PARP1 mRNA Research Articles

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Cytosine–phosphate–guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis

BackgroundRadiotherapy can cause kidney injury in patients with cervical cancer. This study aims to investigate the possible molecular mechanisms by which CpG-ODNs (Cytosine phosphate guanine-oligodeoxynucleotides) regulate the PARP1 (poly (ADP-ribose) polymerase 1)/XRCC1 (X-ray repair cross-complementing 1) signaling axis and its impact on radiation kidney injury (RKI) in cervical cancer radiotherapy.MethodsThe GSE90627 dataset related to cervical cancer RKI was obtained from the Gene Expression Omnibus (GEO) database. Bioinformatics databases and R software packages were used to analyze the target genes regulated by CpG-ODNs. A mouse model of RKI was established by subjecting C57BL/6JNifdc mice to X-ray irradiation. Serum blood urea nitrogen (BUN) and creatinine levels were measured using an automated biochemical analyzer. Renal tissue morphology was observed through HE staining, while TUNEL staining was performed to detect apoptosis in renal tubular cells. ELISA was conducted to measure levels of oxidative stress-related factors in mouse serum and cell supernatant. An in vitro cell model of RKI was established using X-ray irradiation on HK-2 cells for mechanism validation. RT-qPCR was performed to determine the relative expression of PARP1 mRNA. Cell proliferation activity was assessed using the CCK-8 assay, and Caspase 3 activity was measured in HK-2 cells. Immunofluorescence was used to determine γH2AX expression.ResultsBioinformatics analysis revealed that the downstream targets regulated by CpG-ODNs in cervical cancer RKI were primarily PARP1 and XRCC1. CpG-ODNs may alleviate RKI by inhibiting DNA damage and oxidative stress levels. This resulted in significantly decreased levels of BUN and creatinine in RKI mice, as well as reduced renal tubular and glomerular damage, lower apoptosis rate, decreased DNA damage index (8-OHdG), and increased levels of antioxidant factors associated with oxidative stress (SOD, CAT, GSH, GPx). Among the CpG-ODNs, CpG-ODN2006 had a more pronounced effect. CpG-ODNs mediated the inhibition of PARP1, thereby suppressing DNA damage and oxidative stress response in vitro in HK-2 cells. Additionally, PARP1 promoted the formation of the PARP1 and XRCC1 complex by recruiting XRCC1, which in turn facilitated DNA damage and oxidative stress response in renal tubular cells. Overexpression of either PARP1 or XRCC1 reversed the inhibitory effects of CpG-ODN2006 on DNA damage and oxidative stress in the HK-2 cell model and RKI mouse model.ConclusionCpG-ODNs may mitigate cervical cancer RKI by blocking the activation of the PARP1/XRCC1 signaling axis, inhibiting DNA damage and oxidative stress response in renal tubule epithelial cells.

Highly Selective MIF Ketonase Inhibitor KRP-6 Diminishes M1 Macrophage Polarization and Metabolic Reprogramming.

Macrophage polarization is highly involved in autoimmunity. M1 polarized macrophages drive inflammation and undergo metabolic reprogramming, involving downregulation of mitochondrial energy production and acceleration of glycolysis. Macrophage migration inhibitory factor (MIF), an enigmatic tautomerase (ketonase and enolase), was discovered to regulate M1 polarization. Here, we reveal that KRP-6, a potent and highly selective MIF ketonase inhibitor, reduces MIF-induced human blood eosinophil and neutrophil migration similarly to ISO-1, the most investigated tautomerase inhibitor. We equally discovered that KRP-6 prevents M1 macrophage polarization and reduces ROS production in IFN-γ-treated cells. During metabolic reprogramming, KRP-6 improved mitochondrial bioenergetics by ameliorating basal respiration, ATP production, coupling efficiency and maximal respiration in LPS+IFN-γ-treated cells. KRP-6 also reduced glycolytic flux in M1 macrophages. Moreover, the selective MIF ketonase inhibitor attenuated LPS+IFN-γ-induced downregulation of PARP-1 and PARP-2 mRNA expression. We conclude that KRP-6 represents a promising novel therapeutic compound for autoimmune diseases, which strongly involves M1 macrophage polarization.

