scholarly journals Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Jiacheng Lou ◽  
Yuchao Hao ◽  
Kefeng Lin ◽  
Yizhu Lyu ◽  
Meiwei Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Tumor Suppressor ◽  
P53 Protein ◽  
Circular Rnas ◽  
P53 Expression ◽  
Mirna Sponge ◽  
Rna Fish ◽  
In Cells

Abstract Background Inactivation of the tumor suppressor p53 is critical for pathogenesis of glioma, in particular glioblastoma multiforme (GBM). MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. Hitherto, it is unclear whether the stability of the p53/MDM2 complex is affected by lncRNAs, in particular circular RNAs that are usually abundant and conserved, and frequently implicated in different oncogenic processes. Methods RIP-seq and RIP-qPCR assays were performed to determine the most enriched lncRNAs (including circular RNAs) bound by p53, followed by bioinformatic assays to estimate the relevance of their expression with p53 signaling and gliomagenesis. Subsequently, the clinical significance of CDR1as was evaluated in the largest cohort of Chinese glioma patients from CGGA (n = 325), and its expression in human glioma tissues was further evaluated by RNA FISH and RT-qPCR, respectively. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of CDR1as and p53, followed by CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, p53/MDM2 interaction and p53 ubiquitination in cells in which CDR1as expression was manipulated. After AGO2 or Dicer was knocked-down to inhibit miRNA biogenesis, effects of CDR1as on p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts of CDR1as on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects of CDR1as on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on tumor volume/weight and survival of nude mice xenografted with GBM cells (in vivo). Results CDR1as is found to bind to p53 protein. CDR1as expression decreases with increasing glioma grade and it is a reliable independent predictor of overall survival in glioma, particularly in GBM. Through a mechanism independent of acting as a miRNA sponge, CDR1as stabilizes p53 protein by preventing it from ubiquitination. CDR1as directly interacts with the p53 DBD domain that is essential for MDM2 binding, thus disrupting the p53/MDM2 complex formation. Induced upon DNA damage, CDR1as may preserve p53 function and protect cells from DNA damage. Significantly, CDR1as inhibits tumor growth in vitro and in vivo, but has little impact in cells where p53 is absent or mutated. Conclusions Rather than acting as a miRNA sponge, CDR1as functions as a tumor suppressor through binding directly to p53 at its DBD region to restrict MDM2 interaction. Thus, CDR1as binding disrupts the p53/MDM2 complex to prevent p53 from ubiquitination and degradation. CDR1as may also sense DNA damage signals and form a protective complex with p53 to preserve p53 function. Therefore, CDR1as depletion may play a potent role in promoting tumorigenesis through down-regulating p53 expression in glioma. Our results broaden further our understanding of the roles and mechanism of action of circular RNAs in general and CDR1as in particular, and can potentially open up novel therapeutic avenues for effective glioma treatment.

scholarly journals The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites

2000 ◽  
Vol 14 (3) ◽  
pp. 289-300 ◽  
Author(s):  
Sheau-Yann Shieh ◽  
Jinwoo Ahn ◽  
Katsuyuki Tamai ◽  
Yoichi Taya ◽  
Carol Prives
Keyword(s):  
Dna Damage ◽  
P53 Protein ◽  
Kinase Domain ◽  
Human Homolog ◽  
P53 Expression ◽  
Checkpoint Kinase ◽  
Checkpoint Kinases ◽  
Amino Terminal

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. The human homolog of the second S. pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.

scholarly journals A circular RNA derived from PLXNB2 as a valuable predictor of the prognosis of patients with acute myeloid leukaemia

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Leilei Lin ◽  
Yu Wang ◽  
Sicheng Bian ◽  
Lili Sun ◽  
Zhibo Guo ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Regulatory Networks ◽  
Clinical Samples ◽  
Circular Rnas ◽  
Qrt Pcr ◽  
Rna Fish ◽  
Acute Myeloid

