Apoptosis In Renal Cell Carcinoma Cells Research Articles

AB026. Loss of KCNJ15 expression promotes malignant phenotypes and correlates with poor prognosis in renal carcinoma

Potassium inwardly rectifying channel, subfamily J, member 15 (KCNJ15) belongs to the inwardly rectifying potassium channel (KIR) family. Although members of the KIR family have been proven to play important roles in a variety of developmental processes, the expression, biological functions and molecular mechanisms of KCNJ15 in cancers remain unclear. Here, we first found that KCNJ15 was significantly downregulated in renal cell carcinoma (RCC), and this low expression was an independent prognostic factor for clear cell RCC. Moreover, KCNJ15 was associated with advanced TNM stage (n=150, P=0.014) and histologic grade (n=150, P=0.045). Furthermore, KCNJ15 overexpression significantly inhibited RCC cell proliferation, migration, and colony formation, arrested the cell cycle and induced apoptosis of RCC cells in vitro. The inhibitory effect of KCNJ15 overexpression may be regulated by its effects on the epithelial mesenchymal transition process and matrix metalloproteinase (MMP)-7 and p21 expression. These findings indicate that KCNJ15 may be a tumour suppressor in RCC and a possible target for RCC therapy.

Long non-coding RNA HOTTIP promotes renal cell carcinoma progression through the regulation of the miR-615/IGF-2 pathway.

Emerging evidence has indicated that long non‑coding RNA (lncRNA) HOXA transcript at the distal tip (HOTTIP) regulates cell growth, differentiation, apoptosis and cancer progression. However, the expression and function of HOTTIP in the progression of renal cell carcinoma(RCC) remain largely unknown. In this study, we investigated the role of the lncRNA HOTTIP in RCC. The expression levels of HOTTIP in RCC tissues and cell lines were determined by RT‑qPCR. The association between HOTTIP expression and clinicopathological characteristics and prognosis was analyzed in patients with RCC from the TCGA database. Loss‑of‑ function assays were designed and conducted to verify the oncogenic function of HOTTIP in RCC progression. Luciferase assay was performed to explore the mechanisms of the miRNA‑lncRNA sponge. The results revealed that HOTTIP expression was upregulated in RCC. An increased HOTTIP expression in RCC was associated with a larger tumor size and a higher clinical stage, lymph node metastasis and vascular invasion. Additionally, patients RCC with a high HOTTIP expression had a significantly shorter overall survival(OS) and disease‑free survival(DFS). HOTTIP knockdown significantly inhibited cell proliferation, migration and invasion, and increased the apoptosis of RCC cells invitro. Mechanistic analyses revealed that HOTTIP functioned as a competing endogenous RNA (ceRNA) for hsa‑miR‑615‑3p, and led to the derepression of its endogenous target, insulin‑like growth factor-2(IGF‑2), which is a protein hormone that exerts a stimulatory effect on tumor cell growth. miR‑615 inhibition reversed the suppressive effects of HOTTIP knockdown on RCC cell progression. HOTTIP regulated IGF‑2 expression in a miR‑615‑dependent manner in RCC cells. In addition, IGF‑2 expression was significantly upregulated in the RCC specimens and a positive association between the expression of HOTTIP and IGF‑2 in RCC tissues was detected. The effect of HOTTIP was abolished by the siRNA‑mediated silencing of IGF-2 in RCC cells. On the whole, this study demonstrates, for the first time, at least to the best of our knowledge, that the HOTTIP/miR‑615/IGF‑2 axis plays an important role in RCC progression and potentially contributes to the improvement of RCC diagnosis and therapy.

