Basic Investigation| Volume 364, ISSUE 2, P220-228, August 2022

Potential biomarkers and the molecular mechanism associated with DLL4 during renal cell carcinoma progression



      Delta-like canonical notch ligand 4 (DLL4) is considered a potential prognostic gene for renal cell carcinoma (RCC). We assessed the molecular mechanisms and novel biomarkers associated with DLL4 during RCC development.


      Four gene expression profiles were downloaded from the GEO database. Differentially expressed genes (DEGs) were identified between RCC and normal renal samples, including common DEGs (co-DEGs). Thereafter, RCC-associated gene exploration was performed and a PPI network was constructed to identify the core genes. Survival analysis of core genes in the high expression group (H group) and low expression group (L group) was also performed. The key genes related to the core genes were investigated, and the miRNA-target genes and TFs-target genes were analyzed. Finally, the expression levels of VEGFA, FLT1, EGLN3, and DLL4 in RCC and paracancerous tissues were determined.


      A total of 11,867 DEGs and 622 co-DEGs were identified in this study, and 67 RCC-associated genes that were mainly enriched in signal transduction and angiogenesis function were further explored. VEGFA was identified as the core gene. Further, 30 DEGs and 9 DE-miRNAs were identified between the H and L groups. VEGFA was positively correlated with 19 genes, including EGLN3, FLT1, and DLL4. A total of 18 miRNA-target interactions, including miR-134-5p-DLL4, were obtained. VEGFA, FLT1, EGLN3, and DLL4 were significantly expressed in RCC tissues compared with paracancerous tissues.


      DLL4 may contribute to the development of RCC by participating in signal transduction and angiogenesis. VEGFA, FLT1, EGLN3, DLL4, and miR-134-5p may be novel biomarkers for RCC.