Abstract Background As a common haematological malignancy, acute myeloid leukaemia (AML), particularly with extramedullary infiltration (EMI), often results in a high mortality rate and poor prognosis. Circular RNAs (circRNAs) regulate biological and pathogenic processes, suggesting a potential role in AML. We have previously described the overall alterations in circRNAs and their regulatory networks between patients with AML presenting with and without EMI. This study aims to find new prognostic and therapeutic targets potentially associated with AML. Methods qRT-PCR was performed on samples from 40 patients with AML and 15 healthy controls. The possibility of using circPLXNB2 (circRNA derived from PLXNB2) as a diagnostic and prognostic biomarker for AML was analysed with multiple statistical methods. In vitro, the function of circPLXNB2 was studied by lentivirus transfection, CCK-8 assays, flow cytometry, and Transwell experiments. Western blotting and qRT-PCR were performed to detect the expression of related proteins and genes. The distribution of circPLXNB2 in cells was observed using RNA fluorescence in situ hybridization (RNA-FISH). We also investigated the role of circPLXNB2 by establishing AML xenograft models in NOD/SCID mice. Results By analysing the results of qRT-PCR detection of clinical samples, the expression of the circPLXNB2 and PLXNB2 mRNAs were significantly increased in patients with AML, more specifically in patients with AML presenting with EMI. High circPLXNB2 expression was associated with an obviously shorter overall survival and leukaemia-free survival of patients with AML. The circPLXNB2 expression was positively correlated with PLXNB2 mRNA expression, as evidenced by Pearson’s correlation analysis. RNA-FISH revealed that circPLXNB2 is mainly located in the nucleus. In vitro and in vivo, circPLXNB2 promoted cell proliferation and migration and inhibited apoptosis. Notably, circPLXNB2 also increased the expression of PLXNB2, BCL2 and cyclin D1, and reduced the expression of BAX. Conclusion In summary, we validated the high expression of circPLXNB2 and PLXNB2 in patients with AML. Elevated circPLXNB2 levels were associated with poor clinical outcomes in patients with AML. Importantly, circPLXNB2 accelerated tumour growth and progression, possibly by regulating PLXNB2 expression. Our study highlights the potential of circPLXNB2 as a new prognostic predictor and therapeutic target for AML in the future.

scholarly journals Cleavage and Inactivation of ATM during Apoptosis

1999 ◽  
Vol 19 (9) ◽  
pp. 6076-6084 ◽  
Author(s):  
Graeme C. M. Smith ◽  
Fabrizio d’adda di Fagagna ◽  
Nicholas D. Lakin ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Dna Damage Signalling ◽  
In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

scholarly journals Anti-apoptosis mechanism of triptolide based on network pharmacology in focal segmental glomerulosclerosis rats

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Yayu Li ◽  
Xue Jiang ◽  
Litao Song ◽  
Mengdie Yang ◽  
Jing Pan
Keyword(s):  
Focal Segmental Glomerulosclerosis ◽  
Cell Apoptosis ◽  
P53 Protein ◽  
Network Pharmacology ◽  
Tripterygium Wilfordii ◽  
P53 Expression ◽  
Chemical Structures ◽  
Segmental Glomerulosclerosis

Abstract Triptolide (TPL), the active component of Tripterygium wilfordii, exhibits anti-cancer and antioxidant functions. We aimed to explore the anti-apoptosis mechanism of TPL based on network pharmacology and in vivo and in vitro research validation using a rat model of focal segmental glomerulosclerosis (FSGS). The chemical structures and pharmacological activities of the compounds reported in T. wilfordii were determined and used to perform the network pharmacology analysis. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was then used to identify the network targets for 16 compounds from Tripterygium wilfordii. Our results showed that 47 overlapping genes obtained from the GeneCards and OMIM databases were involved in the occurrence and development of FSGS and used to construct the protein–protein interaction (PPI) network using the STRING database. Hub genes were identified via the MCODE plug-in of the Cytoscape software. IL4 was the target gene of TPL in FSGS and was mainly enriched in the cell apoptosis term and p53 signaling pathway, according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. TPL inhibited FSGS-induced cell apoptosis in rats and regulated IL4, nephrin, podocin, and p53 protein levels via using CCK8, TUNEL, and Western blot assays. The effects of IL4 overexpression, including inhibition of cell viability and promotion of apoptosis, were reversed by TPL. TPL treatment increased the expression of nephrin and podocin and decreased p53 expression in rat podocytes. In conclusion, TPL inhibited podocyte apoptosis by targeting IL4 to alleviate kidney injury in FSGS rats.

Immunologically distinct p53 molecules generated by alternative splicing

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals Prolyl hydroxylase 3 stabilizes the p53 tumor suppressor by inhibiting the p53–MDM2 interaction in a hydroxylase-independent manner

2019 ◽  
Vol 294 (25) ◽  
pp. 9949-9958 ◽  
Author(s):  
Yiming Xu ◽  
Qiang Gao ◽  
Yaqian Xue ◽  
Xiuxiu Li ◽  
Liang Xu ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Tumor Suppressor ◽  
P53 Protein ◽  
Prolyl Hydroxylase ◽  
Hydroxylase Activity ◽  
Independent Manner ◽  
P53 Expression ◽  
Genetic Ablation ◽  
Protein Levels

Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo. These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner.

scholarly journals Immunologically distinct p53 molecules generated by alternative splicing.