Disruption of caspase-independent cell proliferation pathway on spheroids (HeLa cells) treated with curcumin

ABSTRACT Curcumin is an antiproliferative phytochemical extracted from Curcuma longa L and which has been studied in preclinical drug screening using cell monolayers and animal models. However, several limitations of these culture systems may be overcome by performing screening with three-dimensional (3-D) cell culture. The aim of this study was to investigate the effects of curcumin on cytotoxicity and genotoxicity as well as spheroid growth using cervical adenocarcinoma HeLa cell spheroids by performing RT-PCR mRNA expression of genes involved in cell death (CASP3, CASP8, CASP9, PARP1, BBC3, BIRC5, BCL2, TNF), autophagy (BECN1, SQSTM1), cell cycle regulation (TP53, C-MYC, NF-kB, CDKN1A, m-TOR, TRAF-2), DNA damage repair (H2AFX, GADD45A, GADD45G), oxidative stress (GPX1), reticulum stress (EIF2AK3, ERN1), and invasion (MMP1, MMP9) was investigated. Curcumin was cytotoxic in a concentration-dependent manner. Curcumin-treated spheroids exhibited lower proliferative recovery and cell proliferation attenuation, as observed in the clonogenic assay. Further, no marked genotoxicity was detected. Curcumin-treated spheroids displayed reduced expression of BECN1 (2.9×), CASP9 (2.1×), and PARP1 (2.1×) mRNA. PARP1 inhibition suggested disruption of essential pathways of proliferation maintenance. Downregulated expression of CASP9 mRNA and unchanged expression of CASP3/8 mRNA suggested caspase-independent cell death, whereas downregulated expression of BECN1 mRNA indicated autophagic disruption. Therefore, curcumin exhibits the potential for drug development with antiproliferative activity to be considered for use in cancers.

Effect of postharvest nicotinamide treatment on NAD+ metabolism and redox status in strawberry fruit during storage

The increased activity of PARP enzymes is associated with a deficiency of NAD+, as well as with a loss of NADPH and ATP, and consequent deterioration of the redox state in fruits. In this study, we checked whether treatment with nicotinamide (NAM) would affect PARP-1 expression and NAD+ metabolism in strawberry fruit during storage. For this purpose, strawberry fruits were treated with 10 mM NAM and co-treated with NAM and UV-C, and then stored for 5 days at 4 °C. Research showed that nicotinamide contributes to reducing oxidative stress level by reducing PARP-1 mRNA gene expression and the protein level resulting in higher NAD+ availability, as well as improving energy metabolism and NADPH levels in fruits, regardless of whether they are exposed to UV-C. The above effects cause fruits treated with nicotinamide to be characterised by higher anti-radical activity, and a lower level of reactive oxygen species in the tissue.

Edible coating enriched with cinnamon oil reduces the oxidative stress and improves the quality of strawberry fruit stored at room temperature.

The present study aimed to assess the impact of a starch/gelatine coating containing cinnamon oil on selected quality attributes and redox status in strawberry fruit stored at room temperature (72 h). Research showed that the application of cinnamon oil to an edible coating allows an improvement of the quality of strawberry fruit stored at room temperature. The cinnamon oil coating inhibits the development of yeast and mould, and reduces loss of soluble solids and ascorbic acid during 72 h storage at room temperature. Moreover, the coating with cinnamon oil clearly reduced the level of oxidative stress, which was manifested by a lower level of reactive oxygen species, as well as a lower activity of antioxidant enzymes. The elimination of oxidative stress in the cinnamon oil-coated fruit also contributed to lower PARP1 mRNA expression, inhibiting the metabolism of NAD+ and reducing ATP losses. The coating of strawberry fruit with a starch/gelatine biofilm containing cinnamon oil is an effective method for delaying postharvest senescence of fruit and the storage degradation of tissue. © 2023 Society of Chemical Industry.