HMGB1/RAGE axis mediates the apoptosis, invasion, autophagy, and angiogenesis of the renal cell carcinoma

BackgroundHigh mobility group box 1 protein (HMGB1) is a sort of non-histone protein in chromatin, which plays an important role in tumor proliferation, invasion, and immune escape. HMGB1-RAGE (receptor for advanced glycation end products) interactions have been reported to be important in a number of cancers.MethodsCCK8, flow cytometry and qRT-PCR were used to detected cell viability, apoptosis and gene expression, respectively.ResultsIn the present study, we demonstrated that HMGB1/RAGE axis regulated the cell proliferation, apoptosis, and invasion of the renal cell carcinoma (RCC). Further, we discovered that HMGB1/RAGE axis increased the expression of autophagic proteins LC3 and Beclin-1 in RCC. Finally, we used a coculture model of human umbilical vein endothelial cells with RCC cell lines to find out that HMGB1 also increased the expression of VEGF and VEGFR2 in human umbilical vein endothelial cells. An in vivo study further confirmed that HMGB1 knockdown inhibited RCC tumor growth.ConclusionOur results illustrated that HMGB1/RAGE axis mediated RCC cell viability, apoptosis, invasion, autophagy, and angiogenesis, which provides a novel theoretical basis for using HMGB1 as the target in RCC.

UCA1 promotes cell proliferation and invasion and inhibits apoptosis through regulation of the miR129-SOX4 pathway in renal cell carcinoma.

BackgroundRenal cell carcinoma (RCC) is the most common cancer in kidney malignancies. UCA1 has been identified as an oncogenic lncRNA in multiple cancers, including RCC. However, the underlying molecular mechanism of UCA1 involved in RCC progression is far from being addressed.MethodsReverse-transcription quantitative polymerase chain reaction (RT-qPCR) assays were used to measure expressions of UCA1, miR129, and SOX4 mRNA. Western blot assays were employed to detect SOX4 protein expression. Cell proliferation, invasion, and apoptosis were assessed by CCK-8, Matrigel invasion, and annexin–fluorescein isothiocyanate (FITC) apoptosis-detection assays, respectively. The interaction between UCA1 and miR129 was demonstrated by luciferase, RNA pull-down, and RNA-immunoprecipitation (RIP) assays. Luciferase assays were also used to explore whether UCA1 was able to act as a molecular sponge of miR129 to affect the interplay of miR129 and SOX4.ResultsUCA1 expression was upregulated in RCC tissue and cells, and higher UCA1 expression was associated with advanced pathogenic status and poor prognosis of RCC patients. UCA1 knockdown suppressed proliferation and invasion and induced apoptosis in RCC cells. UCA1 inhibited miR129 expression by direct interaction in RCC cells. miR129 overexpression inhibited cell proliferation and invasion and promoted apoptosis. Moreover, miR129 downregulation abrogated UCA1 knockdown-mediated antiproliferation, anti-invasion, and proapoptosis effects in RCC cells. Furthermore, UCA1 acted as a ceRNA of miR129 to enhance target-gene SOX4 expression in RCC cells.ConclusionUCA1 promoted cell proliferation and invasion and inhibited apoptosis by regulating SOX4 via miR129 in RCC, offering a promising therapeutic target and prognosis marker for RCC patients.

