A positive feed-forward loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling promotes proliferation and metastasis of clear cell renal cell carcinoma
The aberrant expression of long noncoding RNAs (lncRNAs) has recently emerged as key molecules in human cancers; however, whether lncRNAs are implicated in the progression of clear cell renal cell carcinoma (ccRCC) remains unclear.
Candidate lncRNAs were selected using microarray analysis and quantitative real-time PCR (qRT-PCR) was performed to detect lncRNAs expression in human ccRCC tissues. Overexpression and knocking down experiments in vivo and in vitro were performed to uncover the biological roles of lncRNA-URRCC on ccRCC cell proliferation and invasion. Microarray, chromatin immunoprecipitation, Luciferase reporter assay and western blot were constructed to investigate the molecular mechanisms underlying the functions of lncRNA-URRCC.
The microarray analysis and qRT-PCR identified a new lncRNA, URRCC, whose expression is upregulated in RCC samples and associated with poor prognosis, leading to promote ccRCC cell proliferation and invasion. Mechanistically, URRCC enhances the expression of EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, activation of P-AKT signaling, and suppressing P-AKT downstream gene, FOXO3. In return, FOXO3 could inhibit the transcription of URRCC via binding to the special region on the promoter of URRCC.
Our data suggests that targeting this newly identified feed-back loop between LncRNA-URRCC and EGFL7/P-AKT/FOXO3 signaling may enhance the efficacy of existing therapy and potentially imparts a new avenue to develop more potent therapeutic approaches to suppress RCC progression.
KeywordsClear cell renal cell carcinoma Long noncoding RNA Proliferation Invasion EGFL7 FOXO3
Clear cell renal cell carcinoma
Competing endogenous RNA
Fluorescent in situ hybridization
Gene Expression Omnibus
In vivo imaging systems
Kidney renal clear cell carcinoma
Long noncoding RNA
Quantitative real-time PCR
Renal cell carcinoma
Short hairpin RNA
Short interfering RNA
The Cancer Genome Atlas
Transcriptional start site
Up-regulation in clear cell renal cell carcinoma
Renal cell carcinoma (RCC) is one of the most aggressive human genitourinary cancers and accounts for approximately 4% of adult malignancies [1, 2]. The most common histologic subtype clear cell RCC (ccRCC) derives from the epithelial cells of the proximal renal tubule and associates with high metastatic rate and poor prognosis. Accumulating evidence has demonstrated that ccRCC resists radiotherapy and chemotherapy [3, 4]. Anti-angiogenesis drugs targeting VEGF signaling, such as multiple kinase inhibitors (Sunitinib or Pazopanib) have been recently approved and used for the treatment of advanced or metastatic ccRCC. However, the therapeutic effects are limited for a short time and the patients eventually relapse . Therefore, the detailed mechanism exploring ccRCC tumorigenesis is needed to be testified.
Long non-coding RNAs (lncRNAs) represent a class of non-protein coding RNA species longer than 200 nucleotides in length [6, 7]. Functionally, lncRNAs emerge as the important players in carcinogenesis and have various biological effects such as cell apoptosis, growth, migration, invasion, metastasis and so forth [8, 9, 10, 11]. Mechanistically, lncRNAs involve miRNA binding sites and serve as sponges to arrest miRNA function . Besides, lncRNAs directly interacts with proteins to augment or attenuate their function . Furthermore, lncRNAs impart their functions in new available strategies for early clinical cancer diagnosis and therapy [14, 15, 16]. In renal cancer, the aberrant expression signatures of lncRNAs have been revealed, contributing to new insights into the exploration and discovery of genitourinary malignancies [17, 18]. However, the validation of lncRNAs for clinical biomarkers and the identification of lncRNAs for molecular mechanisms in ccRCC are yet to be elucidated.
Epidermal growth factor-like domain-containing protein 7 (EGFL7), which is up-regulated in several cancers, is involved in neoplasm growth and metastasis [19, 20, 21]. Not only EGFL7 plays a key role in the promotion of hepatocellular carcinoma metastasis and glioma angiogenesis [19, 22], but also its stimulating effect on fibroblast proliferation and migration may provide a useful strategy for wound healing . Furthermore, EGFL7 is reported to be epigenetic modified in gastric cancer and esophageal squamous cell carcinoma [24, 25]. Therefore, as a vital tumor-inducer, EGFL7 involving the epigenetic network in ccRCC progression holds promise for ccRCC-targeted therapy.
