UHRF1 Research Articles

The dopamine receptor D1 inhibitor, SKF83566, suppresses GBM stemness and invasion through the DRD1-c-Myc-UHRF1 interactions.

Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

UHRF1-Mediated Epigenetic Reprogramming Regulates Glycolysis to Promote Progression of B-Cell Acute Lymphoblastic Leukemia

Background Acute lymphoblastic leukemia (ALL) is a hematologic malignant disease with high heterogeneity, including B-ALL and T-ALL. The 5-year overall survival rate of children ALL has been significantly improved, which is over 90%. However, the prognosis of adult patients is not satisfactory, and the 5-year OS is only 30%-50%. Among them, the prognosis of patients with relapsed/refractory B-ALL is very poor. Exploring the molecular mechanism of disease progression is of great significance to improve the outcome. Methods Application of 10× Genomics chromium sequencer reveals the single-cell transcriptome profiling of peripheral blood or bone marrow MNC cells, which were from 6 patients with B-ALL（3 new diagnosed patients and 3 relapsed patients） and 3 healthy donors. The expression of key genes in 84 clinical blood or bone marrow samples was detected, and the prognosis was analyzed in TARGET database by bioinformatics technology. The molecular mechanism was investigated by RNAseq, 850k methylation microarray, qRT-PCR, Western blot, BSP, CHIP-qPCR and flow cytometry. Additionally, we identified a UHRF1 inhibitor using virtual compounds screening, and demonstrated its therapeutic efficacy both in vitro and vivo. Results Using Seurat method to cluster and dimension reduction of scRNA-seq data, 29 subclusters were obtained from 9 clinical samples. It mainly includes eight progenitor B cells, one B cell, six T cells, two NK cells, three monocytes, two neutrophils and three primordial erythrocytes. Nine samples were divided into three groups: Newly Diagnosis group (ND), Relapsed/Refractory group (RR) and Healthy Donors group (HD). The proportion of three subpopulations (cluster 2,5,16) in progenitor B cell group showed a decreasing trend of RR group > ND group > HD group. The marker genes of these three subgroups were enriched by GO and KEGG using ClusterProfiler R package. Cluster 2 was enriched into cancer signal pathway, cluster 5 was enriched into DNA replication process, and the marker genes of cluster 16 was mainly involved in metabolic pathway. The differential expression of these three subgroups between RR group and ND group was analyzed by FindMarkers function. The results showed that cluster 16 was the main differential cluster. The intergroup differential genes of cluster 16 mainly involve metabolic pathway, cancer pathway, cell cycle and p53 signaling pathway (Figure 1). The three groups were compared in pairs. After the intersection of differential genes in cluster 16 and all progenitor B cell groups, four common differential genes (HSPB1, UHRF1, H1F0 and IGFBP2) were screened. The results showed that the expression of UHRF1 gene was up-regulated in acute lymphoblastic leukemia patients, especially in patients with relapsed/refractory. Moreover, UHRF1 overexpression is an independent prognostic risk factor for B-ALL patients in our clinical data, and it is also a molecular marker of poor prognosis in the TARGET dataset. In vitro experiments, knockout of gene UHRF1 promoted the cycle arrest and apoptosis of B-ALL tumor cell line (Reh and nalm6), inhibited the proliferation of cells, and improved the drug sensitivity of vindesine. RNAseq and methylation microarray showed that the gene SFRP5 was demethylated and the expression of mRNA was overexpressed in UHRF1 knockout cell lines. This was verified by subsequent qRT-PCR, Western Blot and Bisulfite sequencing. Further studies showed that UHRF1 was involved in glycolysis by regulating the SFRP5-p38 MAPK-HK2 signal axis. Subsequently, through overexpressing SFRP5, we confirmed that upregulating SFRP5 can inhibit the P38 MAPK-HK2 signaling pathway, thereby suppressing glycolytic levels. Moreover, virtual compound screening identified UM164 as an inhibitor of UHRF1, which significantly inhibits P38 protein phosphorylation, downregulates HK2 expression, and reduces the production of glycolysis-related metabolites. Furthermore, UM164 demonstrated the ability to inhibit the proliferation of B-ALL cells both in vitro and in vivo, leading to prolonged survival in a B-ALL mouse model. In conclusion, we have unraveled the molecular mechanisms of epigenetic and metabolic rearrangements in relapsed/refractory B-ALL, and have provided potential targeted therapeutic strategies to improve its unfavorable prognosis.