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        • Capitanio U
        • Bensalah K
        • Bex A
        • et al.
        Epidemiology of renal cell carcinoma.
        Eur Urol. 2019; 75: 74-84
        • Pfannschmidt J
        • Hoffmann H
        • Muley T
        • et al.
        Prognostic factors for survival after pulmonary resection of metastatic renal cell carcinoma.
        Ann Thoracic Surg. 2002; 74: 1653-1657
        • Motzer RJ
        • Jonasch E
        • Agarwal N
        • et al.
        Kidney cancer, version 2.2017, NCCN clinical practice guidelines in oncology.
        J Natl Compr Cancer Netw. 2017; 15: 804-834
        • Barata PC
        • Rini BI
        Treatment of renal cell carcinoma: current status and future directions.
        CA: Cancer J Clin. 2017; 67: 507-524
        • Calvo E
        • Schmidinger M
        • Heng DY
        • et al.
        Improvement in survival end points of patients with metastatic renal cell carcinoma through sequential targeted therapy.
        Cancer Treatm Rev. 2016; 50: 109-117
        • Rini BI
        • Atkins MB
        Resistance to targeted therapy in renal-cell carcinoma.
        Lancet Oncol. 2009; 10: 992-1000
        • Peach CJ
        • Mignone VW
        • Arruda MA
        • et al.
        Molecular pharmacology of VEGF-A isoforms: binding and signalling at VEGFR2.
        Int J Mol Sci. 2018; 19: 1264
        • Gupta S
        • Johnson SH
        • Vasmatzis G
        • et al.
        TFEB-VEGFA (6p21. 1) co-amplified renal cell carcinoma: a distinct entity with potential implications for clinical management.
        Mod Pathol. 2017; 30: 998-1012
        • Pan X-W
        • Xu D
        • Chen W
        • et al.
        USP39 promotes malignant proliferation and angiogenesis of renal cell carcinoma by inhibiting VEGF-A 165b alternative splicing via regulating SRSF1 and SRPK1.
        Cancer Cell Int. 2021; 21: 486
        • Wang X
        • Zhang J
        • Wang Y
        • et al.
        Upregulated VEGFA and DLL4 act as potential prognostic genes for clear cell renal cell carcinoma.
        OncoTargets Therapy. 2018; 11: 1697
        • Asnaghi L
        • Tripathy A
        • Yang Q
        • et al.
        Targeting Notch signaling as a novel therapy for retinoblastoma.
        Oncotarget. 2016; 7: 70028
        • Kuhnert F
        • Kirshner JR
        • Thurston G
        Dll4-Notch signaling as a therapeutic target in tumor angiogenesis.
        Vasc Cell. 2011; 3: 20
        • Zhang X
        • Yamashita M
        • Uetsuki H
        • et al.
        Angiogenesis in renal cell carcinoma: evaluation of microvessel density, vascular endothelial growth factor and matrix metalloproteinases.
        Int J Urol. 2002; 9: 509-514
        • Gumz ML
        • Zou H
        • Kreinest PA
        • et al.
        Secreted frizzled-related protein 1 loss contributes to tumor phenotype of clear cell renal cell carcinoma.
        Clin Cancer Res. 2007; 13: 4740-4749
        • Yusenko MV
        • Kuiper RP
        • Boethe T
        • et al.
        High-resolution DNA copy number and gene expression analyses distinguish chromophobe renal cell carcinomas and renal oncocytomas.
        BMC Cancer. 2009; 9: 152
        • Von Roemeling CA
        • Radisky DC
        • Marlow LA
        • et al.
        Neuronal pentraxin 2 supports clear cell renal cell carcinoma by activating the AMPA-selective glutamate receptor-4.
        Cancer Res. 2014; 74: 4796-4810
        • Smyth GK limma
        Linear models for microarray data.
        (editors)in: Gentleman R Carey VJ Huber W Bioinformatics and Computational Biology Solutions Using R and Bioconductor. NY: Springer New York, New York2005: 397-420
        • Benjamini YHY
        Controlling the false discovery rate: a practical and powerful approach to multiple testing.
        J R Stat Soc Ser. 1995; B 57: 289-300
        • Huang DW
        • Sherman BT
        • Lempicki RA
        Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
        Nat Protocols. 2008; 4: 44-57
        • Ashburner M
        • Ball CA
        • Blake JA
        • et al.
        Gene ontology: tool for the unification of biology.
        Nat Genet. 2000; 25: 25-29
        • Kanehisa M
        • Goto S
        KEGG: kyoto encyclopedia of genes and genomes.
        Nucl Acids Res. 2000; 28: 27-30
        • Szklarczyk D
        • Franceschini A
        • Wyder S
        • et al.
        STRING v10: protein–protein interaction networks, integrated over the tree of life.
        Nucl Acids Res. 2014; 43: 447-452
        • Yu Tang ML
        • Jianxin W
        • Yi P
        • et al.
        CytoNCA: a cytoscape plugin for centrality analysis and evaluation of biological networks.
        BioSystems. 2014; 11 (005)
        • Bland JM
        • Altman DG
        Survival probabilities (the Kaplan-Meier method).
        Bmj. 1998; 317: 1572
        • Sticht C
        • De La Torre C
        • Parveen A
        • et al.
        miRWalk: an online resource for prediction of microRNA binding sites.
        PloS One. 2018; 13e0206239
        • Han H
        • Cho J-W
        • Lee S
        • et al.
        TRRUST v2: an expanded reference database of human and mouse transcriptional regulatory interactions.
        Nucl Acids Res. 2018; 46: D380-D386
        • Dizman N
        • Philip EJ
        • Pal SK
        Genomic profiling in renal cell carcinoma.
        Nat Rev Nephrol. 2020; : 1-17
        • Huang QB
        • Ma X
        • Li HZ
        • et al.
        Endothelial Delta-like 4 (DLL4) promotes renal cell carcinoma hematogenous metastasis.
        Oncotarget. 2014; 5: 3066
        • Lobov I
        • Mikhailova N
