A mitochondria-related genes associated neuroblastoma signature - based on bulk and single-cell transcriptome sequencing data analysis, and experimental validation.

Background: Neuroblastoma (NB), characterized by its marked heterogeneity, is the most common extracranial solid tumor in children. The status and functionality of mitochondria are crucial in regulating NB cell behavior. While the significance of mitochondria-related genes (MRGs) in NB is still missing in key knowledge. Materials and methods: This study leverages consensus clustering and machine learning algorithms to construct and validate an MRGs-related signature in NB. Single-cell data analysis and experimental validation were employed to characterize the pivotal role of FEN1 within NB cells. Results: MRGs facilitated the classification of NB patients into 2 distinct clusters with considerable differences. The constructed MRGs-related signature and its quantitative indicators, mtScore and mtRisk, effectively characterize the MRGs-related patient clusters. Notably, the MRGs-related signature outperformed MYCN in predicting NB patient prognosis and was adept at representing the tumor microenvironment (TME), tumor cell stemness, and sensitivity to the chemotherapeutic agents Cisplatin, Topotecan, and Irinotecan. FEN1, identified as the most contributory gene within the MRGs-related signature, was found to play a crucial role in the communication between NB cells and the TME, and in the developmental trajectory of NB cells. Experimental validations confirmed FEN1's significant influence on NB cell proliferation, apoptosis, cell cycle, and invasiveness. Conclusion: The MRGs-related signature developed in this study offers a novel predictive tool for assessing NB patient prognosis, immune infiltration, stemness, and chemotherapeutic sensitivity. Our findings unveil the critical function of FEN1 in NB, suggesting its potential as a therapeutic target.

The endonuclease FEN1 mediates activation of STAT3 and facilitates proliferation and metastasis in breast cancer.

BACKGROUND: The metastasis accounts for most deaths from breast cancer (BRCA). Understanding the molecular mechanisms of BRCA metastasis is urgently demanded. Flap Endonuclease 1 (FEN1), a pivotal factor in DNA metabolic pathways, contributes to tumor growth and drug resistance, however, little is known about the role of FEN1 in BRCA metastasis. METHODS AND RESULTS: In this study, FEN1 expression and its clinical correlation in BRCA were investigated using bioinformatics, showing being upregulated in BRCA samples and significant relationships with tumor stage, node metastasis, and prognosis. Immunohistochemistry (IHC) staining of local BRCA cohort indicated that the ratio of high FEN1 expression in metastatic BRCA tissues rose over that in non-metastatic tissues. The assays of loss-of-function and gain-of-function showed that FEN1 enhanced BRCA cell proliferation, migration, invasion, xenograft growth as well as lung metastasis. It was further found that FEN1 promoted the aggressive behaviors of BRCA cells via Signal Transducer and Activator of Transcription 3 (STAT3) activation. Specifically, the STAT3 inhibitor Stattic thwarted the FEN1-induced enhancement of migration and invasion, while the activator IL-6 rescued the decreased migration and invasion caused by FEN1 knockdown. Additionally, overexpression of FEN1 rescued the inhibitory effect of nuclear factor-κB (NF-κB) inhibitor BAY117082 on phosphorylated STAT3. Simultaneously, the knockdown of FEN1 attenuated the phosphorylation of STAT3 promoted by the NF-κB activator tumor necrosis factor α (TNF-α). CONCLUSIONS: These results indicate a novel mechanism that NF-κB-driven FEN1 contributes to promoting BRCA growth and metastasis by STAT3 activation.

FEN1 Inhibition as a Potential Novel Targeted Therapy against Breast Cancer and the Prognostic Relevance of FEN1.

To improve breast cancer treatment and to enable new strategies for therapeutic resistance, therapeutic targets are constantly being studied. Potential targets are proteins of DNA repair and replication and genomic integrity, such as Flap Endonuclease 1 (FEN1). This study investigated the effects of FEN1 inhibitor FEN1-IN-4 in combination with ionizing radiation on cell death, clonogenic survival, the cell cycle, senescence, doubling time, DNA double-strand breaks and micronuclei in breast cancer cells, breast cells and healthy skin fibroblasts. Furthermore, the variation in the baseline FEN1 level and its influence on treatment prognosis was investigated. The cell lines show specific response patterns in the aspects studied and have heterogeneous baseline FEN1 levels. FEN1-IN-4 has cytotoxic, cytostatic and radiosensitizing effects, expressed through increasing cell death by apoptosis and necrosis, G2M share, senescence, double-strand breaks and a reduced survival fraction. Nevertheless, some cells are less affected by the cytotoxicity and fibroblasts show a rather limited response. In vivo, high FEN1 mRNA expression worsens the prognosis of breast cancer patients. Due to the increased expression in breast cancer tissue, FEN1 could represent a new tumor and prognosis marker and FEN1-IN-4 may serve as a new potent agent in personalized medicine and targeted breast cancer therapy.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair.