The transcription factor HBP1 promotes ferroptosis in tumor cells by regulating the UHRF1-CDO1 axis.

The induction of ferroptosis in tumor cells is one of the most important mechanisms by which tumor progression can be inhibited; however, the specific regulatory mechanism underlying ferroptosis remains unclear. In this study, we found that transcription factor HBP1 has a novel function of reducing the antioxidant capacity of tumor cells. We investigated the important role of HBP1 in ferroptosis. HBP1 down-regulates the protein levels of UHRF1 by inhibiting the expression of the UHRF1 gene at the transcriptional level. Reduced levels of UHRF1 have been shown to regulate the ferroptosis-related gene CDO1 by epigenetic mechanisms, thus up-regulating the level of CDO1 and increasing the sensitivity of hepatocellular carcinoma and cervical cancer cells to ferroptosis. On this basis, we constructed metal-polyphenol-network coated HBP1 nanoparticles by combining biological and nanotechnological. MPN-HBP1 nanoparticles entered tumor cells efficiently and innocuously, induced ferroptosis, and inhibited the malignant proliferation of tumors by regulating the HBP1-UHRF1-CDO1 axis. This study provides a new perspective for further research on the regulatory mechanism underlying ferroptosis and its potential role in tumor therapy.

The ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) gene promotes ovarian development in Exopalaemon carinicauda

Ridgetail white prawn, Exopalaemon carinicauda, with high reproductive capacity can reproduce up to three or four generations a year. In exploring the genetic factors affecting the gonadal development of E. carinicauda, we cloned and studied the function of the ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) gene. The open reading frame of uhrf1 gene was 2286 bp, encoding a total of 761 amino acids. The Uhrf1 has UBL, TTD, PhD, SRA, and ring finger domains that are associated with genome methylation, histone recognition and chromatin modification. According to the phylogenetic analysis, the amino acid sequence of Ec-uhrf1 has the highest similarity with Litopenaeus vannamei uhrf1, and it is a branch of Penaeus monodon and L. vannamei. The real-time quantitative PCR showed that the expression level of uhrf1 was highest in ridgetail white prawn ovarian tissue, which was significantly higher than that of other tissues (P < 0.05). In addition, the expression level gradually increased in stages I-IV of ovarian development, with its expression level in stage IV being significantly higher than that in other stages (P < 0.05). RNAi results showed that a knockdown of uhrf1 gene expression significantly reduced the expression of the cell proliferation-related gene pcna, the DNA methylation-related gene dnmt1 and yolk synthesis related genes vg, indicating that uhrf1 regulates the expression of pcna and dnmt1. The in situ hybridization tests further indicated that the uhrf1 gene plays an important role in oocyte proliferation and accumulation of exogenous nutrients.

Association of UHRF1 gene polymorphisms with oligospermia in Chinese males.

UHRF1 plays an important role in maintaining DNA methylation patterns during spermatogenesis. This study was performed to evaluate the association between UHRF1 gene variations and infertility in males with oligozoospermia in a Chinese population. In this case-control study of 735 Chinese men, single-nucleotide polymorphism (SNP) genotypes and alleles in the UHRF1 gene were assessed by direct sequencing. The effects of the mutations on UHRF1 transcription were investigated using a dual-luciferase reporter gene assay. We identified 24 SNPs, including nine SNPs in the promoter region, three in the 5' untranslated region, five in introns, and seven in exons. Interestingly, the genotype frequencies of SNP rs2656927 (P = 0.014) and rs8103849 (P < 0.001) significantly differed between men with oligozoospermia in case group 1 and normozoospermic men. Moreover, four variants (three were novel) were detected only in the patient group, with two in introns and the others in the promoter region. The results of the luciferase assay showed that the -1615C>T-C and -1562A>G-A alleles increased luciferase activity compared with the -1615C>T-T and -1562A>G-G alleles. We detected two SNPs in the UHRF1 gene showing a significant difference between the case and control groups. Two screened SNPs affected UHRF1 promoter activity, improving the understanding of the pathophysiology of oligozoospermia.