LncRNA ANRIL promotes HR repair through regulating PARP1 expression by sponging miR-7-5p in lung cancer

BackgroundRadiotherapy is an important treatment for lung cancer, mainly by triggering DNA double-strand breaks to induce cell death. Blocking DNA damage repair can increase the radiosensitivity of tumor cells. Recent studies have identified long noncoding RNAs as key regulators in DNA damage repair. The lncRNA ANRIL was previously shown to be involved in homologous recombination (HR) repair, but its specific mechanism has not been fully elucidated.MethodsThe downstream interacting miRNAs of ANRIL were predicted according to miRanda software. Fluorescence quantitative PCR was used to detect the expression levels of ANRIL and candidate miRNAs. Clone formation experiment and cell viability assays detect cell viability after ionizing radiation. Apoptosis assay was used to detect the apoptosis of cells after 8 h of ionizing radiation. Western blot analysis and immunofluorescence assays verified the protein expression levels of the downstream target molecule PARP1 of miR-7-5p and key molecules in the HR pathway. Fluorescent reporter gene experiments were used to verify the interaction between ANRIL and miR-7-5p and between miR-7-5p and PARP1.ResultsBioinformatics analysis and qPCR validation suggested that miR-7-5p might be a downstream molecule of ANRIL. The expression of miR-7-5p was up-regulated after knockdown of ANRIL, and the expression of miR-7-5p was down-regulated after overexpression of ANRIL. Meanwhile, there was a negative correlation between ANRIL and miR-7-5p expression changes before and after ionizing radiation. The luciferase reporter gene assay confirmed the existence of ANRIL binding site with miR-7-5p, and found that transfection of miR-7-5p inhibitor can reduce the radiation sensitivity of ANRIL-KD cells. A downstream target molecule of miR-7-5p related to HR repair, PARP1, was screened through website prediction. Subsequently, it was confirmed by Western blot and luciferase reporter assays that miR-7-5p could down-regulate the expression of PARP1, and there was a miR-7-5p binding site on the 3'UTR of PARP1 mRNA. This suggests that ANRIL may act as a competitive endogenous RNA to bind miR-7-5p and upregulate the expression of PARP1. Western blot and immunofluorescence staining were used to detect the expression changes of HR repair factors in ANRIL-KD cells after ionizing radiation, and it was found that knockdown of ANRIL can inhibit the expression of PARP1, BRCA1 and Rad51, hinder radiation-induced HR repair, and eventually result in resensitizing ANRIL-KD cells to ionizing radiation.ConclusionsOur findings provide evidence that ANRIL targets the miR-7-5p/PARP1 axis to exert its regulatory effect on HR repair, suggesting that altering ANRIL expression may be a promising strategy to overcome radiation resistance.

Molecular modeling of PARP1 inhibition through miRNA gene silencing for the treatment of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a category of diseases that are characterized by the death of rod photoreceptors and subsequent death of cone receptors. Parthanatos, a form of regulated necrosis, has been reported as being the underlying mechanism of cell death in RP. Parthanatos in RP occurs with the downstream expression of poly-ADP-ribose-polymerase (PARP) and the subsequent nuclear translocation of the mitochondrial apoptosis-inducing factor (AIF), leading to condensing of chromatin and DNA fragmentation. Extracellular vesicles (EVs) are small-scale liposomes that carry miRNAs and the RNA-induced silencing complex (RISC) associated with gene regulation. miRNAs, loaded on the argonaute protein 2 (Ago-2) bind to the mature mRNA and initiate the recruitment of the RISC complex triggering the degradation of the mRNA, dramatically scaling down gene expression. In vitro validation of gene silencing is a time expensive process and computational resources provide faster and cheaper options. Here, we identified the miRNAs target sequences on the mature PARP1 mRNA construct from the TargetScan database and obtained their complementary miRNA sequences from the miRbase. Using the human Ago-2 crystal structure deposited to the PDB database (PDB ID: 4W5Q) as a template, mutational studies were conducted on the protein/mRNA/miRNA complex to reproduce PARP1 mRNA seed pairing to miRNAs. Upon initial geometry optimization, further conformational search studies were done for thorough exploration of the conformational space of the protein/RNA complex by implementing conformational flooding (CF) molecular dynamics (MD) simulations. CF MD introduces a flooding potential to the force field, thus lowering energy barriers and facilitating observations of macroscale conformational changes. Clustering of the CF trajectories reveal the stable conformations of the Ago-2/miRNA/mRNA complexes. Energetically favorable miRNAs discovered in silico will further be validated with in vitro silencing assays.