In this study, we focus our attention on the clinical outcomes, biological function and molecular mechanisms of a new identified lncRNA-URRCC leading to ccRCC progression. Upregulated URRCC expression is sufficiently associated with tumor size, clinical T stage, metastasis and reduced overall survival of ccRCC patients. Mechanistically, histone H3 acetylation of EGFL7 promoter caused by URRCC could increase AKT signaling pathway and suppress P-AKT downstream FOXO3 signaling, enhancing cell proliferation and invasion in vitro and in vivo. Interestingly, FOXO3 could in turn downregulate URRCC expression via directly binding to its promoter region, thus forming a novel feedback loop which could provide a promising therapeutic target for ccRCC.
Materials and methods
Microarray analysis for the expression of lncRNAs was performed by Shanghai Gminix Biological Information Company (Shanghai, China), applying a pipeline as previously described [26, 27] to identify the probe sets uniquely mapped to lncRNAs from the Affymetrix array, in which special way can evaluate the lncRNA expressions in RCC gene expression data. The accession numbers for the microarray data are Gene Expression Omnibus database GEO: GSE46699 and GSE53757 [28, 29]. Gene expression profiles of the 786-O cells with or without URRCC downregulation were determined using Human Cancer Focus mRNA PCR Array following the manufacturer’s instructions. Microarray experiments were performed by KangChen Bio-tech, Shanghai, China. The differentially expressed genes with statistical significance were identified using volcano plot filtering. The threshold we used to screen upregulated or downregulated genes is fold change > = 2.0 and a p-value <= 0.05.
A total of 116 tumor samples and paired non-cancerous renal tissues were obtained from patients who underwent partial or radical nephrectomy in Department of Urology, Shanghai Tenth People’s Hospital, Tongji University (Shanghai, China). The fresh tissues were frozen in liquid nitrogen to protect the protein or RNA away from degradation. The use of human tissues was approved by the ethics committee of Shanghai Tenth People’s Hospital.
The human RCC cell lines A498, 786-O, CaKi-1, ACHN, 769-P and OSRC-2 and human normal renal tubular epithelial cell line HK-2 were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). A498 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) plus 10% Fetal Bovine Serum (FBS, Hyclone, USA) with 1% Penicillin/Streptomycin (P/S, Gibco, USA). 786-O, CaKi-1, ACHN, 769-P and OSRC-2 cells were cultured in RMPI 1640 (Gibco, USA) plus 10% Fetal Bovine Serum (FBS, Hyclone, USA) with 1% Penicillin/Streptomycin (P/S, Gibco, USA). HK-2 cells were cultured in Keratinocyte Medium (KM, ScienCell, USA) plus 1% Keratinocyte Growth Supplement (KGS, ScienCell, USA) with 1% Penicillin/Streptomycin (P/S, ScienCell, USA). All cells described above were cultured at 37° in 5% CO2.
Cell transfection and vector construction
According to the manufacturer’s protocol, short interfering RNA (siRNA) si-EGFL7/si-FOXO3/si-NC (IBS Solutions Co. Ltd., China) were transiently transfected in A498 cells using Lipofectamine 3000 (Invitrogen, USA) at a final concentration of 100 nM. Lentiviral sh-control/sh-URRCC and oe-URRCC/oe-FOXO3 were purchased from Shanghai Integrated Biotech Solutions Co., Ltd. Lentiviral infection was performed as previously described [30, 31]. In order to avoid the off-target effects of shRNA, we designed two different shRNAs for lncRNA-URRCC and two different siRNAs for EGFL7 and FOXO3. The sequences of oligonucleotides used in this experiment were listed in Additional file 1: Table S1 and Additional file 2: Table S2.
Fluorescent in situ hybridization (FISH)
FISH was performed as previously described . It is a powerful technique that uses non-toxic fluorescent DNA probes to target any given sequence within a nucleus, resulting in colored signals that are detected with a fluorescence microscope and was performed at Biosense Co. Ltd. Paraffin-embedded tissue blocks were retrospectively retrieved from renal cancer patients. Quantum dot fluorescent in situ hybridization (QD-FISH) was performed to detect the presence of URRCC using a digoxin-labeled oligonucleotide probe indirectly labeled with digoxin-antibody-conjugated quantum dots.
Isolation of cytoplasmic and nuclear RNA
Cytoplasmic and nuclear RNA were isolated and purified using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA) according to the manufacturer’s instructions.
