuhrman grade. C1QBP significantly increased ROS level, apoptosis, and the expression of apoptotic proteins such as cleaved caspase-3 and bax/bcl2 via regulating XDH.Conclusion: C1QBP promotes the catabolism of hypoxanthine and elevates the apoptosis of RCC cells by modulating XDH-mediated ROS generation.

HIF-1α Induces HECTD2 Up-Regulation and Aggravates the Malignant Progression of Renal Cell Cancer via Repressing miR-320a.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system. It has been found that hypoxia mediates the malignant evolvement of RCC. Here, we probe the impact and potential mechanism of HECT domain E3 ubiquitin-protein ligase 2 (HECTD2) and HIF-1α on regulating RCC evolvement. RCC tissues and adjacent normal tissues were collected, and the association between the expression profiles of HECTD2 and HIF-1α and the clinicopathological features was analyzed. Additionally, we constructed HECTD2/HIF-1α overexpression and knockdown models in RCC cell lines to ascertain the impacts of HECTD2 and HIF-1α on RCC cell proliferation, apoptosis, migration, and growth in vivo. We applied bioinformatics to predict the upstream miRNA targets of HECTD2. Meanwhile, RNA immunoprecipitation (RIP), and the dual-luciferase reporter assays were employed to clarify the targeting association between HECTD2 and miR-320a. The effect of miR-320a on HECTD2-mediated RCC progression was investigated. The results suggested that both HIF-1α and HECTD2 were up-regulated in RCC (compared with adjacent non-tumor tissues), and they had positive relationship. Moreover, higher level of HECTD2 and HIF-1α is associated with poorer overall survival of RCC patients. HECTD2 overexpression heightened RCC cell proliferation and migration, and weakened cell apoptosis. On the other hand, the malignant phenotypes of RCC cells were signally impeded by HECTD2 or HIF-1α knockdown. Moreover, miR-320a targeted the 3′-untranslated region of HECTD2 and suppressed HECTD2 expression. The rescue experiments showed that miR-320a restrained HECTD2-mediated malignant progression in RCC, while up-regulation of HIF-1α hampered miR-320a expression. Collectively, HIF-1α mediated HECTD2 up-regulation and aggravated RCC progression by attenuating miR-320a.

LncRNA SNHG17 promotes tumor progression and predicts poor survival in human renal cell carcinoma via sponging miR-328-3p.

Accumulating data shows that dysregulation of long non-coding RNAs (lncRNAs) are involved in human tumors' occurrence and progression. Small nucleolar RNA host genes (SNHGs) are recently revealed to play a carcinogenic role in various human neoplasms. However, the functions and underlying mechanisms of lncRNA SNHG17 in renal cell carcinoma (RCC) are still elusive. We analyzed the relationship between SNHG17 expression levels and clinicopathologic characteristics and prognosis in patients with RCC according to TCGA RNA-sequencing data and our cohort data. Loss-of-function and gain-of-function experiments were conducted to examine the biological behaviors of SNHG17 on RCC cell proliferation, migration, invasion, apoptosis, and tumor growth in vivo. The interaction between SNHG17, miR-328-3p, and Histone’sH2Avariant (H2AX) was verified by bioinformatics, dual-luciferase reporter gene, and RNA immunoprecipitation (RIP). Highly expressed SNHG17 was evident in RCC tissue samples and cell lines, and SNHG17 overexpression was related to advanced TNM stage and reduced relapse-free and overall survival of patients with RCC. Knockdown of SNHG17 prohibited malignant phenotypes, whereas ectopic SNHG17 expression showed the opposite effects. More importantly, SNHG17 could upregulate the expression of H2AX by acting as a miR-328-3p sponge. In vivo experiments confirmed that SNHG17 promoted the growth of RCC tumors. SNHG17/miR-328-3p/H2AXaxis might be involved in RCC progression, which provided a potential therapeutic target for RCC.