MP28-12 SUPPRESSION OF ONCOGENIC MICRORNA-1274A INDUCES CELL APOPTOSIS THROUGH BMPR1B ACCELERATION IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Epidemiology & Evaluation/Staging I1 Apr 2018MP28-12 SUPPRESSION OF ONCOGENIC MICRORNA-1274A INDUCES CELL APOPTOSIS THROUGH BMPR1B ACCELERATION IN RENAL CELL CARCINOMA Hideki Enokida, Hirofumi Yoshino, Satoshi Sugita, Takashi Sakaguchi, Youichi Osako, Shuichi Tatarano, Takashi Yamane, and Masayuki Nakagawa Hideki EnokidaHideki Enokida More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Satoshi SugitaSatoshi Sugita More articles by this author , Takashi SakaguchiTakashi Sakaguchi More articles by this author , Youichi OsakoYouichi Osako More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , Takashi YamaneTakashi Yamane More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.912AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is considerable evidence that normal microRNA (miRNA) regulatory mechanisms are disrupted in cancer cells. However, the regulatory mechanisms by which upregulated miRNAs exert their effects has not been often understood compared to downregulated miRNAs. Our previous studies of the miRNA expression signature in renal cell carcinoma (RCC) indicated that miR-1274a was significantly upregulated in clinical specimens, suggesting that miR-1274a might act as an oncogenic miRNA in RCC. The aim of this study was to investigate the functional roles of miR-1274a and identify downstream tumor suppressive targets regulated by miR-1274a in RCC cells. METHODS Expression levels of miR-1274a and its target gene were determined by qRT-PCR. Functional studies of miR-1274a were carried out by anti-miRNA to investigate cell proliferation and apoptosis by using A498, ACHN, and Caki1 RCC cell lines. Putative target genes were determined by in-silico analyses with Gene Expression Omnibus (GEO) database and the microarray study of miR-1274a-transfected ACHN. In addition, statistical analyses were performed based on the TCGA database of RCC patients. RESULTS The expression levels of miR-1274a were significantly upregulated in RCC in comparison with normal kidneys (P < 0.0001). Suppression of miR-1274a significantly inhibited cell proliferation and induced apoptosis in RCC cells (P < 0.0001 and P < 0.05, respectively). In-silico analysis of the gene expression data and luciferase reporter assays demonstrated that miR-1274a directly regulated bone morphogenetic protein receptor type 1B (BMPR1B), a member of BMP receptor family of transmembrane serine/threonine kinases. It is known that the ligands of this receptor BMPs, members of the TGF-beta superfamily, bind their receptor including BMPR1B, which lead to apoptosis thorough activation of the SMAD-dependent pathway subsequently through phosphorylation of SMAD1/5/8. Moreover, TCGA database as well as the immunohistochemistry showed that low expression of BMPR1B was observed in RCC clinical specimens compared to normal kidneys (P < 0.0001 and P = 0.0015, respectively). CONCLUSIONS We conclude that loss of oncogenic miR-1274a reduced cancer cell proliferation and induced apoptosis in RCC through targeting tumor suppressive BMPR1B. Our data demonstrating molecular pathways and a target gene regulated by oncogenic miR-1274a will provide new insight into the potential mechanisms of RCC oncogenesis. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e361 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Hideki Enokida More articles by this author Hirofumi Yoshino More articles by this author Satoshi Sugita More articles by this author Takashi Sakaguchi More articles by this author Youichi Osako More articles by this author Shuichi Tatarano More articles by this author Takashi Yamane More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MiR-488 inhibits cell growth and metastasis in renal cell carcinoma by targeting HMGN5.

PurposemicroRNAs are thought to play crucial roles in tumorigenesis. Dysregulation of miR-488 has been implicated to be involved in several cancer progressions. However, the biological functions of miR-488 in renal cell carcinoma (RCC) remain unclear. This study aimed to explore the molecular mechanism underlying the role of miR-488 in RCC development.Materials and methodsThe expression levels of miR-488 were detected in 38 paired RCC tumor samples and cell lines by quantitative real-time polymerase chain reaction method. miR-488 was upregulated by mimics transfection in RCC cell lines. MTT, colony formation, transwell assay, flow cytometry assay, and a xenograft model were performed to determine cell proliferation, invasion, migration, epithelial-to-mesenchymal transition, and apoptosis in vitro and in vivo. Moreover, the potential target of miR-488 was verified by dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blot. The correlation between miR-488 expression and its target gene expression was confirmed by Spearman’s correlation analysis in 38 selected RCC tissue samples.ResultsWe found that miR-488 was remarkably downregulated in human RCC samples and cell lines compared with paired normal tissues and cell lines. Functional investigations revealed that overexpression of miR-488 significantly suppressed cell proliferation, invasion, and migration, and promoted cell apoptosis in RCC cells. Nucleosome binding protein 1 (high-mobility group nucleosome binding domain 5 [HMGN5]) was identified as a direct target of miR-488, and an inverse relationship was found between miR-488 expression and HMGN5 mRNA levels in RCC specimens. Rescue experiments suggested that restoration of HMGN5 partially abolished miR-488-mediated cell proliferation and invasion inhibition in RCC cells through regulating phosphatidylinositol 3-kinase/protein kinase B/the mammalian target of rapamycin and epithelial-to-mesenchymal transition signaling pathways.ConclusionThese data indicated that miR-488 acted as a tumor suppressor in RCC proliferation and invasion by targeting HMGN5, which might provide potential therapeutic biomarker for RCC patients.