RCC cell proliferation assay
RCC cells were seeded in 24-well plates (3000 cells/well) and cultured for 0, 2, 4, or 6 days. Cells were harvested and cell numbers were calculated using MTT agent. DMSO was used as the control. We added 250 μl of 5 mg/ml MTT to each well, incubated for 2 h in incubator at 37 °C, removed the media and added 150 μl DMSO. We then covered with tinfoil and agitated cells on orbital shaker for 15 min and then read the absorbance at 570 nm.
RCC cell invasion assay
The invasive capability of RCC cells was determined by the Transwell assay. The upper chamber of Transwll was pre-covered with Matrigel (BD356230, CORNING, USA). RCC cells were harvested and seeded with serum-free DMEM into the upper chambers at 5 × 104 cells/well, and the bottom chambers contained DMEM with 10% FBS, and then transwells incubated for 24 or 36 h at 37 °C. Following incubation, the invasive cells attached to the lower surface of the membrane were fixed by 4% paraformaldehyde and stained with 1% toluidine blue. Cell numbers were counted in five randomly chosen microscopic fields (100×) per membrane.
Xenograft subcutaneous implantation and tail vein implantation
Six-week-old male BALB/c nude mice were purchased from ShanghaiSipper-BK laboratory animal Company (Shanghai, China). After 5 days of adapting to the environment, a total of 1 × 106 A498 cells (stably transfected with sh-URRCC and sh-control) and OSRC-2 cells (stably transfected with oe-URRCC and mock) were injected hypodermically into the right armpit or tail vein of each mouse, respectively (n = 8 mice/group). Cells were also transduced with luciferase for the non-invasive in vivo imaging system (IVIS) (NightOWL II, LB983, Berthold Technologies, Germany) that was performed once a week. The tumor volume and the weight of each mouse were measured per week. The mice were killed on the 60th days after injection. We also measured and recorded the weight of each tumor. All animal studies were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s hospital.
RNA isolation and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from frozen tissues or cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Reverse transcription was performed using a PrimeScript RT reagent kit (TaKaRa, Japan), and qRT–PCR was performed with KAPA SYBR FAST qPCR Kit (Kapa Biosystems, USA) using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Japan). The mRNA and lncRNA levels were normalized against β-actin in cell and tissue lysates. The miRNA levels were normalized against U6. Data were analyzed using the 2-ΔΔCt method. The primer sequences were listed in the Additional file 1: Table S1.
Western blot (WB)
Protein extracts were separated from cells or human tissues by RIPA buffer containing protease inhibitors. 30 μg of protein extracts were loaded to 8–10% sodium dodecylsulfate–polyacrylamide gel electrophoresis gels and transferred onto nitrocellulose membranes. The membranes were hybridized with a primary antibody at 4 °C overnight, and incubated with a secondary antibody in 1 h at room temperature. The expression of β-actin was used as loading control. The intensity of the fluorescence was scanned by the Odyssey scanner (LI-COR Biosciences, USA). The information of antibodies was listed as follow: EGFL7 (1:500, proteintech, 19,291–1-AP), AKT (1:10000, Abcam, ab179463), P-AKT (1:1000, Abcam, ab131443), FOXO3 (1:500, Abcam, ab12162), H3K27ac (1:1000, Abcam, ab4729), H3 (1:1000, Abcam, ab1791), β-actin (11,000, Abcam, ab8226).
Immunohistochemistry for the target molecules was performed on paraffin sections using a primary antibody against EGFL7 (1:50, proteintech, 19,291–1-AP), FOXO3 (1:500, Abcam, ab12162), P-AKT (1:50, Abcam, ab131443), Ki67 (1:500, Abcam, ab92742) and the proteins in situ were visualized with 3, 3-diaminobenzidine. For histological scoring, 3 high power fields (magnification, × 400) were randomly selected from renal cancer tissues and normal renal tissues. The reactivity degree was assessed by at least two pathologists without knowledge of the clinicopathological features of the tumors. The degree of positivity was initially classified according to scoring both the proportion of positive staining tumor cells and the staining intensities. Scores representing the proportion of positively stained tumor cells were graded as: 0 (< 10%); 1 (11–25%); 2 (26–50%); 3 (51–75%) and 4 (> 75%). The intensity of staining was determined as: 0 (no staining); 1 (weak staining = light yellow); 2 (moderate staining = yellow brown); and 3 (strong staining = brown). The staining index (SI) was calculated as the product of staining intensity × percentage of positive tumor cells, resulting in scores of 0, 1, 2, 3, 4, 6, 8, 9 and 12. Only cells with clear tumor cell morphology were scored.