SREBP1 site 1 protease inhibitor PF-429242 suppresses renal cell carcinoma cell growth

Renal cell carcinoma (RCC) cells have increased lipogenesis and cholesterol synthesis. Sterol regulatory element-binding protein-1 (SREBP1) is cleaved by site 1 protease (S1P) to release the transcriptionally active amino-terminal domain. PF-429242 is a potent and competitive S1P inhibitor. We here tested its activity in RCC cells. In established and primary human RCC cells, PF-429242 potently inhibited cell proliferation, migration, and invasion. The S1P inhibitor provoked apoptosis activation in RCC cells. Furthermore, shRNA-mediated S1P silencing or CRISPR/Cas9-induced S1P knockout led to RCC cell growth inhibition and apoptosis activation. Conversely, ectopic overexpression of SREBP1 or S1P augmented RCC cell proliferation and migration. Daily i.v. injection of a single dose of PF-429242 robustly inhibited RCC xenograft growth in severe combined immunodeficiency mice. Additionally, intratumoral injection of S1P shRNA lentivirus inhibited RCC xenograft growth in mice. SREBP1, S1P, and its target gene low density lipoprotein receptor (LDLR) were significantly elevated in human RCC tissues. These results suggest that targeting S1P by PF-429242 inhibited RCC cell growth in vitro and in vivo.

The compound AST-003 could effectively promote apoptosis of renal cell carcinoma cells in vitro.

BackgroundThe toxicity of Sunitinib limits its clinical application. A new compound AST-003 was designed and synthesized based on the structure of Sunitinib, and previous study has confirmed that AST-003 has the same efficacy and less toxicity as Sunitinib. We conducted this study to further verify the effect of AST-003 on renal cell carcinoma (RCC) cells in vitro and its mechanism.MethodsFive RCC cell lines, A498, 786-0, SW-13, Caki-1 and ACHN, were used. Cells were treated with different concentrations of AST-003, and the effect of AST-003 on cell viability was detected by MTT assay and IC50 was determined. Blank control group and AST-003 group were set to evaluate the effect of AST-003 on cell apoptosis and cell cycle. The effect of AST-003 on cell protein expression was detected by western blot.ResultsAfter treatment with AST-003, the viability of the above five RCC cells was significantly inhibited. The IC50 of AST-003 on A498, 786-0, SW-13, Caki-1 and ACHN were 7.396, 6.592, 3.803, 12.05 and 3.422 µmol/L, respectively. Compared with the blank control group, the apoptotic rates were 52.37% and 13.34% in A498 cells (P<0.001), 59.23% and 6.66% in 786-0 cells (P<0.001), 45.67% and 4.19% in SW13 cells (P<0.001), 51.67% and 2.33% in Caki-1 cells (P<0.001), 55.40% and 5.50% in ACHN cells (P<0.001). Flow cytometry revealed that the proportion of cells in G0/G1 phase was significantly increased and the proportion of cells in S phase was significantly decreased in AST-003 group compared with blank control group, suggesting that AST-003 could block cells in G0/G1 phase. Western blot indicated that AST-003 could induce the up-regulation of the protein expression levels of apoptotic genes Caspase3, Caspase8, Caspase9, Bax and tumor suppressor p53, and inhibit the phosphorylation of STAT3, mTOR, AMPK and ERK, as well as the expression of Bcl-2, MKK, MKKK, c-jun and Ras proteins.ConclusionsWe found that AST-003 could effectively promote the apoptosis of RCC cells in vitro and block the cells in the intercellular phase, and its mechanism was similar to that of Sunitinib, suggesting that AST-003 has the value for further research.

Ginsenoside compound K exerts antitumour effects in renal cell carcinoma via regulation of ROS and lncRNA THOR.

Renal cell carcinoma (RCC) is a common type of kidney cancer that lacks effective therapeutic options. Ginsenoside compound K (CK), an active metabolite of ginsenosides, has been reported to induce apoptosis in various types of cancer cells. However, the effects of CK in RCC remain to be elucidated. Thus, the aim of the present study was to investigate the antitumor effects of CK on RCC cells. The effects of CK on the proliferation, migration, invasion, cell cycle and apoptosis of RCC cell lines (Caki-1 and 768-O) were investigated using MTT, wound healing, Transwell and flow cytometry assays, respectively. Changes in the expression levels of long non-coding RNAs (lncRNAs) and proteins were measured via reverse transcription-quantitative PCR and western blotting, respectively. Transfections with testis associated oncogenic (THOR) small interfering RNA and pcDNA were performed to knock down and overexpress lncRNA THOR, respectively. It was found that CK could effectively inhibit the proliferation, migration and invasion of RCC cells. CK also induced cell cycle arrest and caspase-dependent apoptosis in RCC cells. Furthermore, the generation of reactive oxygen species and inhibition of the lncRNA THOR played important roles in the antitumour effects of CK in RCC cells. The present data revealed that CK was a potent antitumour agent against RCC.