MP72-11 TARGETING SUNITINIB RESISTANT CELLS BY TUMOR SUPPRESSIVE MIRNA-99A-3P THROUGH RIBONUCLEOTIDE REDUCTASE REGULATION IN CLEAR CELL RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology II1 Apr 2018MP72-11 TARGETING SUNITINIB RESISTANT CELLS BY TUMOR SUPPRESSIVE MIRNA-99A-3P THROUGH RIBONUCLEOTIDE REDUCTASE REGULATION IN CLEAR CELL RENAL CELL CARCINOMA Youichi Osako, Hirofumi Yoshino, Takashi Sakaguchi, Satoshi Sugita, Shuichi Tatarano, Takashi Yamane, Hideki Enokida, and Masayuki Nakagawa Youichi OsakoYouichi Osako More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Takashi SakaguchiTakashi Sakaguchi More articles by this author , Satoshi SugitaSatoshi Sugita More articles by this author , Shuichi TataranoShuichi Tatarano More articles by this author , Takashi YamaneTakashi Yamane More articles by this author , Hideki EnokidaHideki Enokida More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2295AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sunitinib is the most common targeting drug for metastatic renal cell carcinoma (RCC), however resistance to sunitinib is a large problem in clinical aspect. In this study, we focused on miR-99a-3p, which showed decreased expression in sunitinib-resistant RCC based on previous screening analyses, and we investigated the mechanism of sunitinib tolerance associated with miR-99a-3p and the molecular network controlled by miR-99a-3p. METHODS Expression levels of miR-99a-3p and its candidate target genes in the RCC cell lines (786-o, ACHN, and our sunitinib-resistant cell (SU-R-786-o)) and RCC clinical specimens were evaluated by qRT-PCR. For the gain of function study, cell proliferation, apoptosis, and colony formation assay were performed in miR-99a-3p transfected RCC cell lines. Putative target genes were determined by the analyses with publically available database such as GEO and TargetScan, as well as RNA sequencing expression analysis using SU-R-786-o. Loss of functional studies of the target gene was performed using si-RNA. Overall survival between high and low expression of the target gene was analyzed by the Kaplan-Meier method using the database of The Cancer Genome Atlas (TCGA). RESULTS The expression levels of miR-99a-3p were down regulated in RCC clinical specimens and the RCC cell lines compared to normal kidneys. In addition, its expression in SU-R-786-o was much lower compared to the parent cell. Restoration of miR-99a-3p in the RCC cells including SU-R-786-o significantly inhibited cell proliferation (p<0.0001) through induction of apoptosis (p<0.0001). RRM2, involved in the synthesis of deoxynucleotides that is necessary for polymerization and repair of DNA, was identified as a direct target by miR-99a-3p based on target analyses. Loss of functional studies using si-RRM2 showed that cell proliferation was significantly inhibited through induction of apoptosis especially in SU-R-786-o (p<0.0001). Kaplan-Meier analysis revealed that the patients (n= 534) with high expression of RRM2 showed lower overall survival time than those with low expression according to the TCGA database (p<0.0001). CONCLUSIONS miR-99a-3p might have tumor suppressive function through induction of cell apoptosis in sunitinib-resistant RCC cells. To the best of our knowledge, this is the first report demonstrating that miR-99a-3p directly regulate RRM2. The identification of novel target gene regulated by tumor-suppressive miR-99a-3p in sunitinib resistant RCC cells may lead to a better understanding of resistant mechanism and the development of new therapeutic strategies. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e956 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Youichi Osako More articles by this author Hirofumi Yoshino More articles by this author Takashi Sakaguchi More articles by this author Satoshi Sugita More articles by this author Shuichi Tatarano More articles by this author Takashi Yamane More articles by this author Hideki Enokida More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Dauricine inhibits viability and induces cell cycle arrest and apoptosis via inhibiting the PI3K/Akt signaling pathway in renal cell carcinoma cells.