C/D box small nucleolar RNA SNORD104 promotes endometrial cancer by regulating the 2ʹ-O-methylation of PARP1

BackgroundSmall nucleolar RNAs (snoRNAs) are dysregulated in many cancers, although their exact role in tumor genesis and progression remains unclear.MethodsThe expression profiles of snoRNAs in endometrial cancer (EC) tissues were analyzed using data from The Cancer Genome Atlas, and SNORD104 was identified as an upregulated snoRNA in EC. The tumorigenic role of SNORD104 in EC was established in CCK8, colony formation, EdU, apoptosis, Transwell, and in vivo xenograft experiments. The molecular mechanisms of SNORD104 were analyzed by RNA immunoprecipitation (RIP), Nm-seq, RTL-P assay, RNA stability assay, qRT-PCR, and western blotting.ResultsAntisense oligonucleotide (ASO)-mediated knockdown of SNORD104 in Ishikawa cells significantly inhibited their proliferation, colony formation ability, migration, and invasion in vitro and increased apoptosis. On the other hand, overexpression of SNORD104 promoted EC growth in vivo and in vitro. RIP assay showed that SNORD104 binds to the 2ʹ-O-methyltransferase fibrillarin (FBL), and according to the results of Nm-seq and RTL-P assay, SNORD104 upregulated PARP1 (encoding poly (ADP-ribose) polymerase 1) 2ʹ-O-methylation. The binding of FBL to PARP1 mRNA was also verified by RIP assay. Furthermore, SNORD104 expression was positively correlated with PARP1 expression in EC tissues. In the presence of actinomycin D, SNORD104 increased the stability of PARP1 mRNA and promoted its nuclear localization. Finally, silencing FBL or PARP1 in the HEC1B cells overexpressing SNORD104 inhibited their proliferative and clonal capacities and increased apoptosis rates.ConclusionsSNORD104 enhances PARP1 mRNA stability and translation in the EC cells by upregulating 2ʹ-O-methylation and promotes tumor growth.

Exploring heterometallic bridged Pt(II)-Zn(II) complexes as potential antitumor agents

The four novel complexes [{cis-PtCl(NH3)2(μ-4,4′-bipyridyl)ZnCl(terpy)}](ClO4)2 (C1), [{trans-PtCl(NH3)2(μ-4,4′-bipyridyl)ZnCl(terpy)}](ClO4)2 (C2), [{cis-PtCl(NH3)2(μ-pyrazine)ZnCl(terpy)}](ClO4)2 (C3) and [{trans-PtCl(NH3)2(μ-pyrazine)ZnCl(terpy)}](ClO4)2 (C4) (where terpy = 2,2′:6′,2′′-terpyridine) were synthesized and characterized. Acid–base titrations and concentration dependent kinetic measurements for the reactions with biologically relevant ligands such as guanosine-5′-monophosphate (5′-GMP), inosine-5′-monophosphate (5′-IMP) and glutathione (GSH), were studied at pH 7.4 and 37 °C. The binding of the heterometallic bridged cis- or trans-Pt(II)-Zn(II) complexes to calf thymus DNA (CT-DNA) was studied by UV absorption and fluorescence emission spectroscopy and molecular docking. The results indicated that the complexes bind strongly to DNA, through groove binding, hydrogen bonds, and hydrophobic or electrostatic interaction. The possible in vitro DNA protective effect of cis- and trans-Pt-L-Zn complexes has shown that C3 had significant dose-dependent DNA-protective effect and the same ability to inhibit peroxyl as well as hydroxyl radicals. Antiproliferative effect of the complexes, mRNA expression of apoptosis and repair-related genes after treatment in cancer cells indicated that newly synthesized C2 exhibited highly selective cytotoxicity toward colon carcinoma HCT116 cells. Only treatment with trans analog C2 induced effect similar to the typical DNA damaging agent such as cisplatin, characterized by p53 mediated cell response, cell cycle arrest and certain induction of apoptotic related genes. Both cis- and trans-isomers C1 and C2 showed potency to elicit expression of PARP1 mRNA and in vitro DNA binding.