Mechanism of gypenosides of Gynostemma pentaphyllum inducing apoptosis of renal cell carcinoma by PI3K/AKT/mTOR pathway

Ethnopharmacological relevanceGynostemma pentaphyllum (Thunb.) Makino is a traditional medicine commonly used in China, East Asia and Southeast Asia. In clinic, it is mainly used for hyperlipidemia and antitumor. Its antitumor activity was first recorded in “Illustrated Catalogue of Plants”. Gypenosides were the main active ingredients of G. pentaphyllum. The anticancer activity of gypenosides in vivo and in vitro had been widely reported. However, the mechanism of gypenosides in renal cell carcinoma (RCC) still unclear. Aim of the studyIn this study, we tried to investigate the active constituents from G. pentaphyllum and potential mechanisms in RCC treatment through network pharmacology and in vitro experiments. Material/methodsActive compounds and their targets were evaluated and screened through TCMSP and Swiss Target Prediction database. Notably, nine preliminary screened components obtained from database were identified by LC-MS and LC-MS/MS. The targets associated with RCC were obtained from OMIM, TTD and GeneCards database. The PPI network and active component/target/pathway networks were constructed to identify the potential drug targets using String database and Cytoscape software. The functions and pathways of targets were analyzed through DAVID database. Finally, AutoDockTools 1.5.6 was used for molecular docking to assess the binding ability between compounds and targets. To support our prediction, we then explore the antitumor effect and mechanism of gypenosides by vitro experiments. CCK8 and flow cytometry were performed to evaluate cell death treated with gypenosides. Quantitative real-time PCR and Western blot were conducted to detect the changes of PI3K/AKT/mTOR signaling pathway. ResultsNine saponins and 68 targets have been screened. The hub targets covered PIK3CA, VEGFA, STAT3, JAK2, CCND1 and MAPK3. Enrichment analysis showed that the pathways mainly contained PI3K/Akt/mTOR, HIF-1, TNF, JAK-STAT and MAPK signaling pathways. Gypenosides extracted from G. pentaphyllum showed strong activity against 786-O and Caki-1 cells, and cell apoptosis were detected through Annexin V/PI dual staining assay. RT-qPCR showed that gypenosides downregulated the levels of PIK3CA, Akt and mTOR in Caki-1 and 786-O cells. Mechanistically, gypenosides induced apoptosis of RCC cells through regulating PI3K/Akt/mTOR signaling pathway which was implemented though decreasing the phosphorylation level of Akt and mTOR. ConclusionsGypenosides induced apoptosis of RCC cells by modulating PI3K/Akt/mTOR signaling pathway.

Nobiletin downregulates the SKP2-p21/p27-CDK2 axis to inhibit tumor progression and shows synergistic effects with palbociclib on renal cell carcinoma.

Objective:Natural extracts, including nobiletin, have been reported to enhance the efficacy and sensitivity of chemotherapeutic drugs. However, whether and how nobiletin affects tumor growth and progression in renal cell carcinoma (RCC) are still unclear.Methods:Cell proliferation, cell cycle and apoptosis analyses, colony-formation assays, immunoblotting analysis, and qRT-PCR analysis were performed to investigate how nobiletin affected RCC cell proliferation in vitro. The nude mouse model was used to test the efficacy of nobiletin alone or in combination with palbociclib.Results:Nobiletin inhibited cell proliferation by inducing G1 cell cycle arrest and cell apoptosis in RCC cells. Mechanistically, nobiletin decreased SKP2 protein expression by reducing its transcriptional level. The downregulated SKP2 caused accumulation of its substrates, p27 and p21, which further inhibited the activity of the G1 phase-related protein, CDK2, leading to inhibition of cell proliferation and tumor formation. A higher SKP2 protein level indicated less sensitivity to the CDK4/6 inhibitor, palbociclib. A combination of nobiletin and palbociclib showed a synergistic tumor inhibition in vitro and in an in vivo model.Conclusions:Nobiletin downregulated the SKP2-p21/p27-CDK2 axis to inhibit tumor progression and showed synergistic tumor inhibition effects with the CDK4/6 inhibitor, palbociclib, on RCC, which indicates a potential new therapeutic strategy.