Renal cell carcinoma (RCC), which is derived from the proximal tubules of nephrons, is one of the most common solid cancers. Due to its inherent insensitivity to radiotherapy and chemotherapy, surgery remains the only curative strategy for RCC. Therefore, a novel strategy for treating RCC is urgently needed. This study aims to investigate the effects of dauricine, a bisbenzylisoquinoline alkaloid, in RCC cells and the underlying mechanisms of its action. The effects of dauricine on viability, cell cycle distribution and apoptosis in RCC cells were determined invitro by MTT assay, flow cytometry and nucleosome ELISA assay, respectively. Mechanism studies were performed by analyzing related proteins using western blotting assays. We show that dauricine effectively inhibits the viability of four RCC cell lines (786‑O, Caki‑1, A‑498 and ACHN). In addition, dauricine induces cell cycle arrest at the G0/G1phase in RCC cells. Dauricine also induces apoptosis via the intrinsic pathway, since caspase‑9 and caspase‑3 but not caspase‑8 activation was detected after the treatment. Moreover, dauricine was able to inhibit the PI3K/Akt signaling pathway. Our findings suggest inhibitory effects of dauricine in renal cancer cells and provide a better understanding of its underlying mechanism. Our findings suggest that dauricine could be a potential therapeutic agent for treating RCC.

Overexpression of ATP citrate lyase in renal cell carcinoma tissues and its effect on the human renal carcinoma cells in vitro.

ATP citrate lyase (ACLY) is a key enzyme of lipogenesis in cells. However, ACLY expression in renal cell carcinoma (RCC) and its association with clinicopathological parameters remain unclear. The present study aimed to evaluate ACLY expression levels in RCC and adjacent normal tissues. This study included 33 patients with clear cell RCC (ccRCC). ACLY protein was assayed using immunohistochemistry and western blotting methods. ACLY mRNA expression was determined by reverse transcription-quantitative polymerase chain reaction. Serum ACLY concentrations were measured using the ELISA. Compared with adjacent normal tissues, significantly higher levels of ACLY protein expression were observed in all of the ccRCC tissues (P<0.05). ACLY protein levels were positively associated with the T stage and nuclear grade of RCC. ACLY immunostaining was located in the cytoplasm and nucleus. ACLY protein levels and ACC1 mRNA expression in RCC tissues were significantly higher compared with that in adjacent normal tissues (P<0.05). There were no significant differences in serum ACLY concentrations between patients with RCC and health controls (P>0.05). Preliminary evaluation of ACLY function showed that ACLY small interfering RNA downregulation inhibited RCC cell proliferation and migration, but promoted RCC cell apoptosis. ACLY may be a novel biomarker for the evaluation of biological aggressiveness and may be a potential target for RCC treatment.

Tumor suppressor microRNA-136-5p regulates the cellular function of renal cell carcinoma.

MicroRNAs (miRs) are involved in diverse physiological and developmental processes at the post-transcriptional level in cells. Previous studies have demonstrated that miR-136-5p is involved in certain types of cancer. However, the function of miR-136-5p in renal cell carcinoma (RCC) remains to be fully elucidated. In present study, miR-136-5p expression levels were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and MTT assays, CCK-8 assays, Transwell assays, wound healing assays and flow cytometry were performed to investigate the function of miR-136-5p in RCC. RT-qPCR revealed that the expression of miR-136 was significantly lower in RCC tissues and cells compared with adjacent non-tumor tissues and cells in vitro. miR-136-5pwas also demonstrated to be associated with RCC cell proliferation, viability, migration, invasion and apoptosis. miR-136-5p may therefore function as a tumor suppressor in RCC. Further studies are required to elucidate the molecular mechanisms and signaling pathways underlying these functions of miR-136-5p, to investigate the potential function of miR-136-5p as a biomarker for the early detection and prognosis of RCC, and its potential as a therapeutic target for the treatment of RCC.