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

FEN1 promotes cancer progression of cholangiocarcinoma by regulating the Wnt/ß-catenin signaling pathway.

PURPOSE: Cholangiocarcinoma (CHOL) comprises a cluster of highly heterogeneous malignant biliary tumors. Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. This study aimed to explore the biological functions and mechanisms of FEN1 in CHOL. METHODS: FEN1 expression was analyzed in tissues of patients with CHOL and FEN1 mutations. We observe the influence of FEN1 on cellular proliferation, migration, and invasion, as well as on DNA damage repair and glycolysis. Western blotting was performed to determine the regulatory mechanism of FEN1 in CHOL progression. RESULTS: FEN1 was highly expressed in the cancer tissues of CHOL patients. The high mutation rate of FEN1 in CHOL tissues was mainly due to the amplified repeats. FEN1 promotes the proliferation, migration, and invasion of HUCCT1 and QBC939 cells. In addition, FEN1 induced DNA damage repair and aerobic glycolysis in CHOL cells. FEN1 also promoted xenograft tumor growth in vivo. Moreover, we showed that FEN1 mediated the epithelial-mesenchymal transition (EMT) of CHOL. FEN1-mediated EMT was found to be transduced by the Wnt/ß-catenin signaling pathway. CONCLUSION: FEN1 was significantly overexpressed in CHOL tissues, and FEN1 regulates the progression of CHOL through the Wnt/ß-catenin signaling pathway.

FEN1 inhibitor SC13 promotes CAR-T cells infiltration into solid tumours through cGAS-STING signalling pathway.

It is well known that chimeric antigen receptor T-cell immunotherapy (CAR-T-cell immunotherapy) has excellent therapeutic effect in haematological tumours, but it still faces great challenges in solid tumours, including inefficient T-cell tumour infiltration and poor functional persistence. Flap structure-specific endonuclease 1 (FEN1), highly expressed in a variety of cancer cells, plays an important role in both DNA replication and repair. Previous studies have reported that FEN1 inhibition is an effective strategy for cancer treatment. Therefore, we hypothesized whether FEN1 inhibitors combined with CAR-T-cell immunotherapy would have a stronger killing effect on solid tumours. The results showed that low dose of FEN1 inhibitors SC13 could induce an increase of double-stranded broken DNA (dsDNA) in the cytoplasm. Cytosolic dsDNA can activate the cyclic GMP-AMP synthase-stimulator of interferon gene signalling pathway and increase the secretion of chemokines. In vivo, under the action of FEN1 inhibitor SC13, more chemokines were produced at solid tumour sites, which promoted the infiltration of CAR-T cells and improved anti-tumour immunity. These findings suggest that FEN1 inhibitors could enable CAR-T cells to overcome poor T-cell infiltration and improve the treatment of solid tumours.

Inhibition of FEN1 promotes DNA damage and enhances chemotherapeutic response in prostate cancer cells.

Prostate cancer (PCa) refers to epithelial malignancies occurring in prostate and is the most commonly diagnosed cancer among men. Flap structure-specific endonuclease 1 (FEN1) is one of the major base excise repair enzymes and is abnormally expressed in a variety of cancers, which contributes to cancer progression. Targeting FEN1 serves as a potent strategy for cancer therapy. However, how FEN1 acts on PCa cell proliferation and its role in chemotherapeutic response remain largely unknown. In this study, we show that knockdown of FEN1 by CRISPR/Cas9 system impedes the proliferation and migration of PCa cells. FEN1 Inhibitor SC13 induced DNA damage accumulation and further resulted in apoptosis of PCa cells. Furthermore, genetic knockdown of FEN1 or inhibition of FEN1 by SC13 promoted DNA damage and enhanced docetaxel (DTX)-induced chemotherapeutic response in PCa cells. Collectively, these findings demonstrate the importance of FEN1 in PCa cell proliferation and implicate FEN1 as a promising target for monotherapy or combination therapeutic strategy in PCa treatment.

Identification of VPS34-PI(3)P-FEN1-mediated DNA repair pathway as a potential drug target to overcome chemoresistance.