Silencing circular RNA circ_0054537 and upregulating microRNA-640 suppress malignant progression of renal cell carcinoma via regulating neuronal pentraxin-2 (NPTX2)

ABSTRACT Hsa_circ_0054537 (circ_0054537) is a novel tumor-related circular RNA in renal cell carcinoma (RCC), and we intended to ascertain its dysregulation and functions in RCC malignant progression, as well as the underlying mechanism via serving as competing endogenous RNA (ceRNA). In this research, using real-time quantitative PCR, we found circ_0054537 was upregulated in RCC tissues and cells, and distributed throughout the cytoplasm. Then, functional effects of circ_0054537 in RCC were detected using cell counting kit-8, transwell, flow cytometry and glycolysis stress test and adenosine Triphosphate (ATP) assays. The results uncovered that circ_0054537 knockdown inhibited cell proliferation, migration, invasion, autophagy and glycolysis, but promoted apoptosis in RCC cells. Notably, circ_0054537 was identified as a ceRNA for microRNA (miR)-640, and miR-640 could target neuronal pentraxin-2 (NPTX2), as evidenced by dual-luciferase reporter assay and RNA immunoprecipitation assay. Besides, miR-640 downregulation or NPTX2 overexpression partly overturned the tumor suppressor function of circ_0054537 silence and miR-640 overexpression in RCC cells. Additionally, RCC cell growth in vivo was retarded by circ_0054537 silence. In conclusion, circ_0054537/miR-640/NPTX2 ceRNA pathway regulated RCC malignant progression in vitro and curbed RCC tumor growth in vivo, which could be a potential diagnosis and therapeutic target of RCC.

CircAMOTL1L Suppresses Renal Cell Carcinoma Growth by Modulating the miR-92a-2-5p/KLLN Pathway.

Accumulating evidence indicates that the dysregulation of circular RNAs (circRNAs) contributes to tumor progression; however, the regulatory functions of circRNAs in renal cell carcinoma (RCC) remain largely unknown. In this study, the function and underlying mechanism of circAMOTL1L in RCC progression were explored. qRT-PCR showed the downregulation of circAMOTL1L in RCC tissues and cell lines. The decrease in circAMOTL1L expression correlated with the tumor stage, metastasis, and poor prognosis in patients with RCC. Functional experiments revealed that circAMOTL1L inhibited cell proliferation and increased apoptosis in RCC cells. Subcutaneous implantation with circAMOTL1L-overexpressing cells in nude mice decreased the growth ability of the xenograft tumors. Mechanistically, circAMOTL1L served as a sponge for miR-92a-2-5p in upregulating KLLN (killin, p53-regulated DNA replication inhibitor) expression validated by bioinformatics analysis, oligo pull-down, and luciferase assays. Further, reinforcing the circAMOTL1L–miR-92a-2-5p–KLLN axis greatly reduced the growth of RCC in vivo. Conclusively, our findings demonstrate that circAMOTL1L has an antioncogenic role in RCC growth by modulating the miR-92a-2-5p–KLLN pathway. Thus, targeting the novel circAMOTL1L–miR-92a-2-5p–KLLN regulatory axis might provide a therapeutic strategy for RCC.

Up-Regulation of miR-26a-5p Inhibits E2F7 to Regulate the Progression of Renal Carcinoma Cells.

BackgroundMetastasis is the main cause of renal cell carcinoma (RCC) tumor death, and effective inhibition of RCC metastasis is an essential means to meliorate the prognosis of RCC patients. MicroRNAs (miRs) have been proved to be stable and important biomarkers for several malignancies. This study is therefore set out to explore the metastasis-related miR and its mechanism in RCC.MethodsThe expression of miR-26a-5p in RCC was analyzed using the expression profile in the Cancer Genome Atlas (TCGA). MiR-26a-5p and E2F transcription factor 7 (E2F7) in RCC patients were detected by qRT-PCR. Cell Counting Kit-8 (CCK-8) was adopted to assess cell proliferation, Transwell was utilized to evaluate migration and invasion, and flow cytometry (FC) was used to determine apoptosis. Mouse cell-derived and patient-derived xenotransplantation models were established to evaluate the effect of miR-26a-5p on tumor growth and metastasis in vivo. The molecular mechanism of miR-26a-5p was analyzed by dual-luciferase reporter (DLR) gene analysis, qRT-PCR, and Western blot (WB) both in vivo and in vitro.ResultsMiR-26a-5p was reduced in renal carcinoma cells and may serve as a biomarker for renal cancer metastasis and prognosis. MiR-26a-5p up-regulation inhibited migration and invasion in renal cell lines and tumor metastasis in vivo. Bioinformatics target prediction and RNA-seq results showed that E2F7 was among the targets of miR-26a-5p and was significantly inhibited by miR-26a-5p in vivo and in vitro.ConclusionMiR-26a-5p presents low expression in RCC and promotes RCC cell apoptosis and prevents cells from proliferating and invading by targeting E2F7, which is a promising therapeutic target for RCC.

BRD4 inhibition sensitizes renal cell carcinoma cells to the PI3K/mTOR dual inhibitor VS-5584.