PARP1 and OGG1 in Medicated Patients With Depression and the Response to ECT.

Oxidative stress and oxidation-induced DNA damage may contribute to the pathophysiology of depression. Two key mediators of base excision repair (BER) in response to oxidative damage of DNA are OGG1 and PARP1. Few studies have examined changes in OGG1 or PARP1 mRNA in patients with depression or following antidepressant treatment. We examined PARP1 and OGG1 mRNA levels in patients with depression at baseline/pre-electroconvulsive therapy (baseline/pre-ECT) vs in healthy controls and in patients following a course of ECT. PARP1 and OGG1 were examined in whole blood samples from medicated patients with depression and controls using quantitative real-time polymerase chain reaction. Exploratory subgroup correlational analyses were performed to determine associations between PARP1 and OGG1 and mood (Hamilton Depression Rating Scale 24-item version) scores as well as with vitamin B3, SIRT1, PGC1α, and tumor necrosis factor alpha levels, as previously reported on in this cohort. PARP1 levels were reduced in samples from patients with depression vs controls (P = .03), though no difference was noted in OGG1. ECT had no effect on PARP1 or OGG1. Higher baseline PARP1 weakly correlated with greater mood improvement post ECT (P = .008). Moreover, PARP1 positively correlated with SIRT1 at baseline and post ECT, and positive correlations were noted between change in PARP1 and change in OGG1 with change in tumor necrosis factor alpha post ECT. To our knowledge, this is the first study to examine the effect of ECT on BER enzymes. A better understanding of BER enzymes and DNA repair in depression could unearth new mechanisms relevant to the pathophysiology of this condition and novel antidepressant treatments.

Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC

High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported an increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent non-malignant stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are elevated in tumors relative to normal tissue in the fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in the omentum. We also showed that PARP1 expression is increased in a malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed since PARP inhibitors are currently being used in the treatment of patients with OC.

High Expression of PARP1 in Tumor and Stroma Cells Predicts Different Prognosis and Platinum Resistance in Patients With Advanced Epithelial Ovarian Cancer.

ObjectiveThis study aimed to explore the roles of PARP1 mRNA and protein expression in platinum resistance and prognosis of EOC patients, and reveal the different roles of PARP1 protein in epithelial tumor and stroma cells.MethodsThe PARP1 mRNA expression of the EOC tissues was examined by RT-qPCR. The impacts of PARP1 expression on prognosis were measured by Kaplan-Meier and Cox regression. Receiver operating characteristic (ROC) curve analysis was employed for calculating the diagnostic value of PARP1 on platinum resistance. The microarray of formalin-fixed, paraffin-embedded (FFPE) tissues was processed for multiplex immunofluorescence to detect the protein levels of PARP1 and cytokeratin (CK).ResultsThe PARP1mRNA expression of EOC patients was higher in the platinum-resistant group compared with the sensitive group (P<0.01). Kaplan-Meier analysis demonstrated that high PARP1 mRNA expression was associated with poor survival of EOC patients. In Cox regression analyses, high PARP1 mRNA expression independently predicted poor prognosis (P=0.001, HR=2.076, 95%CI=1.373-3.140). The area under the ROC curve of PARP1 mRNA for predicting the platinum resistance in EOC patients was 0.649, with a sensitivity of 0.607 and specificity of 0.668. Furthermore, the protein expression of PARP1 was higher in the platinum-resistant group than in the sensitive group (P<0.01) and associated with a worse prognosis. Additionally, according to CK labeling, we observed that enhanced expression of PARP1 in the CK+ region was associated with platinum resistance and lower survival, but in CK- region, it predicted a good prognosis and platinum sensitivity.Conclusion PARP1 may be a potential biomarker to predict platinum resistance and prognosis for EOC patients, exerting different roles on epithelial tumor and stromal cells.