Intrinsic or acquired chemoresistance represents a major obstacle in cancer treatment. Multiple mechanisms can contribute to cancer cells' resistance to chemotherapy. Among them, an aberrantly strengthened DNA repair mechanism is responsible for a large proportion of drug resistance to alkylating agents and radiation therapy. In cancer cells, damping overactivated DNA repair system can overcome survival advantages conferred by chromosomal translocations or mutations and lead to cytostatic effects or cytotoxic. Therefore, selectively targeting DNA repair system in cancer cells holds promise for overcoming chemoresistance. In this study, we revealed that the endonuclease Flap Endonuclease 1 (FEN1), essential for DNA replication and repair, directly interacts with phosphatidylinositol 3-phosphate [PI(3)P], and FEN1-R378 is the primary PI(3)P-binding site. PI(3)P-binding deficient FEN1 mutant (FEN1-R378A) cells exhibited abnormal chromosomal structures and were hypersensitized to DNA damage. The PI(3)P-mediated FEN1 functionality was essential for repairing DNA damages caused by multiple mechanisms. Furthermore, VPS34, the major PI(3)P synthesizing enzyme, was negatively associated with patients' survival in various cancer types, and VPS34 inhibitors significantly sensitized chemoresistant cancer cells to genotoxic agents. These findings open up an avenue for counteracting chemoresistance by targeting VPS34-PI(3)P-mediated DNA repair pathway, and call for assessing the efficacy of this strategy in patients suffering from chemoresistance-mediated cancer recurrence in clinical trials.

Label-free and low-background FEN1 sensing based on cleavage-induced ligation of bifunctional dumbbell DNA and in-situ signal readout.

Flap endonuclease 1 (FEN1) is overexpressed in various types of human tumor cells and has been recognized as a promising biomarker for cancer diagnosis in recent years. In this work, a label-free fluorescent nanosensor for FEN1 detection was developed based on cleavage-induced ligation of bifunctional dumbbell DNA and in-situ signal readout by copper nanoparticles (CuNPs). The dumbbell DNA was rationally designed with a FEN1 cleavable 5' flap for target recognition and AT-riched stem-loop template for CuNPs formation. In the presence of FEN1, 5' overhanging DNA flap of dumbbell DNA was effectively removed to form a linkable nick site. After the ligation by T4 DNA ligase, the dumbbell DNA changed to exonuclease-resisted closed structure which enabled in-situ generation of fluorescent CuNPs that served as signal source for target quantification. The low background attributed to synergic digestion by exonucleases facilitated the highly sensitive detection of FEN1 with limit of detection of 0.007 U/mL. Additionally, the sensor was extended to the assay of FEN1 inhibitor (aurintricarboxylic acid) with reasonable results. Last but not least, the normal cells and tumor cells were distinguished unambiguously by this sensor according to the detected concentration difference of cellular FEN1, which indicates the robustness and practicability of this nanosensor.

Programmable CRISPR-Cas12a and self-recruiting crRNA assisted dual biosensing platform for simultaneous detection of lung cancer biomarkers hOGG1 and FEN1.

Human 8-oxoguanine DNA glycosylase (hOGG1) and flap endonuclease 1 (FEN1) are recognized as potential biomarkers in lung cancer investigations. Developing analytical platforms of simultaneously targeting hOGG1 and FEN1 with high selectivity, sensitivity, especially programmability and universality is highly valuable for clinical research. Herein, we established a signal-amplified platform for simultaneously detecting hOGG1 and FEN1 on the basis of cleavage-induced ligation of DNA dumbbell probes, rolling circle transcription (RCT) and CRISPR-Cas12a. A hOGG1 cleavable site and FEN1 cleavable flap were dexterously designed at the 5' end of DNA flapped dumbbell probes (FDP) for hOGG1 and FEN1. After cleavage, the resulting nick sites with juxtaposition of 5' phosphate and 3' hydroxyl terminus could be linked to closed DNA dumbbell probes (CDP) by DNA ligase. The CDP served as a template for RCT, producing plentiful crRNA repeats to activate the trans-cleavage activity of CRISPR-Cas12a which could cleave fluorophores (TAMRA and FAM) and quenchers (BHQ2 and BHQ1) double-labeled ssDNA reporters. Then, hOGG1 and FEN1 could be detected by the recovered fluorescence signal, allowing for the highly sensitive calculated detection limits of 0.0013 and 0.0052 U/mL, respectively. Additionally, this method made it possible to evaluate the inhibitory effects, even to measure hOGG1 and FEN1 activities at the single-cell level. This novel target enzyme-initiated, circles-transcription without promoters, real-time generation, and self-assembly features of FDP-RCT-Cas12a system suppressed nonspecific background remarkably and relieved rigorous requirement of protospacer adjacent motif site. Hence, the universality of FDP-RCT-Cas12a system toward various disease-related non-nucleic acid targets which are tested without using aptamers was extremely improved.