MiR-506 Targets UHRF1 to Inhibit Colorectal Cancer Proliferation and Invasion via the KISS1/PI3K/NF-κB Signaling Axis.

BackgroundThe UHRF1 gene is an epigenetic modification factor that mediates tumor suppressor gene silencing in a variety of cancers. Related studies have reported that UHRF1 can inhibit the expression of the KISS1 gene. However, the regulatory mechanism underlying UHRF1 expression in colorectal cancer (CRC) is still unclear. The aim of this study was to gain a better understanding of the regulation of UHRF1 expression in CRC and to determine whether it regulates the mechanism by which KISS1 promotes CRC metastasis.MethodsIn the present study, the levels of miR-506, UHRF1 and KISS1 expression in CRC tissues and in human CRC cell lines were studied using quantitative real-time PCR (qRT-PCR) and Western blotting. Cell proliferation, migration, and invasion assays are used to detect cell proliferation, migration, and invasion. A dual-luciferase reporter system was used to confirm the target gene of miR-506.ResultsThis study found that UHRF1 protein is highly expressed in CRC tissues and negatively correlated with KISS1 protein expression. UHRF1 overexpression activates the PI3K/NF-κB signaling pathway by inhibiting the mRNA expression levels of pathway mediators. Bioinformatics analysis and luciferase reporter gene assays confirmed that miR-506 targets UHRF1.ConclusionThis study identified the regulation of UHRF1 expression in CRC and the mechanism of CRC metastasis. UHRF1 may be a new potential target molecule for future CRC metastasis treatment.

Abstract 2479: A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines

Abstract DNA methylation has a powerful influence on cellular processes. It plays important roles in cancer initiation and progression. Cancer cells exhibit frequent epigenomic alterations that affect expression of genes involved in apoptosis, cell proliferation, and DNA repair. We used expression information of known methylating and demethylating genes in cancer cell lines to analyze their association with methylation levels of individual probes and median epigenome-wide methylation levels. We utilized RNA-seq gene expression measures and Illumina Infinium HumanMethylation450 BeadChip DNA methylation data of 645 cancer cell lines from 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer resources. We analyzed 73 genes, products of which are involved in methylation and demethylation processes, and examined correlations between expression levels of these genes and methylation status of individual probes and epigenome-wide. In a combined analysis across all cancer types, we observed statistically significant correlations between expression of global DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, and probe methylation levels in 566, 909, and 230 genes, respectively. In particular, DNMT3A expression was associated with methylation status of growth factor and receptor genes FGF1, FGFR4, and EGFR. Expression of hydroxymethylating genes TET1, TET2, and TET3 was correlated with methylation status of probes in 363, 1, and 1055 genes, respectively, including associations of TET3 expression with methylation of probes in the FGF1, KIT, FHIT, IGF1R, IGF2BP2, and PARP3 genes, which may affect growth, proliferation, and DNA repair of tumor cells. APOBEC1, CBX2, UHRF1 and ZBTB38, products of which are involved in various molecular steps of DNA methylation or demethylation, had the largest numbers of associations in different cancer types. For example, in chronic lymphocytic leukemia cell lines we observed a strong negative correlation between ZBTB38 expression, the product of which binds methylated DNA, and methylation levels of probes in 204 genes. We also found a strong correlation between UHRF1 gene expression and methylation levels of probes in 150 genes in chronic lymphocytic leukemia cell lines, in agreement with reported roles of the product of this gene in binding DNA methyltransferases and affecting DNMT1 methylation activity. These results provide a better understanding of molecular mechanisms of epigenetic dysregulation in cancer cells and may provide strategies for future analysis of molecular targets of drug response that may be affected via epigenetic processes. Citation Format: Suleyman Vural, Julia Krushkal. A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2479.

Role of UHRF1 in malignancy and its function as a therapeutic target for molecular docking towards the SRA domain.

