Abstract
Heparanase has been implicated in many pathological conditions, especially inflammation and cancer, attributed to its degradation of heparan sulfate, a crucial component maintaining the integrity of the extracellular matrix. By silencing the heparanase gene (HPSE) in MDA‐MB‐435s melanoma cells, we investigated the impact of this protein on gene transcription. Transcriptome sequencing yielded a list of 279 differentially expressed genes, of which 140 were up‐regulated and 239 down‐regulated. The 140 up‐regulated genes were classified into a substantial set of gene ontology defined functions, for example, positive regulation of cell death, apoptotic process, response to cytokine, while 239 down‐regulated genes classify only into the two categories: nucleosome and nucleosome assembly. Our focus was drawn to an array of 28 pro‐apoptotic genes regulated by heparanase: real‐time PCR experiments further validated up‐regulation of EGR1, TXNIP, AXL, CYR61, LIMS2 and TNFRSF12A by at least 1.5‐fold, among which EGR1, CYR61, and TNFRSF12A were confirmed on protein level. We demonstrated significantly increased apoptotic cells by TUNEL staining upon HPSE silencing, mediated by activation of caspase 3/PARP1 pathway. The pro‐apoptotic gene expression and observation of apoptosis were extended to another melanoma cell line, MV3 cells, thus consolidating the anti‐apoptosis effect of heparanase in melanoma cells.
Highlights
Heparanase is the only endo‐β‐D‐glucuronidase that cleaves gluco‐ siduronic bonds between glucuronate/iduronate and glucosamine residues of heparan sulfate (HS) chains resulting in release of oli‐ gosaccharides with shortened HS chains remaining on proteogly‐ cans (PGs) on the cellsurface and in the extracellular matrix (ECM).[1]
We found a substantial set of genes that were up‐regulated in heparanase gene (HPSE) silenced cells, which suggests that heparanase could act as a negative regulator of transcription; those genes classify in the Gene ontology (GO) term of positive regulation of cell death, apoptotic process, re‐ sponse to cytokine, response to external stimuli, response to stimuli (Figure 3A)
Except binding to a plethora of cytokines including the members of the fibroblast growth factor family, transforming growth factors, bone morphogenetic proteins and interleukins, HS interacts with ECM molecules like collagens, laminin, fibronectin, to contrib‐ ute to the structural integrity of the ECM, assists in preservation of proper tissue organization and inhibits cellular invasion by promot‐ ing cell–cell and cell–ECM interactions.[34,35]
Summary
Heparanase is the only endo‐β‐D‐glucuronidase that cleaves gluco‐ siduronic bonds between glucuronate/iduronate and glucosamine residues of heparan sulfate (HS) chains resulting in release of oli‐ gosaccharides with shortened HS chains remaining on proteogly‐ cans (PGs) on the cellsurface and in the extracellular matrix (ECM).[1]. SONG and SPILLMANN innate immune responses through interactions with toll‐like recep‐ tor 4.6 HPSE expression is enhanced in almost all malignant tumours examined including various carcinomas, sarcomas and haematologi‐ cal malignancies.[7,8,9,10,11] Heparanase regulates pleiotropic biological ac‐ tivities that promote tumour growth, angiogenesis and metastasis. Numerous clinical studies have consistently demonstrated that up‐ regulation of HPSE expression correlates with increased tumour size, tumour angiogenesis, enhanced metastasis and poor prognosis.[12,13,14] In contrast, silencing of HPSE or treatments of tumour‐bearing mice with heparanase inhibiting compounds, markedly attenuate tumour progression.[15,16,17,18] the mechanisms underlying these effects remain to be further investigated. Focusing on its regulation on cell apoptosis, we were able to validate the up‐regulation of pro‐apoptotic genes and display apoptosis in cell culture after silencing HPSE
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