Activation of the PI3K/AKT/mTOR pathway promotes the progression of renal cell carcinoma (RCC). This study tested the anti-RCC cell activity of the PI3K/mTOR dual inhibitor, VS-5584. We show that VS-5584 inhibited PI3K/AKT/mTORC1/2 activation in established (786-O and A498 lines) and primary RCC cells, thereby suppressing cell survival, proliferation, migration and cell cycle progression. VS-5584 induced significant apoptosis in RCC cells. A daily single oral dose of VS-5584 (20 mg/kg) significantly inhibited 786-O tumor growth in vivo. VS-5584 treatment of 786-O tumor xenografts and RCC cells resulted in feedback upregulation of bromodomain-containing protein 4 (BRD4). Furthermore, BRD4 inhibition (by JQ1 and CPI203), knockdown or complete knockout potentiated VS-5584-induced RCC cell death and apoptosis. Conversely, forced overexpression of BRD4 attenuated the cytotoxicity of VS-5584 in 786-O cells. Collectively, VS-5584 potently inhibits RCC cell proliferation and survival. Its anti-tumor activity is further enhanced by the targeted inhibition of BRD4.

MiR-4461 Inhibits Tumorigenesis of Renal Cell Carcinoma by Targeting PPP1R3C.

Background: Renal cell carcinoma (RCC) is one of the most common and malignant tumors in the urinary system. The aim of this research was to investigate the mechanism and clinical significance of miR-4461 in the RCC progression. Materials and Methods: Twenty-eight (28) paired RCC tissue samples and adjacent nontumor tissue samples, as well as RCC cell lines were used to measure the expression of miR-4461 and protein phosphatase 1 regulatory subunit 3C (PPP1R3C) transcript by real-time quantitative PCR. The target relationship between miR-4461 and PPP1R3C was predicted by TargetScan and further verified by dual-luciferase reporter gene assay and RNA pull-down assay. Cell Counting Kit-8 (CCK-8) assay and BrdU ELISA assay were performed to measure RCC cell viability and proliferation. In addition, caspase-3 activity assay and cell adhesion assay were implemented to measure RCC cell apoptosis and adhesion. Results: MiR-4461 was lowly expressed both in RCC tissues and cells, while upregulated PPP1R3C was tested in RCC tissues and cells. In addition, miR-4461 was validated to directly target PPP1R3C, thereby negatively regulating PPP1R3C. Particularly, miR-4461 exerted a clear inhibitory effect on the malignant phenotypes of RCC cells by binding and inhibiting PPP1R3C. Conclusion: MiR-4461, which served as a tumor suppressor, inhibited RCC progression by targeting and downregulating PPP1R3C.

Holliday junction‑recognition protein modulates apoptosis, cell cycle arrest and reactive oxygen species stress in human renal cell carcinoma.

Holliday junction recognition protein (HJURP) is involved in the regulation of mortality in various cell types, including renal cell carcinoma (RCC) cells. The specific mechanisms by which HJURP regulates RCC cell apoptosis and the cell cycle have not been previously investigated, to the best of our knowledge. In the present study, the expression of HJURP in RCC tissues and adjacent paracancerous renal tissue, as well as in RCC cell lines, was analyzed using reverse transcription‑quantitative PCR and western blot analysis. The A498 RCC cells were transfected with an HJURP overexpression vector, which resulted in reduced proliferation, as demonstrated using immunofluorescence staining, a Cell Counting Kit‑8 assay and a colony formation assay. Flow cytometry and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling assays were used to determine the effect of HJURP on the cell cycle and apoptosis of RCC cells. Proteins associated with the reactive oxygen species (ROS) status were analyzed using western blot analysis. The expression of HJURP was lower in RCC tissues and cells compared with that in the adjacent paracancerous renal tissues and control cells. Furthermore, overexpression of HJURP resulted in a decrease in cell viability and proliferation invitro. Overexpression of HJURP resulted in cell cycle arrest at the G0/G1 phase, cell apoptosis and an increase in ROS stress. In addition, the phosphorylated/total sirtuin 1 (SIRT1) protein ratio was decreased, whereas the expression of peroxisome proliferator‑activated receptor (PPAR)γ was increased in the HJURP‑overexpressing RCC cells. In clinical practice, decreased HJURP expression may be associated with poor prognosis in patients with RCC. These results suggest that HJURP may regulate cell apoptosis and proliferation in RCC cells and this may be mediated by PPARγ/SIRT1. Thus, HJURP may be used as a predictor of prognosis in patients with RCC.