The figure describes the location of UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1) gene, mRNA and protein synthesis in the tumor cell and its structural domains with a focus on the docking of Naphthazarin on the SRA domain of UHRF1, resulting in reduction of tumor size.

Silencing UHRF1 Inhibits Cell Proliferation and Promotes Cell Apoptosis in Retinoblastoma Via the PI3K/Akt Signalling Pathway.

This study aimed to investigate the effect of silencing ubiquitin-like with PHD and RING finger domains 1 (UHRF1) on the proliferation and apoptosis of retinoblastoma (RB) cells and to clarify the molecular mechanism of the UHRF1 gene in the development of RB. Human RB WERI-Rb-1 cells were selected and assigned into a blank group (WERI-Rb-1 cells with no transfection), NC-shRNA group (WERI-Rb-1 cells infected with NC-shRNA virus) and UHRF1-shRNA group (WERI-Rb-1 cells infected with pGC-UHRF1-shRNA-LV-GFP# (39-1) virus). The mRNA and protein expression of UHRF1 was detected by RT-qPCR and Western blot analysis. The effect of silencing UHRF1 on the proliferation and apoptosis of WERI-Rb-1 cells was assessed by MTT assay, EdU assay, flow cytometry, and Hoechst staining. Furthermore, the expression of cell cycle-related factor (cyclin D1), apoptosis-related factors (caspase-9, Bcl-2 and Bax), and PI3K/Akt signalling pathway-related factors (p-PI3K, PI3K, p-Akt and Akt) were measured via Western blot analysis. The RNA interference plasmid UHRF1-shRNA was successfully constructed. After WERI-Rb-1 cells were infected with UHRF1-shRNA, decreased mRNA and protein expression of UHRF1 was found. WERI-Rb-1 cells infected with UHRF1-shRNA showed inhibited proliferative ability and increased apoptosis. In the UHRF1-shRNA group, more cells arrested at the G0/G1 phase and less cells at the S and G2/M phases. WERI-Rb-1 cells infected with UHRF1-shRNA had increased expression of caspase-9 and Bax and decreased expression of Bcl-2 expression and decreased levels of p-PI3K and p-Akt. In conclusion, our study demonstrated that silencing UHRF1 could inhibit the proliferation of RB cells and promote apoptosis. The mechanism may be caused by the downregulation of the proportion of Bcl-2/Bax expression and the promotion of the expression of caspase-9 through the PI3K/Akt signalling pathway.

Abstract A41: Role of UHRF1 in tumorigenesis and metastasis of osteosarcoma

Abstract Osteosarcoma is the eighth most common form of childhood cancer, with very limited advances in treatment over the past three decades. Osteosarcoma is a highly metastatic cancer, with 15-20% of osteosarcoma patients diagnosed after the cancer has already metastasized, causing the survival rate to be little over 50%. Common sites of metastasis include lung and liver. Loss of the retinoblastoma protein (RB) is associated with poor prognosis in osteosarcoma, as defined by shorter life span and heavier tumor burden. Ubiquitin Like with PHD and Ring Finger Domains 1 (UHRF1) has been identified as a critical downstream effector of the RB/E2F pathway that is overexpressed in various cancers. We have confirmed using human mesenchymal stem cells (MSCs) and osteosarcoma cell lines that UHRF1 is directly regulated by the RB/E2F pathway in the bone lineage, and found that the UHRF1 gene is upregulated and its protein overexpressed in osteosarcoma. Using gain/loss-of-function assays to study the role of UHRF1 in osteosarcoma, we observed that UHRF1 is involved in promoting cell migration. To study the role of UHRF1 in osteosarcoma tumor development, we generated Uhrf1 conditional knockout mice. In this system, we use an Osterix-driven Cre recombinase (Osx-cre) to facilitate preosteoblast-specific loss of various combinations of genes. Osx-cre Tp53; Rb1 double knockout mice give rise to osteosarcoma and have a median survival of 26.7 weeks. Additional loss of Uhrf1 in Osx-cre Tp53; Rb1 double knockout mice (Osx-cre Tp53; Rb1; Uhrf1 triple knockout mice) significantly delays the age of tumor onset to &amp;gt;47 weeks. Furthermore, triple knockout mice have a decreased frequency and number of lung and liver metastasis. In conclusion, we have strong evidences both in vitro and in vivo that UHRF1 is a crucial component in aiding osteosarcoma progression, and have identified its novel role in tumor metastasis. The high metastatic rate and low 5-year survival rate following metastasis of osteosarcoma make UHRF1 a promising target for therapeutics. Further studies on the mechanism by which UHRF1 overexpression contributes to osteosarcomagenesis, including tumor growth and dissemination, will strongly aid the process of drug development for anticancer treatment. Citation Format: Stephanie C. Wu, Claudia A. Benavente. Role of UHRF1 in tumorigenesis and metastasis of osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A41.