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239 ◽  
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals High Expression of NEK2 Mediated By p53 Contributes to Progression and Relapse of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 192-192
Author(s):  
Wen Zhou ◽  
Fenghuang Zhan
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
P53 Protein ◽  
High Expression ◽  
Hek293 Cells ◽  
Reporter System ◽  
P53 Expression

Abstract High expression of NEK2 mediated by p53 contributes to progression and relapse of multiple myeloma Xiangling Feng1,2, Jiaojiao Guo1, Bowen Ouyang2, Yinghong Zhu1,Gang An3, Hao Zhen1, Jiliang Xia1, Yongjun Guan1, Xinying Zhao2, Lugui Qiu3, Jiaxi Zhou3, Fenghuang Zhan4,Wen Zhou1 1, Cancer Research Institute,Central South University, Changsha 410078, China. 2Xiang Ya School of Public Health, Central South University, Changsha, Hunan, China. USA. 3State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin, China. 4Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, University of Iowa, Iowa City, USA. E-mail: [email protected]. Background: Loss of p53 is an independent prognostic factor in patients with multiple myeloma (MM). Our previous studies found abnormal high expression of NEK2 was closely related to the poor prognosis and drug resistance of myeloma patients. However, it's unclear how NEK2 was up-regulated in MM. Through bioinformatics analysis, the binding site of p53 protein is found in NEK2 promoter, but the relationship and function of p53 and NEK2 in MM are poorly understood. Materials and Methods: In this study, p53-/- MM cell lines (ARP1 and KMS11) and p53 p53+/+ MM cell lines (MM1S and H929) were used for investigating the role of NEK2 in p53-/- MM cell. FISH was performed on interphase nuclei of MM primary cells to detect p53 and NEK2 copy numbers. Chromatin immunoprecipitation and fluorescence reporter system were applied for examining the binding site of p53 protein in the distal NEK2 promoter. CGH and RNA-seq were performed to validate copy number changes and variations in the expression of several transcripts. Results: The top 10% of MM patients with the highest NEK2 expression and lowest p53 had a significantly inferior OS (P<0.001) in TT2 and TT3 patients (GSE2658) and the expression of NEK2 increased significantly in myeloma cells during chemotherapy(GSE19554), while p53 decreased with the disease progression, suggesting a strong relationship with drug resistance. Single cell PCR showed increased NEK2 expression correlated with decreased p53 expression in single CD138+ plasma cell. FISH confirmed the loss of p53 in CD138+ plasma cells with amplification of NEK2 copies. Furthermore, NEK2 was also high expressed in p53 low expressed MM cells by Immunofluorescence (IF) (P<0.01). In addition, NEK2 was upregulated in p53-/- MM cell lines and HEK293 cells by deleted p53 gene with CRISPR technique both on mRNA and protein level (P<0.01), suggesting a negative correlation between the p53 and the expression level of NEK2. Meanwhile, when p53 deletion and NEK2 overexpression occur simultaneously, the phenomena of asymmetric mitosis and multipolar division are more obvious (P<0.001), suggested that the double hit of p53 deletion and NEK2 overexpression increases the chromosomal instability. Further in vivo study indicated the subcutaneous tumorigenesis in p53 deletion and NEK2 overexpression group was significantly greater than that of the single overexpression of NEK2 and deletion of p53 group (P<0.001), suggested that NEK2 overexpression and p53 deletion enhances the tumorigenic ability in vivo. While down-regulation of NEK2 by shRNA in p53 deletion cells, cell growth was inhibited in vitro and in vivo.To explore the relationship between p53 and NEK2, chromatin immunoprecipitation and fluorescence reporter system showed that p53 could bind to the promoter region of NEK2 and regulate its transcriptional expression. Further CGH analysis of the deletion of p53 expression in HEK293 cells can cause 1q21.4-44 amplification of the chromosome region of the NEK2 directly, which further confirmed by FISH. Finally, RNA-seq revealed several chromosome instability genes were abnormal expressed in NEK2 overexpression and p53 deletion double-hit group. Conclusion: In summary, p53 deletion and NEK2 overexpression induced cancer cell drug resistance, proliferation and chromosomal instability. p53 could bind to the promoter region of NEK2 and cause NEK2 amplification. Down-regulation of NEK2 by shRNA in p53 deletion cells inhibited cell growth in vitro and in vivo. Thus,The significance of this study will provide the pre-clinical application of the NEK2 inhibitor to overcome the drug resistance induced by p53 in MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circ_0030998 Promotes Tumor Proliferation and Angiogenesis by Sponging miR-567 to Regulate VEGFA in Colorectal Cancer

2020 ◽  
Author(s):  
Longyang Jin ◽  
Chao Han ◽  
Tianyu Zhai ◽  
Xiaoyu Zhang ◽  
Chun Chen ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Circular Rnas ◽  
Potential Therapeutic Target ◽  
Bioinformatic Tools ◽  
Qrt Pcr ◽  
In Vivo Assays ◽  
Rna Fish ◽  
Expression Of Genes