MiR‑539 suppresses proliferation and induces apoptosis in renal cell carcinoma by targeting high mobility group A2

Renal cell carcinoma (RCC) is one of the most common urinary malignancies with a high rate of morbidity. MicroRNAs (miRNAs) have been shown to be critical post-transcriptional regulators in tumorigenesis. The present study aimed to investigate the effect of miRNA (miR)-539 on the proliferation and apoptosis of RCC. The expression of miR-539 and high mobility group AT-hook 2(HMGA2) were examined in clinical RCC specimens. The 786-O RCC cell line was also used and was transfected with miR-539 mimics or inhibitors. The correlation between miR-539 and HMGA2 was confirmed using a luciferase reporter assay. Cell viability and apoptosis were detected using MTT and flow cytometry assays. The protein levels of HMGA2, AKT, phosphorylated (p)-AKT, mammalian target of rapamycin (mTOR) and p-mTOR were analyzed using western blot analysis. The results revealed that miR-539 was negatively correlated with the expression of HMGA2 in clinical RCC specimens. Further experiments identified HMGA2 as a direct target of miR-539. The overexpression of miR-539 downregulated the expression of HMGA2, reduced cell proliferation and promoted cell apoptosis, whereas the knockdown of miR-539 led to the opposite results. miR-539 also suppressed the phosphorylation of AKT and mTOR, without altering the levels of total AKT and mTOR. Taken together, the results of the present study indicated that miR-539 negatively regulated the expression of HMGA2 in clinical specimens and in vitro. miR539 inhibited cell proliferation and induced apoptosis in RCC cells. This regulatory effect of miR-539 may be associated with the AKT signaling pathway. Therefore, miR-539 may be used as a biomarker for predicting the progression of RCC.

Knockdown of MAGEA6 Activates AMP-Activated Protein Kinase (AMPK) Signaling to Inhibit Human Renal Cell Carcinoma Cells

Background/Aims: Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC). Methods: MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo. Results: MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues. Conclusion: MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling.

GV1001 Induces Apoptosis by Reducing Angiogenesis in Renal Cell Carcinoma Cells Both In Vitro and In Vivo

To investigate the anticancer effects of GV1001 and its biological mechanism of action in renal cell carcinoma (RCC). The effects of GV1001 on cell survival and apoptosis in RCC cells were examined in vitro using cell viability assay, fluorescence-activated cell sorting, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. To evaluate the effect of GV1001 on migration, invasion, and angiogenesis, we used wound healing, invasion, endothelial cell tube formation assay, and western blot analysis. Furthermore, we used an RCC xenograft model with either phosphate buffered saline or GV1001 to confirm the anticancer effect of GV1001 in vivo. Tumor volume was monitored during treatment, and tumor weight was measured after animals were killed. Apoptosis and angiogenesis of the tumor tissue were assessed using hematoxylin and eosin staining, immunohistochemistry, and western blot analysis. GV1001 reduced cell viability and induced apoptosis in RCC cells in vitro. Furthermore, GV1001 suppressed the migration and invasion of RCC cells through regulation of matrix metalloproteinases and tissue inhibitors of metalloproteinases. In addition, GV1001 reduced angiogenesis via regulation of hypoxia-inducible factor 1α. In xenograft mouse model experiment, GV1001 reduced tumor growth and induced apoptosis. As in the in vitro results, GV1001 significantly reduced angiogenesis through regulation of hypoxia-inducible factor 1α in vivo. Our data demonstrated that GV1001 induced apoptosis through suppression of angiogenesis in RCCs both in vitro and in vivo, which suggests that GV1001 may be a potential therapeutic target for RCC.

MiR-411 functions as a tumor suppressor in renal cell cancer.

Recent studies have revealed that microRNAs (miRNAs) play important roles as oncogenes or tumor suppressors in tumorigenesis and tumor development, by negatively regulating protein expression. A previous study of microarrays identified that miR-411 was down-regulated in renal cell carcinoma (RCC), while few studies investigating the role of miR-411 in the pathogenesis of RCC have been performed. We assessed the miR-411 expression in RCC and paired adjacent normal tissues, as well as in RCC cell lines and a normal renal cell line, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Furthermore, the effects of miR-411 on RCC and normal renal cell proliferation, apoptosis and migration were determined using MTT assay, CCK-8 assay, flow cytometry and scratch wound assay following restoration of miR-411 with synthetic mimics. Results of qRT-PCR indicated that the expression of miR-411 was down-regulated in RCC tissues and cell lines when compared with adjacent normal tissues and a normal renal cell line. Further, results of CCK-8, MTT, cell scratch and transwell assay showed that over-expression of miR-411 suppressed RCC cell (786-O and ACHN) proliferation and migration. Flow cytometry assay revealed that miR-411 could induce RCC cell apoptosis. However, overexpression of miR-411 had no obvious effect on normal renal cell line 293T. To sum up, miR-411 is significantly down-regulated and plays a role as a tumor suppressor in RCC. Further studies are warranted to determine the mechanisms of miR-411 in RCC pathogenesis and define the target genes of miR-411 in RCC.