Evaluation of UHRF1 and P16INK4A expression levels in newly diagnosed AML patients

Introduction: Gene mutation is an infrequent cause of tumor suppressor gene (TSG) defect in de novo AML patients. Instead, it seems that leukemic cells employ epigenetic tricks to attenuate the negative impacts of intact TSGs. Ordinarily, critical TSGs, such as p16INK4A, is hyper-methylated in AML blasts under the impact of master epigenetic regulators, such as UHRF1. In this study, we investigated the correlation between UHRF1 and p16INK4A gene expression levels in newly diagnosed AML patients.&#x0D; Methods: Bone marrow and peripheral blood samples were obtained from 50 newly diagnosed AML patients and 18 healthy normal control subjects. Gene expression levels of UHRF1 and P16INK4A were surveyed using SYBR Green Quantitative Real-time PCR. Statistical analyses were done using SPSS statistical software 21.0.&#x0D; Results: P16INK4A gene expression showed reduced levels in 80.64% of patients above 45 years of age, while only 32% of patients below 45 years had reduced expression levels. The Spearman correlation test also demonstrated a significant negative correlation between UHRF1 and p16INK4A gene expression levels in AML patients, which was not observed in the control group (r=0.343 and P= 0.015).&#x0D; Conclusion: Regarding the age-related patterns of UHRF1 and p16INK4A gene expression, and also the presence of negative correlation between them, we conclude that UHRF1 may potentially be involved in p16INK4A down-regulation in elderly AML patients, which may subsequently facilitate the progression of AML in older ages.&#x0D;

Expression of ubiquitin-like with PHD and ring finger domains 1 and its clinical significance in hepatocellular carcinoma

Objective To investigate the expression of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in hepatocellular carcinoma (HCC) and its relationship with the prognosis of patients with HCC. Methods Immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of UHRF1 in 50 HCC tissues, and the relationship between the level of UHRF1 expression and the prognosis of patients with HCC was analyzed. Results The expression of UHRF1 mRNA in cancerous tissues was upregulated in 33 patients with HCC compared with para-cancerous tissues (P=0.001), the positive expression rate of UHRF1 protein in cancerous tissues was also higher than that in para-cancerous tissues of 32 patients with HCC (64% vs. 22%, P=0.000), the expression level of UHRF1 were correlated with tumor size, TNM stage, tumor metastasis and relapse, and patients with HCC tended to have a worse prognosis. Conclusion Up-regulated expression of UHRF1 predicts poor prognosis of patients with HCC, and UHRF1 may be a good biomarker for evaluating the prognosis of patients with HCC. Key words: Hepatocellular carcinoma; Ubiquitin-like with PHD and ring finger domains 1; Expression; Prognosis

FOXM1 contributes to taxane resistance by regulating UHRF1-controlled cancer cell stemness

Therapy-induced expansion of cancer stem cells (CSCs) has been identified as one of the most critical factors contributing to therapeutic resistance, but the mechanisms of this adaptation are not fully understood. UHRF1 is a key epigenetic regulator responsible for therapeutic resistance, and controls the self-renewal of stem cells. In the present study, taxane-resistant cancer cells were established and stem-like cancer cells were expanded. UHRF1 was overexpressed in the taxane-resistant cancer cells, which maintained CSC characteristics. UHRF1 depletion overcame taxane resistance in vitro and in vivo. Additionally, FOXM1 has been reported to play a role in therapeutic resistance and the self-renewal of CSCs. FOXM1 and UHRF1 are highly correlated in prostate cancer tissues and cells, FOXM1 regulates CSCs by regulating uhrf1 gene transcription in an E2F-independent manner, and FOXM1 protein directly binds to the FKH motifs at the uhrf1 gene promoter. This present study clarified a novel mechanism by which FOXM1 controls CSCs and taxane resistance through a UHRF1-mediated signaling pathway, and validated FOXM1 and UHRF1 as two potential therapeutic targets to overcome taxane resistance.