HABP4 overexpression promotes apoptosis in goat turbinate bone cells

Hyaluronic acid-binding protein (HABP4) plays important roles in regulating cell cycle and apoptosis. However, its functions in regulating cell apoptosis remain unclear. To reveal the effects of HABP4 on cell proliferation, cell cycle and apoptosis, the HABP4 sequence was cloned, and we investigated the gain and loss functions of HABP4 in goat turbinate bone cells. Our results showed that a 1,496-bp HABP4 sequence was cloned successfully. The interference effect of siRNA1 on HABP4 was the strongest, reducing its mRNA expression level by 83%, decreasing the cells in the G0/G1 and S phases of the cell cycle and inhibiting cell growth and apoptosis. The overexpression of HABP4 produced contrasting results. Furthermore, an HABP4 knockdown caused the up-regulated expression of genes associated with apoptosis, including Bcl-2 and BCL2L11, but the down-regulation of Caspase3, Caspase7, Bax, PARP1, SOCS2 and P53 mRNA levels. Additionally, HABP4 overexpression significantly up-regulated the expression levels of Bax, Caspase3, Caspase7, BCL2L11, P53, SOCS2 and PARP1. However, the expression of Bcl-2 was down-regulated. These data provide an important foundation for further in-depth studies of HABP4 functions.

Role of DNMT3a in Hydroquinone-Induced Hematopoietic Stem Cell Toxicity

To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.

METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability.

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3'-untranslated Region (3'-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

Stat1 confers sensitivity to radiation in cervical cancer cells by controlling Parp1 levels: a new perspective for Parp1 inhibition

Cervical cancer (CC) is the fourth most common cause of cancer-related death in women. According to international guidelines, a standard treatment for locally advanced cervical cancer (LACC) consists of exclusive concurrent chemoradiation treatment (CRT). However, chemoradioresistance and subsequent relapse and metastasis of cancer occur in many patients, and survival for these women has generally remained poor. Therefore, strategies to overcome resistance are urgently needed. We have recently reported a radiosensitizing effect of the signal transducer and activator of transcription 1 (STAT1) in CC, associated with the control of [Poly(ADP-ribose) polymerase −1] PARP1 levels, a key factor in cell response to DNA damage induced by radiation. Here, we sought to decipher the underlying mechanism of STAT1-mediated control of PARP1, elucidating its role as a radiosensitizer in CC. Functional and molecular biology studies demonstrated that STAT1 may act at both transcriptional and posttranscriptional levels to modulate PARP1 expression in CC cells. In light of these results, we tested the effect of Olaparib in sensitizing CC cells to radiation and investigated signaling pathways involved in the activity observed. Results showed that PARP1 inhibition, at clinically achievable doses, may indeed selectively improve the sensitivity of resistant CC cells to DNA-damaging treatment. The translational relevance of our findings was supported by preliminary results in a limited patient cohort, confirming that higher PARP1 levels are significantly associated with a radioresistant phenotype. Finally, bioinformatics analysis of GEPIA and TCGA databases, demonstrated that PARP1 mRNA is higher in CC than in normal tissues and that increased PARP1 mRNA expression levels are associated with poor prognosis of LACC patients. Overall, our data open new opportunities for the development of personalized treatments in women diagnosed with CC.

Bone Marrow Levels of PARP1 mRNA Predict Response to Treatment with 5-Azacytidine in Patients with Myelodysplastic Syndrome