        The role of Dll4/Notch signaling in normal and pathological ocular angiogenesis: Dll4 controls blood vessel sprouting and vessel remodeling in normal and pathological conditions.
        J Ophthalmol. 2018; 20183565292
        • Wang W
        • Yu Y
        • Wang Y
        • et al.
        Delta‑like ligand 4: A predictor of poor prognosis in clear cell renal cell carcinoma.
        Oncol Lett. 2014; 8: 2627-2633
        • Lobov I
        • Renard R
        • Papadopoulos N
        • et al.
        Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.
        Proc Natl Acad Sci. 2007; 104: 3219-3224
        • Benedito R
        • Roca C
        • Sörensen I
        • et al.
        The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.
        Cell. 2009; 137: 1124-1135
        • Lobov IB
        • Renard RA
        • Papadopoulos N
        • et al.
        Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.
        Proc Natl Acad Sci USA. 2007; 104: 3219-3224
        • Hu G-H
        • Liu H
        • Lai P
        • et al.
        Delta-like ligand 4 (Dll4) predicts the prognosis of clear cell renal cell carcinoma, and anti-Dll4 suppresses tumor growth in vivo.
        Int J Clin Exp Pathol. 2014; 7: 2143
        • Sakai I
        • Miyake H
        • Fujisawa M
        Acquired resistance to sunitinib in human renal cell carcinoma cells is mediated by constitutive activation of signal transduction pathways associated with tumour cell proliferation.
        BJU Int. 2013; 112: E211-E220
        • Lee D
        • Kim D
        • Choi YB
        • et al.
        Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.
        mAbs. 2016; 8: 892-904
        • Li J-L
        • Sainson RC
        • Oon CE
        • et al.
        DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo.
        Cancer Res. 2011; 71: 6073-6083
        • Kim DH
        • Lee S
        • Kang HG
        • et al.
        Synergistic antitumor activity of a DLL4/VEGF bispecific therapeutic antibody in combination with irinotecan in gastric cancer.
        BMB Rep. 2020; 53: 533-538
        • Pulkkinen HH
        • Kiema M
        • Lappalainen JP
        • et al.
        BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF.
        Angiogenesis. 2021; 24: 129-144
        • Krispin S
        • Stratman AN
        • Melick CH
        • et al.
        Growth differentiation factor 6 promotes vascular stability by restraining vascular endothelial growth factor signaling.
        Arterioscler, Thromb Vasc Biol. 2018; 38: 353-362
        • Claesson-Welsh L
        • Welsh M
        VEGFA and tumour angiogenesis.
        J Internal Med. 2013; 273: 114-127
        • Xin Z
        • Ye F
        • Li Z
        • et al.
        FP075 Hypoxic renal tubular epithelial cells derived exosomes promoted peritubular endothelial cell proliferation via transfer of VEGFA in ischemic kidney injury.
        Nephrol Dialysis Transplant. 2019; 34 (gfz106FP075)
        • Crona DJ
        • Skol AD
        • Leppänen V-M
        • et al.
        Genetic variants of VEGFA and FLT4 are determinants of survival in renal cell carcinoma patients treated with sorafenib.
        Cancer Res. 2019; 79: 231-241
        • Zeng F-C
        • Zeng M-Q
        • Huang L
        • et al.
        Downregulation of VEGFA inhibits proliferation, promotes apoptosis, and suppresses migration and invasion of renal clear cell carcinoma.
        OncoTargets Therapy. 2016; 9: 2131
        • Azimi G
        • Ranjbaran F
        • Arsang-Jang S
        • et al.
        Upregulation of VEGF-A and correlation between VEGF-A and FLT-1 expressions in Iranian multiple sclerosis patients.
        Neurol Sci. 2020; : 1-7
        • Fischer C
        • Mazzone M
        • Jonckx B
        • et al.
        FLT1 and its ligands VEGFB and PlGF: drug targets for anti-angiogenic therapy?.
        Nat Rev Cancer. 2008; 8: 942-956
        • Yegin Z
        • Duran T
        • Yildirim I
        Thymoquinone down-regulates VEGFA and up-regulates FLT1 transcriptional levels in human breast cancer cells.
        Int J Hum Genet. 2020; 20: 19-24
        • Funakoshi T
        • Lee C-H
        • Hsieh JJ
        A systematic review of predictive and prognostic biomarkers for VEGF-targeted therapy in renal cell carcinoma.
        Cancer Treatm Rev. 2014; 40: 533-547
        • Lin L
        • Cai J
        Circular RNA circ-EGLN3 promotes renal cell carcinoma proliferation and aggressiveness via miR-1299-mediated IRF7 activation.
        J Cell Biochem. 2020;
        • Liu Y
        • Tao Z
        • Qu J
        • et al.
        Long non-coding RNA PCAT7 regulates ELF2 signaling through inhibition of miR-134-5p in nasopharyngeal carcinoma.
        Biochem Biophys Res Commun. 2017; 491: 374-381
        • Chi J
        • Liu T
        • Shi C
        • et al.
        Long non-coding RNA LUCAT1 promotes proliferation and invasion in gastric cancer by regulating miR-134-5p/YWHAZ axis.
        Biomed Pharmacother. 2019; 118109201
        • Shi F
        • Dong Z
        • Li H
        • et al.
        MicroRNA-137 protects neurons against ischemia/reperfusion injury through regulation of the Notch signaling pathway.
        Exp Cell Res. 2017; 352: 1-8
        • Goradel NH
        • Mohammadi N
        • Haghi-Aminjan H
        • et al.
        Regulation of tumor angiogenesis by microRNAs: state of the art.
        J Cell Physiol. 2019; 234: 1099-1110
        • Zohny SF
        • Zamzami MA
        • Al-Malki AL
        • et al.
        Highly expressed DLL4 and JAG1: their role in incidence of breast cancer metastasis.
        Arch Med Res. 2020; 51: 145-152
        • Sales CB
        • Buim ME
        • de Souza RO
        • et al.
        Elevated VEGFA mRNA levels in oral squamous cell carcinomas and tumor margins: a preliminary study.
        J Oral Pathol Med. 2016; 45: 481-485