Expression of UHRF1 protein in breast cancer tissue and its correlation with local recurrence

Objective To investigate the expression of UHRF1 protein in breast cancer and adjacent normal tissues, and its relationship with local recurrence. Methods Immunohistochemistry (IHC) was applied to detect the expression of UHRF1 protein in 69 specimens of breast cancer and 33 specimens of corresponding adjacent normal tissues; Chi-square test was used to analyze the relationship between UHRF1 protein expression and clinical factors. Results The positive rate expression of UHRF1 protein in breast cancer was 55.1% (38/69), and UHRF1 protein expression was not found in adjacent normal tissues. The positive expression rate of UHRF1 protein in stage III was higher than that in stageⅠ-Ⅱ [68.4% (26/38) vs. 38.7% (12/31), P 0.05). Conclusions The positive expression rate of UHRF1 protein is obviously higher than that in adjacent normal tissue. Besides, UHRF1 protein is related to the stage and chest wall of local recurrence after radiotherapy. Key words: Breast neoplasms; Radiation tolerance; UHRF1 protein

Expression of ubiquitin-like with PHD and ring finger domain 1 in breast cancer and its significance

Objective To observe the expression and significance of ubiquitin-like with PHD and ring finger domain 1 (UHRF1) in breast cancer and adjacent normal tissue. Methods The expression level of UHRF1 mRNA and protein was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Relationships between UHRF1 protein positive expression and clinicopathological characteristics were analyzed. Results The UHRF1 mRNA expression level in breast cancer (1.34±0.03) was higher than in adjacent normal tissue (0.61±0.03). And the UHRF1 protein expression level in breast cancer was also higher than in adjacent normal tissue. The UHRF1 protein positive expression in breast cancer tissue was significantly correlated with clinical stages and lymphatic metastasis. Conclusion UHRF1 protein positive expression is correlated to the occurrence of breast cancer. Key words: Breast cancer; Ubiquitin-like with PHD and ring finger domain 1

Overexpression of UHRF1 gene correlates with the major clinicopathological parameters in urinary bladder cancer.

ABSTRACTIntroduction Recently, expression of the UHRF1 gene was found to be up-regulated in numerous neoplasms, including the urinary bladder transitional cell carcinoma (TCC).Objective The aim of our study was to determine if the expression levels of UHRF1 gene correlates with the major pathological characteristics of the tumor and patients’ clinical outcome.Materials and Methods In our study, we have analyzed the tissue samples derived from group of 70 patients with histologically confirmed TCC of the urinary bladder, while normal urinary bladder mucosa obtained from 40 patients with nonmalignant diseases was used as a negative control group. Expression of UHRF1 gene in each patient sample was determined using reverse transcriptase-polymerase chain reaction.Results UHRF1 gene expression was found to be app. 2.5 times higher in samples from patients with TCC in comparison with normal epithelium derived from control group patients. Analysis show that gene expression correlates with the malignancy of the tumor. A highly significant differences were found between the expression values of samples from low and high grade TCC, as well as between the high grade and control group. UHRF1 expression was higher in patients with non-muscle invasive disease than in those with muscle invasive disease.Conclusions The result of this study indicates that UHRF1 gene expression levels correlates with the major pathological characteristics of TCC samples and with the clinical outcome of those patients. Determination of UHRF1 gene expression could have a potential to be used as a sensitive molecular marker in patients with urinary bladder cancer.

Effect of dihydroartemisinin on UHRF1 gene expression in human prostate cancer PC-3 cells.