Chromatin immunoprecipitation assay (ChIP)
EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA, USA) and the EpiQuik Tissue Acetyl-Histone H3 and H4 CHIP Kit (Epigentek Group Inc., NY, USA) were used to perform ChIP assays. In brief, cells were crosslinked with 4% formaldehyde for 10 min followed by cell collection and sonication with a predetermined power to yield genomic DNA fragments of 300–1000 bp long. The chromatin was immunoprecipitated using an anti-acetyl-histone H3 or anti-acetyl-histone H4 antibody, or anti-FOXO3 antibody. Normal mouse IgG or normal rabbit IgG were used as the negative control. qRT-PCR was conducted using KAPA SYBR FAST qPCR Kit (Kapa Biosystems, USA). PCR products were analyzed by agarose gel electrophoresis. Specific primer sequences are listed in Additional file 1: Table S1.
Luciferase reporter assay
Cells were co-transfected with psiCHECK2 dual-luciferase reporter and pcDNA3.1-URRCC-promoter (wt or mt) or pcDNA3.1-FOXO3 using Lipofectamine 3000 (Invitrogen, USA). All groups were run in triplicate in 48-well plates. Luciferase activity was measured by Dual-Luciferase Assay (Promega, Madison, WI, USA) according to the manufacturer’s manual after 48 h of transfection. Renilla luciferase activity was normalized against Firefly luciferase activity.
The preprocessed level 3 RNA-seq data and corresponding clinical information of clear cell renal cell carcinoma patients were collected from The Cancer Genome Atlas (TCGA) database (http:// cancergenome.nih.gov/). All statistical analysis was done using IBM SPSS Statistics (Version 20.0, IBM) and Graphpad Prism V6 (GraphPad Software, Inc., 2012). Student’s t-test, one-way ANOVA, LSD-t test, Pearson chi-square test, Log-rank test, linear regression, and Cox Regression Analyses were performed as indicated. All data was considered significantly when *p<0.05.
URRCC is a novel lncRNA involved in renal malignant transformation
To search for the potential lncRNAs involved in ccRCC progression, we systematically analyzed the dis-regulated lncRNAs expression profiles of ccRCC tissues versus matched normal kidney tissues using two published GEO DataSets (GSE46699 and GSE53757). The detailed program was shown in Additional file 3: Figure S1A.
Using Ensemble software, we further confirmed the character of URRCC was from clone DKFZp779P0730, position within chromosome 12: 100,623,715-100,628,286 (Additional file 3: Figure S1E). The full sequence of URRCC, with transcript length 3967 bp, was presented in Additional file 3: Figure S1F. Furthermore, RNA fluorescence in situ hybridization (FISH) on ccRCC tissues detected that URRCC (green) was mainly located in the cytoplasm while the nucleuses were stained with DAPI (blue). Subcellular fraction assay also found that URRCC was mainly located in the cytoplasm, consistent with online software lncLocator (http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/) predicting the location of URRCC (Fig. 1e, f and Additional file 3: Figure S1G).
URRCC associates with ccRCC poor prognosis
To further explore the relationship between URRCC expression and the clinicopathological characteristics of 116 patients with ccRCC, we analyzed URRCC expression in ccRCC tissues. We ranked URRCC mRNA level and selected the median (4.215) of the whole set of data as the cutoff point between the high URRCC group and the low URRCC group (Fig. 1g). Clinicopathological analysis confirmed that high level of URRCC in tumors was associated with tumor size, T stage and metastasis (Additional file 5: Table S4). In the univariate analysis, high URRCC expression in tumors (hazard ratio, HR = 2.59; 95% confidence interval, CI = 1.46–4.33; p = 0.001), Tumor size (HR = 1.59; 95%CI = 1.44–2.74; p = 0.035), T stage (HR = 1.42; 95%CI = 1.25–2.68; p = 0.038) and metastasis (HR = 1.47; 95%CI = 1.05–1.86; p = 0.026) was remarkably correlated with overall survival (Additional file 6: Table S5). Multivariate analysis showed that a higher URRCC expression (HR = 2.44; 95%CI = 1.36–4.40; p = 0.003) were significantly associated with overall survival (Additional file 6: Table S5). In addition, Kaplan-Meier analysis in the 116 patients with ccRCC demonstrated that high URRCC expression level in ccRCC tissues dramatically correlated with a reduction in 5 years overall survival rates (Fig. 1h).