Dual inhibition of BRD4 and PI3K-AKT by SF2523 suppresses human renal cell carcinoma cell growth.

Bromodomain-containing protein 4 (BRD4) and PI3K-AKT are both important for renal cell carcinoma (RCC) development and progression. SF2523 is a BRD4 and PI3K-AKT dual inhibitor. The present study demonstrated that SF2523 was cytotoxic and anti-proliferative to established RCC cell lines (786-O and A498) and primary human RCC cells. SF2523 induced activation of caspase and apoptosis in RCC cells. Further, SF2523 disrupted RCC cell cycle progression and inhibited cell migration in vitro. At the signaling level, SF2523 in-activated PI3K-AKT-mTOR, and downregulated BRD4-dependent proteins, Bcl-2 and Myc, in RCC cells. Remarkably, SF2523 was more efficient than Wortmannin (the PI3K inhibitor) and JQ1 (the BRD4 specific inhibitor) in killing RCC cells. In vivo, SF2523 administration at well-tolerated doses suppressed 786-O xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that concurrent blockage of BRD4 and PI3K-AKT signalings by SF2523 efficiently inhibits RCC cell growth in vitro and in vivo.

MiR-514a-3p functions as a tumor suppressor in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common type of kidney cancer, and the prognosis of metastatic RCC remains poor with a high rate of recurrence and mortality. A previous study has revealed that microRNA (miRNA), which negatively regulates protein expression, serves a role of oncogene or tumor suppressor. The aim of the present study was to investigate the expression and function of miR-514a-3p in RCC. To detect the expression of miR-514a-3p in 32 paired RCC tissues, quantitative polymerase chain reaction (qPCR) was performed. The function of miR-514a-3p in the proliferation, mobility and apoptosis of RCC cells (786-O and ACHN) was assessed by MTT, CCK-8, cell scratch, Transwell, Hoechst 33342 staining and flow cytometry assay. The results of qPCR revealed that miR-514a-3p was significantly downregulated in RCC tissues compared with adjacent normal tissues. Upregulation of miR-514a-3p by transfection of mimics suppressed RCC cell proliferation, migration and invasion, and induced cell apoptosis. The results revealed that miR-514a-3p was significantly downregulated in RCC and may serve a role as tumor suppressor in RCC. Further studies are required, focusing on the possibility of using miR-514a-3p as a biomarker for RCC as well as the pathway of miR-514a-3p in RCC.

Non-coding RNA 886 promotes renal cell carcinoma growth and metastasis through the Janus kinase 2/signal transducer and activator of transcription 3 signaling pathway.

Non-coding RNA 886 (nc886) has been suggested to serve tumor-suppressing roles in several cancer cells. However, the expression pattern of nc886 and its function in renal cell carcinoma (RCC) has not been reported until now. The present study aimed to examine the expression of nc886 in human RCC tissues and to investigate the role of nc886 in RCC cell proliferation, apoptosis and invasion invitro. Furthermore, whether nc886 exerts its function on RCC via Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling was investigated. It was demonstrated that nc886 is overexpressed in human RCC tissues compared with normal tissues, as determined by reverse transcription-quantitative polymerase chain reaction analysis. The nc886 mimic and inhibitor were transfected into the A‑498 cells to overexpress or knock down nc886 expression. Cell proliferation, cell apoptosis rate and cell invasion ability were determined by MTT, flow cytometry and Transwell‑Matrigel invasion assays. The results demonstrated that nc886 overexpression promotes A‑498 cell proliferation and invasion, and inhibits cell apoptosis, while nc886 knockdown resulted in the opposite effects. Furthermore, nc886 could activate the JAK2/STAT3 signaling pathway in A‑498 cells. AG490, an inhibitor of JAK2, could attenuate the effects of nc886 on cell proliferation, apoptosis and invasion. In conclusion, to the best of our knowledge, the present study for the first time revealed the expression profile and the tumor‑promoting role of nc886 in RCC. nc886 affects RCC cell proliferation, apoptosis and invasion at least partially via the activation of JAK2/STAT3 signaling. This study may provide a useful therapeutic target for RCC.