As the second most common cancer in men around the world, prostate cancer is increasingly gaining more attention. Dihydroartemisinin (DHA) has been proven to be a promising anticancer agent in vitro as well as in vivo in accumulating data. However, the detailed mechanisms of how DHA action in human prostate cancer PC-3 cells remain elusive. This study aimed to investigate the effects of DHA, a novel anticancer agent, by inhibiting the expression of ubiquitin like containing PHD and ring finger 1 (UHRF1) in PC-3 cells. The apoptosis and cell-cycle distribution were detected by flow cytometry. Quantitative real-time PCR was performed to examine both UHRF1 and DNA methyltransferase 1 (DNMT1) expressions at mRNA levels, whereas the expressions of UHRF1, DNMT1, and p16 proteins at protein levels were detected by Western blotting. Methylation levels of p16 CpG islands were determined by bisulfite genomic sequencing. We showed that DHA induced the downregulation of UHRF1 and DNMT1, accompanied by an upregulation of p16 in PC-3 cells. Decreased p16 promoter methylation levels in DHA-treated groups were also observed in PC-3 cells. Furthermore, DHA significantly induced apoptosis and G1/S cell-cycle arrest in PC-3 cells. Our results suggested that downregulation of UHRF1/DNMT1 is upstream to many cellular events, including G1 cell arrest, demethylation of p16, and apoptosis. Together, our study provides new evidence that DHA may serve as a potential therapeutic agent in the treatment of prostate cancer.

Abstract P1-08-11: UHRF1 promotes breast cancer progression via suppressing KLF17 expression

Abstract Background: UHRF1, also termed as ICBP90 or Np95, is reported to be an epigenetic regulator of DNA methylation and histone modifications, which takes pivotal functions in cell differentiation and tumorigenesis. Here, we show that UHRF1 is markedly increased in breast cancer tissues and cell lines. And patients with high UHRF1 expression have a poor prognosis compare with those in low UHRF1 expression as showed by Kaplan Meier plotter analysis. However, the underlying mechanisms of UHRF1 in breast cancer remain largely unknown. Thus, the aim of this study is to validate potential mechanisms of UHRF1 in breast cancer cells. Methods: The stable cell lines were constructed using interference and overexpression methods and the efficiency was confirmed by western blot. Then cell proliferation and migration were evaluated by CCK8 and transwell assay. The MDA-MB-231 pCDH and pCDH-UHRF1 cells were inoculate into mouse left flank to further testify UHRF1 function in vivo. Furthermore, microarray and ChIP-seq were implemented to detect potential mechanisms and the candidate genes were assessed by ChIP and qPCR. The BSP or MSP primers were designed by the methprimer database. Genomic DNA was prepared from breast cancer cells and then the genomic DNA was bisulfite modified using EZ DNA Methylation-Gold Kit. The methylation PCR products were cloned into pMD-18T and ten positive clones were sequenced. The data were analyzed using the QUMA analyzer software. Results: We found that UHRF1 was indeed increased in breast cancer tissues and cells. And patients with high levels of UHRF1 were likely to have a shorter disease-free survival than patients with low levels. UHRF1 overexpression can promote cell proliferation and migration while UHRF1 downregulation have inverse functions. In vivo, UHRF1 also accelerate tumor growth on mice. Mechanistic studies revealed that 9 candidate genes were obtained by analysis from ChIP-seq and microarray databases. The further verification showed that Krüppel-like factor 17 (KLF17) is the most significantly upregulated one when UHRF1 gene was depleted, with rich CpG islands on its promoter region. We also observed that an inverse relationship between the expression of UHRF1 and KLF17 both in breast cancer cell lines and tissues.What's more, cells proliferation and migration which were inhibited by UHRF1-depletion can be rescued by KLF17 silence, suggesting UHRF1 promote breast cancer proliferation and migration through downregulating KLF17 expression. Moreover, overexpression of UHRF1 increases the methylation of CpG nucleotides and reduces the expression of KLF17 while depletion of UHRF1 decreases the methylation of CpG nucleotides with elevating expression of KLF17. Conclusion: Our results demonstrate that increased UHRF1 can promote breast cancer cell proliferation and migration by epigenetic silencing of KLF17 expression through CpG islands methylation in its promoter region. These results provide insight into the breast cancer progression process and suggest that making changes in this mechanism represent new therapeutic approaches to block breast cancer development. Citation Format: Gao S-P, Sun H-F, Li L-D, Jin W. UHRF1 promotes breast cancer progression via suppressing KLF17 expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-11.