URRCC promotes the development of progression in ccRCC cell lines
Conversely, we examined the tumor-inducing phenotype in OSRC-2 cells with overexpression of URRCC (oe-URRCC) (Fig. 2d). As expected, upregulating URRCC expression obviously enhanced proliferation of OSRC-2 cells compared with the mock group via MTT assay (Fig. 2e). Consistently, overexpressed URRCC also induced invasion of OSRC-2 cells through transwell invasion assay (Fig. 2f).
Together, results from Fig. 2 substantiated that URRCC could promote cell proliferation and invasion in ccRCC cells.
URRCC enhances the development of progression in ccRCC in vivo
We next generated both cells labeled with firefly luciferase and then injected into tail vein of nude mice to monitor tumor metastasis. A dramatic reduction of metastatic luciferase expression in lungs of A498/sh-URRCC mice was detected by In Vivo Imaging Systems (IVIS) compared with sh-control group (Fig. 3c and d). HE analysis also showed a decrease of pulmonary metastasis in A498/sh-URRCC group compared with sh-control group (Fig. 3f). Contrarily, URRCC overexpression yielded an enhanced pulmonary metastasis of OSRC-2 cells in vivo (Fig. 3c, e and g). In addition, IHC demonstrated the decreased expression of Ki67 from sh-URRCC group compared with sh-control group, while the increased expression of Ki67 from oe-URRCC group compared with the mock group in pulmonary metastasis (Fig. 3h and i).
Together, results from Fig. 3 demonstrated that URRCC functioned as a critical tumor enhancer in vivo by promoting tumor proliferation and metastatic colonization.
URRCC enhances EGFL7 level by mediating histone H3 acetylation of EGFL7 promoter
To investigate the mechanism responsible for the upregulation of EGFL7 in ccRCC, we probed for EGFL7 could be regulated by inhibitors of histone deacetylases according to previous studies revealing that the histone acetylation was the important regulation approach for EGFL7 . qRT-PCR and WB assays indicated that EGFL7 was upregulated via the histone deacetylase inhibitor trichostatin A (TSA) in A498 and OSRC-2 cells (Fig. 4h). Meanwhile, ChIP assay confirmed that sh-URRCC caused a significant decreased level of histone H3 acetylation, but not histone H4 across EGFL7 promoter region (− 2000 to + 50 bp relative to Transcriptional start site (TSS)) in A498 cells (Fig. 4i). Additional ChIP assay indicated that sh-URRCC could not affect the H3 and H4 acetylation of GAPDH promoter in A498 cells (Fig. 4j). Conversely, oe-URRCC increased histone H3 acetylation level across EGFL7 promoter region in OSRC-2 cells (− 2000 to + 50 bp relative to TSS) (Fig. 4k). To further investigate the mechanism that why EGFL7 expression was regulated by histone H3 acetylation, we explored the potential histone acetylation sites from the promoter of EGFL7 via UCSC browser. Interestingly, aberrant enrichment of the H3K27ac histone mark were found across the promoter region of EGFL7 (Fig. 4l). Consistently, we found TSA could increase expression of H3K27ac in protein level in A498 and OSRC-2 cells, which may explain why H4 acetylation was not affected (Fig. 4m). Moreover, ChIP assay showed that TSA could reverse the down-regulation of EGFL7 caused by sh-URRCC both on acetylation and expression of EGFL7 (Additional file 8: Figure S3F-H).
Taken together, all results above illustrated that URRCC enhanced EGFL7 expression by mediating histone H3 acetylation across EGFL7 promoter region.
URRCC is associated with EGFL7/P-AKT/FOXO3 signaling pathway
Altogether, our data suggested that URRCC could enhance renal cancer cell proliferation and invasion through EGFL7/P-AKT/FOXO3 signaling.
FOXO3 inhibits URRCC transcriptional level via binding to its promoter region
Intriguingly, BX649059, named as URRCC in our study, was previously discovered to be downregulated in colorectal cancer and required for obvious proliferation and invasion inhibition in colorectal cancer cells , contradiction our conclusion that the transcriptional level of URRCC was higher in ccRCC samples than in normal renal tissues and functioned as an onco-lncRNA to elevate cell growth and metastasis. The opposite result above suggested that URRCC might be a tissue-specific non-coding RNA in human various tissues and its expression associated with cancer development. Its clinical significance in other human cancers was yet to be elucidated. On the other hand, the expression of lncRNA was involved in clinical significance of tumor outcomes, as diagnostic or prognostic biomarkers [15, 38, 39, 40]. However, the clinical biomarker of lncRNA in the development of RCC remained to be elucidated. In the present study, we observed a negative relationship between URRCC expression and patient overall survival outcome. Statistical analysis further attested that URRCC might be an effective biomarker for helping to identify the prognosis of patients with ccRCC.