IntroductionPoly [ADP-ribose] polymerase 1 (PARP1) is the major member of a family of nuclear enzymes that participate in DNA repair and gene transcription, by recruiting various proteins involved in DNA repair to sites of DNA damage. PARP1 overexpression has been correlated to poor treatment response in children with acute lymphoblastic leukemia, while PARP1-driven apoptosis is important in patients with chronic lymphocytic leukemia. Moreover, PARP1 inhibitors have been tested in vitro in lymphoid malignancies, acute myeloid leukemia, myelodysplastic syndrome (MDS) and acute promyelocytic leukemia.We have recently shown that bone marrow PARP1 mRNA levels correlate to the prognosis of patients with MDS (Diamantopoulos et al. Blood Cancer 2017). Moreover, PARP1 has been related to hypermethylation through DNA methyltransferase-1 (DNMT1) gene upregulation and protein activation (Zampieri et al. PLoS One. 2009). This correlation with DNA methylation is very important, since hypermethylation is a major event in the pathogenesis of high risk MDS, and hypomethylating agents, such as 5-azacytidine, are the most effective available treatment option for patients with higher risk MDS.AimBased on the relation of PARP1 with hypermethylation, we investigated the correlation of bone marrow PARP1 mRNA levels of patients with MDS with treatment response to 5-azacytidine.MethodsBone marrow samples were collected from patients with MDS at diagnosis. Only samples from patients that were eventually treated with 5-azacytidine were selected for analysis. RNA extraction and reverse transcription were performed using standard protocols. Primers for PARP1 and beta-actin were designed using the primer3 software (University of Massachusetts, USA) and a SYBR green based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). PARP1 mRNA levels were expressed as a PARP1/beta-actin ratio. IBM SPSS statistics, version 23.0 (IBM Corporation, North Castle, NY, USA) was used for the analysis of the results.ResultsSixty-seven patients diagnosed with MDS per the WHO 2008 classification and treated with 5-azacytdine were included in the study. The basic characteristics of the patients are shown in Table 1. The median follow-up of the patients was 25.8 (2.6-117.0) months. Response to treatment with 5-azacytidine per the International Working Group (IWG) criteria is shown in Table 1. The median PARP1 mRNA level was 0.0793 (range (0.0000424 - 3.43). Response to treatment differed between patients with high and low PARP1 mRNA levels (Independent samples Kruskal-Wallis test, 2-sided p=0.009). More precisely, the median level of PARP1 mRNA was 4 times higher in responders (partial response, complete response, hematologic improvement) than in non-responders (failure and stable disease), Independent samples Mann-Whitney U test, 2-sided p=0.006. We found no correlation of any other of the studied factors (MDS type, IPSS, IPSS-R, WPSS, karyotype risk, number of cytopenias, marrow blast percentage, hemoglobin level, neutrophil and platelet count, dose reduction of 5-azacytidine) to response to treatment in this cohort of patients. Although there was no correlation of PARP1 mRNA levels with overall survival, survival of patients after 5-azacytidine treatment tended to be longer in patients with higher PARP1 mRNA levels (38.7 versus 19.8 months, log rank test, p=0.136).DiscussionWe found that patients with MDS and high PARP1 mRNA levels have a better response to treatment with 5-azacytidine. This correlation has a theoretical basis, since upregulation of PARP1 is correlated to DNA hypermethylation, and hypomethylating agents may be more effective in cells with higher methylation levels. Moreover, in our cohort of patients, PARP1 level was the only factor affecting treatment response, although this response was not translated into a survival benefit.PAPR1 is the only biological factor and one of very few identified factors to predict response to treatment with hypomethylating agents - low hemoglobin and platelet count were found to be an independent factor of poor response to hypomethylating agents (Jung et al. Oncotarget 2016), and absence of aberrant myeloid progenitor cells were found to predict response to treatment (Alhan et al. Cytometry B Clin Cytom 2014). A possible synergy between PARP1 inhibitors and hypomethylating agents in MDS should be further investigated. [Display omitted] DisclosuresVassilakopoulos:Amgen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Celgene/GenesisPharma: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Association of T2285C polymorphism in PARP1 gene coding region with its expression, activity and NSCLC risk along with prognosis.

Poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair gene, is the crucial player in the maintenance of genome integrity. T2285C polymorphism in coding region of PARP1 has been reported to be associated with susceptibility to tumours. We explored the relationship and mechanism of T2285C polymorphism of PARP1 to its expression and activity along with risk and prognosis in non-small cell lung cancer (NSCLC). mRNA expression was measured using quantitative RT-PCR assay or collected from TCGA dataset. Protein expression was examined with immunoblotting assay. Genotypes were determined by PCR-RFLP and sequencing approaches. PARP1 activity was determined with enzyme activity assay. Regulation of SIRT7 to PARP1 was determined by overexpression and small interference experiment. Association of PARP1 T2285C polymorphism with NSCLC risk was evaluated via multiple logistic regression analysis. Comparison of treatment response and progression-free survival (PFS) of NSCLC patients among different genotypes or regimens was made by chi-square test. Results indicated that mRNA and protein expression of PARP1 dramatically increased in NSCLC tissues in comparison with paired para-carcinoma tissues (P < 0.05). TC/CC mutant genotypes were associated with markedly enhanced PARP1 mRNA level compared with TT genotype (P = 0.011). No significant difference was discovered in PARP1 protein expression among TT, TC or CC genotypes (P > 0.05). Subjects with variant allele C had higher risk of NSCLC in comparison with allele T carriers [odds ratio = 1.560; P = 0.000]. NSCLC patients carrying mutational TC or CC genotypes were correlated with unfavourable response to platinum-based chemotherapy (TT vs. TC vs. CC, P = 0.010), and shorter PFS compared with TT genotype (TT vs. TC vs. CC, P = 0.009). T2285C mutation of PARP1 resulted in the enhancement of its mRNA, but the decrease of enzyme activity in tumour cell. Overexpression of SIRT7 attenuated PARP1 expression and activity. These findings suggest the variant allele C of T2285C polymorphism of PARP1 linked to an increase of NSCLC risk, and unfavourable efficacy and prognosis of NSCLC patients with platinum-based chemotherapy, which might be associated with enhancement of its mRNA expression and the diminishment of activity. Identification of PARP1 T2285C polymorphism and mRNA expression may be the promising way for the individualised treatment of NSCLC.

Low expression of TRAF3IP2-AS1 promotes progression of NONO-TFE3 translocation renal cell carcinoma by stimulating N6-methyladenosine of PARP1 mRNA and downregulating PTEN

BackgroundNONO-TFE3 translocation renal cell carcinoma (NONO-TFE3 tRCC) is one subtype of RCCs associated with Xp11.2 translocation/TFE3 gene fusions RCC (Xp11.2 tRCCs). Long non-coding RNA (lncRNA) has attracted great attention in cancer research. The function and mechanisms of TRAF3IP2 antisense RNA 1 (TRAF3IP2-AS1), a natural antisense lncRNA, in NONO-TFE3 tRCC remain poorly understood.MethodsFISH and qRT-PCR were undertaken to study the expression, localization and clinical significance of TRAF3IP2-AS1 in Xp11.2 tRCC tissues and cells. The functions of TRAF3IP2-AS1 in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay, Transwell assay and apoptosis analysis. The regulatory mechanisms among TRAF3IP2-AS1, PARP1, PTEN and miR-200a-3p/153-3p/141-3p were investigated by luciferase assay, RNA immunoprecipitation, Western blot and immunohistochemistry.ResultsThe expression of TRAF3IP2-AS1 was suppressed by NONO-TFE3 fusion in NONO-TFE3 tRCC tissues and cells. Overexpression of TRAF3IP2-AS1 inhibited the proliferation, migration and invasion of UOK109 cells which were derived from cancer tissue of patient with NONO-TFE3 tRCC. Mechanistic studies revealed that TRAF3IP2-AS1 accelerated the decay of PARP1 mRNA by direct binding and recruitment of N6-methyladenosie methyltransferase complex. Meanwhile, TRAF3IP2-AS1 competitively bound to miR-200a-3p/153-3p/141-3p and prevented those from decreasing the level of PTEN.ConclusionsTRAF3IP2-AS1 functions as a tumor suppressor in NONO-TFE3 tRCC progression and may serve as a novel target for NONO-TFE3 tRCC therapy. TRAF3IP2-AS1 expression has the potential to serve as a novel diagnostic and prognostic biomarker for NONO-TFE3 tRCC detection.