UHRF1 gene silencing inhibits cell proliferation and promotes cell apoptosis in human cervical squamous cell carcinoma CaSki cells

BackgroundUp-regulation of UHRF1 has been observed in a variety of cancers and appears to serve as an independent prognostic factor.ObjectiveTo explore the effect of UHRF1 gene silencing on apoptosis and proliferation of cervical squamous cell carcinoma (CSCC) CaSki cells.MethodsThis study consisted of 47 CSCC tissues and 40 normal cervical tissues. The CaSki cells were assigned into Blank group (CaSki cells not transfected), NC group (CaSki cells transfected with control siRNA), and UHRF1 Silence group (CaSki cells transfected with UHRF1 siRNA). qRT-PCR and Western blot were used for UHRF1 mRNA and protein expressions, CKK-8 assay for cell proliferation, flow cytometry for cell cycle and apoptosis, Western blot for expressions of apoptosis-related proteins. Nude mice tumor transplant experiment was performed.ResultsUHRF1 exhibited higher mRNA and protein expressions in the CSCC tissues than normal cervical tissues (both P < 0.05). The cell proliferation ability in the UHRF1 Silence group was reduced when compared with the Blank group and the NC group, the cells at S-G2M stage in the UHRF1 Silence group were dropped when compared with the Blank group and the NC group (P < 0.05), while the cells at G0/G1 stage were elevated (P < 0.05), and the proportion of Annexin V positive cells in the UHRF1 Silence group was increased in comparison with the Blank group and the NC group (P < 0.05). Nude mice tumor transplant experiment indicated that the growth rate and weight of tumor in the Blank group and NC group was higher and heavier than the UHRF1 Silence group (P < 0.05).ConclusionUHRF1 showed a high expression in CSCC and UHRF1 silencing can reduce proliferation and enhance apoptosis of the CaSki cells.

Expressions of UHRF1, P53 and their clinical significances in colon carcinoma

Objective To investigate the expression levels and clinical significances of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) and P53 in colon carcinoma. Methods The expressions of UHRF1 and P53 in 70 colon cancer tissues and 30 normal colon ones were detected by means of immunohistochemistry to analyze the correlation of these two proteins in the occurrence and development of colon carcinoma, and the relationship between their expressions and clinico-pathological factors as well as prognosis was discussed. Results The expression levels of UHRF1 and P53 in cancer tissues were significantly higher than those of cancer-adjacent tissues (87.1% vs. 56.7%, χ2=11.366, P=0.001; 64.3% vs. 6.7%, χ2=27.988, P=0.000) and showed a positive correlation between the two proteins (r=0.248, P=0.038). Both UHRF1 and P53 were associated with TNM stages (χ2=4.426, P=0.049; χ2=6.000, P=0.016) and the depth of invasion (χ2=12.553, P=0.002; χ2=4.904, P=0.036). However, they were irrelevant with the age (χ2=0.473, P=0.494; χ2=0.090, P=0.799) and gender (χ2=2.297, P=0.166; χ2=0.512, P=0.617) of patients, as well as the size (χ2=0.638, P=0.481; χ2=2.392, P=0.215) and histological grading of tumors (χ2=2.088, P=0.352; χ2=0.303, P=0.859). Kaplan-Meier analysis showed that the median survival time of patients with UHRF1, P53 positive expression was 21.83 months that was lower than patients with UHRF1 positive expression of 24.49 months (Z=-0.624, P=0.533). Both former of which was distinctly lower than that of negative ones of 37.33 months (Z=-2.856, P=0.004; Z=-2.694, P=0.007). Conclusion Both UHRF1 and P53 are highly expressed in colon cancer tissues, which imply that UHRF1 and P53 may be strongly related with colon cancer development and can be a predictor for colon cancer prognosis. Key words: Colon neoplasms; Tumor suppressor protein p53; Ubiquitin-like with PHD and ring finger domains 1