Accumulating evidence shows that lncRNAs could function as ceRNAs to sequester miRNAs, leading to the up-regulation of corresponding miRNA-targeted genes, such as lncRNA-ATB , or serve as molecular scaffolds to facilitate multiple proteins interaction, such as lncRNA-GClnc1 . Interestingly, lncRNA that mainly located in cytoplasm also can regulate mRNA through non-ceRNA mode and lncRNA has rarely been reported to active genes by epigenetic modification [14, 25]. According to this, we sought to explore the mechanisms between lncRNA and mRNA and found that lncRNA-URRCC could enhance the expression of EGFL7 through mediating histone H3 acetylation of EGFL7 promoter. This is different from the two common mechanisms of lncRNAs above and the deeper molecular mechanism might include the interaction between URRCC and enzymes of epigenetic modification, which is worth going deep into research in the future.
The differential expression of EGFL7 in several cancers was associated with epigenetic modification, involving malignant pleural mesothelioma, gastric cancer, and esophageal squamous cell carcinoma [24, 25, 42]. Here, we identified that knocked down URRCC potently abrogated EGFL7 expression and blunted cell growth and invasion while overexpressed URRCC markedly elevated EGFL7 expression and enhanced cell growth and invasion. Mechanistically, URRCC could crosstalk with EGFL7 via mediating histone H3 acetylation of EGFL7 promoter, which is consistent with the previously report [25, 43, 44]. In addition, EGFL7 was reported to active multiply signaling pathways in human tumors [43, 45, 46, 47, 48]. In in vitro system, in vivo system and clinical specimen, we substantiated the upregulated URRCC was potent to promote EGFL7 expression, enhancing AKT pathway, and then contribute to tumorigenesis.
Transcription factor FOXO3 is a member of the forkhead family and its activity is regulated by AKT signaling pathway [34, 35]. In our study, URRCC promoted the phosphorylation of AKT and lead to the inhibition of FOXO3. FOXO3 deregulation plays essential roles in the development of cancer and thus FOXO3 has been classified as a tumor suppressor . Previous studies have demonstrated that FOXO3 could repress lncRNA and be promoted by circular RNA or Foxo3 pseudogene [33, 50]. Interestingly, our ChIP and luciferase assays showed that FOXO3 could depress URRCC expression through directly binding to URRCC promoter. Moreover, recent clinical studies have shown that FOXO3 is a good prognostic marker in some cancers [51, 52, 53]. Additionally, Kaplan–Meier survival analysis of FOXO3 in our work also could support this notion. Taken together, these results suggested that FOXO3 is an important biological factor and could be a potential target for cancer therapy.
In summary, our research reveals that URRCC functions as a tumor-inducer to control the ccRCC progression via modulating URRCC/EGFL7/P-AKT/FOXO3 positive feedback loop and cause cascading effects in ccRCC. The identification and validation of this study is a critical component of research and clinical management of ccRCC. Targeting this newly identified lncRNA-URRCC might help us to broaden the known strategy for ccRCC treatment.
This work was sponsored by the National Natural Science Foundation of China (31570775, 81602216 and 81772705), Natural Science Foundation of Shanghai (16ZR1426500), and Shanghai Pujiang Program (16PJD037). Three Year Plan of Action Program from Shen Kang (16CR3062B), Cultivation Program of Renji Hospital (PYZY16–006).
Availability of data and materials
Data and materials are available from website of Molecular Cancer.
WZ, JM, RZ and DG conducted all experiments and analyzed the data. HZ and WZ provided support with experimental techniques. JM, RZ, DG and HZ provided clinical samples. JM, RZ, DG and HZ collected clinical data. JM and WZ wrote the manuscript. WZ and JM contributed to manuscript revision. JZ, YC, YH, JZ and WX conceived the project and supervised all experiments. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by the Shanghai Tenth People’s hospital ethics committee and written informed consent was obtained from all patients. All animal studies were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s Hospital.
Consent for publication
The authors declare that they have no competing interests.
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