Abstract BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Accumulating studies have demonstrated that circular RNAs (circRNAs), a novel class of noncoding RNAs, are involved in pathological processes, especially in the development of cancers. But the roles of circRNAs in CRC are largely unknown.MethodsThe microarray data GSE138589 was analyzed and qRT-PCR was performed to screen out the circRNA with the highest upregulated level in CRC. QRT-PCR was used to determine the expression of genes in different tissues or cells. RNA-FISH was conducted to identify the subcellular localization of Circ_0030998. In vitro and in vivo assays were performed to determine the effect of Circ_0030998 on the CRC cells proliferation and angiogenesis. The relationships of Circ_0030998, miR-567 and VEGFA were predicted and validated using bioinformatic tools, RIP and luciferase assays.ResultsWe identified a novel circRNA, Circ_0030998, was upregulated in CRC tissues and cells, and its upregulation was related with poor prognosis in CRC patients. Circ_0030998 promoted CRC cells proliferation and angiogenesis. RNA-FISH showed that Circ_0030998 was mainly localized in the cytoplasm of CRC cells. Mechanistic studies demonstrated that Circ_0030998 acted as a miR-567 sponge to relieve its inhibitory effect on VEGFA. Rescue assays validated that Circ_0030998 functioned in CRC cells proliferation and angiogenesis relying on VEGFA.ConclusionsThe present study clarified the Circ_0030998/miR-567/VEGFA regulation axis and indicated that Circ_0030998 could be a potential therapeutic target for CRC.

P5434P300-mediated inactivation of p53 protects against doxorubicin-induced cardiotoxicity

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
M Adam ◽  
K K Singh ◽  
K A Connelly
Keyword(s):  
Dna Damage ◽  
Topoisomerase Ii ◽  
Caspase 3 ◽  
P53 Expression ◽  
Doxorubicin Treatment ◽  
Chemotherapy Drugs ◽  
Pre Treatment ◽  
Induced Apoptosis

Abstract Introduction Doxorubicin is amongst the most widely prescribed chemotherapy drugs due to its effectiveness in cancer treatment. However, progressive treatment using doxorubicin severely increases the risk of congestive heart failure. Mechanistically, researchers have shown that doxorubicin (i) intercalates into DNA and disrupts topoisomerase-II-mediated DNA repair and (ii) generates free radicals which then causes damage to cellular membranes, DNA, and proteins. Ultimately these cellular insults induce cardiomyocyte (CM) death mediated by the tumour suppressor, p53. Currently, there are no clinically applicable preventative treatments for doxorubicin-induced cardiotoxicity and so, extensive research is being done in discovering a potential therapy. One such candidate is curcumin – a natural polyphenol compound non-toxic to humans. We have previously demonstrated that curcumin inhibits lysine acetyltransferase activity of p300, therefore reducing both histone and non-histone protein acetylation. To induce CM death, p53 requires acetylation by p300. Therefore, we hypothesize that curcumin protects against doxorubicin-induced CM death and cardiotoxicity via p300-mediated inactivation of p53. Methods Rat H9c2 cardiomyoblast cells were cultured and treated with a 2.5 μM dose of doxorubicin for 16 hours. One group of cells were pre-treated with curcumin (15μM) 4 hours prior to doxorubicin treatment, and controls were cultured with only diluent added. Following treatment, the cells were harvested for total protein. At end point, we performed immunoblotting to measure protein expression of key proteins involved in DNA damage (y-H2A.X, p53), and apoptosis (cleaved-Caspase 3). Results Our findings show that following doxorubicin treatment, p53 expression was significantly increased (p<0.001), confirming its role in doxorubicin-associated cardiotoxicity. Furthermore, doxorubicin was associated with increased DNA-damage as evidenced by increased expression and activation of DNA double-stranded breaks (DSBs) marker, y-H2A.X (p<0.001). Elevated levels of DNA-damage were further associated with significantly increased doxorubicin-induced apoptosis as measured by immunoblotting for cleaved-Caspase 3 (p<0.001). However, DNA-damage and apoptosis were attenuated by pre-treatment of cells with curcumin. Curcumin treatment led to a significant decrease in both y-H2A.X (p<0.01) and cleaved-Caspase 3 (p<0.01) expression compared to cells treated with only doxorubicin. Conclusions Our data provides the first evidence that curcumin protects against doxorubicin-induced apoptosis in rat cardiomyoblast cells in vitro. Further studies are warranted in vivo to confirm the potential of curcumin as a cardio-protective drug. Curcumin is a natural compound with little to no side-effects in humans, therefore our finding may provide a novel therapeutic target and treatment approach for doxorubicin-associated cardiotoxicity. Acknowledgement/Funding Heart & Stroke Foundation of Canada

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