CMTM5 inhibits renal cancer cell growth through inducing cell-cycle arrest and apoptosis.

CKLF-like MARVEL transmembrane domain-containing 5 (CMTM5) has been reported to function as a potential tumor suppressor in several human cancers. However, the involvement of CMTM5 in human renal cell carcinoma (RCC) remains unclear. The current study aimed to detect its expression pattern in RCC tissues and cells, and to determine its anti-proliferative functions in this malignancy. The mRNA and protein expression levels of CMTM5 in RCC tissues and cells were detected by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. Following the transfection with CMTM5 lentivirus or control lenti-EGFP lentivirus into the RCC cell line ACHN, the viability, migration, apoptosis and cell cycle of these cells were detected by Cell Counting kit-8 assay, Transwell assay and flow cytometry, respectively. Compared with the adjacent non-malignant kidney tissue samples, CMTM5 expression was significantly downregulated in RCC tissues (P<0.05). In addition, enforced expression of CMTM5 could efficiently inhibit the cell growth of ACHN cells, which were arrested in G0/G1 phase. Furthermore, the migration and invasion of ACHN cells were also inhibited by restoration of CMTM5 expression. The present data suggest that CMTM5 may function as a tumor suppressor in human RCC by suppressing the viability of RCC cells, implying its potential as a therapeutic target for this malignancy.

LncRNA CCAT1 inhibits cell apoptosis of renal cell carcinoma through up-regulation of Livin protein.

This study was to investigate the involvement of long non-coding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) in renal cell carcinoma (RCC) and to further uncover its underlying mechanism. In this study, the expression of CCAT1 and Livin of RCC tissues or cells was determined using qRT-PCR (quantitative real-time PCR) and western blot, respectively. RNA pulldown and RIP (RNA-Binding Protein Immunoprecipitation) assays were performed to examine the sequence interaction between CCAT1 and Livin. The viability and apoptosis of RCC cells was assessed by MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and TUNEL (TdT-mediated dUTP nick end labeling) assays, respectively. Mice of tumor animal models were established to observe the effect of CCAT1 on RCC tumor growth. The relative expression of CCAT1 in RCC tissues and cell lines was obviously higher than that of the control. CCAT1 knockdown could reduce cell viability and increase the apoptosis of RCC cells in vitro. Furthermore, Livin was significantly inhibited by CCAT1 silencing; RNA pulldown and RIP assays showed that CCAT1 was physically associated with Livin protein. Moreover, Livin overexpression not only significantly inhibited RCC cell apoptosis and increased cell viability, but completely reversed the si-CCAT1-mediated repression of cell viability. More importantly, CCAT1 silencing could inhibit the growth of RCC in vivo that was accompanied by the reduction of Livin in RCC tissues. CCAT1 inhibits RCC cell apoptosis and increases cell viability through up-regulation of Livin.

MicroRNA‑373 promotes tumorigenesis of renal cell carcinoma in vitro and in vivo.

Renal cell carcinoma (RCC) is the most common type of malignancy in the kidney parenchyma. MicroRNAs (miRNAs) are small non‑coding RNAs that serve a role in various biological processes associated with human cancer. The present study aimed to explore the potential role of miRNA (miR)‑373 in the tumorigenesis of RCC. The effects of miR‑373 on the proliferation and apoptosis of RCC cells were determined using MTT, colony formation and flow cytometry assays invitro. The results demonstrated that miR‑373 was significantly upregulated in RCC tissues and cell lines. Knockdown of miR‑373 expression reduced cell proliferation and promoted cell apoptosis in 786‑O and ACHN cell lines. Furthermore, an invivo tumorigenicity assay revealed that knockdown of miR‑373 expression reduced tumor growth in nude mice. Taken together, these data indicate that miR‑373 may promote tumorigenesis in RCC, suggesting that miR‑373 may act as a potential therapeutic target against RCC.