Lighting-up aptamer transcriptional amplification for highly sensitive and label-free FEN1 detection.

Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.

An ultrasensitive biosensing platform for FEN1 activity detection based on target-induced primer extension to trigger the collateral cleavage of CRISPR/Cas12a.

Flap endonuclease 1 (FEN1), a structure-selective endonuclease essential for DNA replication and repair, has been considered as a new promising marker for early cancer diagnosis. However, reliable, sensitive and convenient biosensors for FEN1 detection are still technically challenging. Herein, a fluorometric biosensor based on target-induced primer extension to initiate the collateral cleavage of CRISPR/Cas12a has been established for ultrasensitive and specific detection of FEN1 activity. Using branched DNA to probe FEN1 activity, the cleaved 5' flap initiated DNA polymerase-mediated primer extension to produce plenty of DNA duplexes containing protospacer adjacent motif (PAM) which act as activators to initiate the collateral cleavage activity of Cas12a protein, producing an significantly amplified fluorescence response for ultrasensitive determination of FEN1 activity. The developed biosensing platform displays excellent analytical performance, with a limit of detection (LOD) down to 8.9 × 10-5 U µL-1, and a wide linear range from 1.0 × 10-4 to 5.0 × 10-1 U µL-1. Moreover, the proposed strategy was successfully used for FEN1 detection in serums and cell lysates and suggests potential clinical applications, which may provide a reliable approach for FEN1 that will allow effective diagnosis in the early stages of related cancer.

The Contribution of Flap Endonuclease 1 Genotypes to Oral Cancer Risk.

BACKGROUND/AIM: Flap endonuclease 1 (FEN1) is a critical protein in DNA repair, genomic stability, and carcinogenesis. Functional polymorphisms in FEN1 promoter -69G>A (rs174538) and 3'UTR 4150G>T (rs4246215), have been associated with the susceptibility to several cancers, including lung, breast, esophageal, gastric, liver, colorectal, and gallbladder cancer, as well as glioma, endometriosis, and leukemia. However, the contribution of FEN1 variant genotypes to oral cancer has never been examined. Thus, we aimed to evaluate the contribution of FEN1 rs174538 and rs4246215 genotypes to oral cancer risk in Taiwan. MATERIALS AND METHODS: The contribution of FEN1 genotypes to oral cancer risk was examined in 958 oral cancer patients and 958 age- and sex-matched healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The percentages of GG, AG, and AA genotypes at FEN1 rs174538 were 34.8%, 46.0%, and 19.2% among oral cancer patients and 37.8%, 45.2%, and 17.0% among healthy controls (p for trend=0.2788). The genotypic percentages of FEN1 rs4246215 were 35.9%, 45.9%, and 18.2% among oral cancer patients and 37.6%, 45.1%, and 17.3% among healthy controls (p for trend=0.7315). Overall, FEN1 rs174538 and rs4246215 were not differently distributed between the oral cancer patient and healthy control groups. The allele frequency analysis confirmed that FEN1 rs174538 and rs4246215 were non-differentially distributed among case and control groups (OR=1.11 and 1.05, 95%CI=0.98-1.27 and 0.93-1.20, p=0.1074 and 0.4491, respectively). CONCLUSION: FEN1 may contribute to oral cancer risk determination via protein expression and/or post-transcription modification, but may not be a practical genetic marker.

Conversion of hepatitis B virus relaxed circular to covalently closed circular DNA is supported in murine cells.

Background & Aims: HBV has a narrow host restriction, with humans and chimpanzees representing the only known natural hosts. The molecular correlates of resistance in species that are commonly used in biomedical research, such as mice, are currently incompletely understood. Expression of human NTCP (hNTCP) in mouse hepatocytes enables HBV entry, but subsequently covalently closed circular (cccDNA) does not form in most murine cells. It is unknown if this blockade in cccDNA formation is due to deficiency in repair of relaxed circular DNA (rcDNA) to cccDNA. Methods: Here, we deployed both in vivo and in vitro virological and biochemical approaches to investigate if murine cells contain a complete set of repair factors capable of converting HBV rcDNA to cccDNA. Results: We demonstrate that HBV cccDNA does form in murine cell culture or in mice when recombinant rcDNA without a protein adduct is directly introduced into cells. We further show that the murine orthologues of core components in DNA lagging strand synthesis, required for the repair of rcDNA to cccDNA in human cells, can support this crucial step in the HBV life cycle. It is worth noting that recombinant HBV rcDNA substrates, either without a protein adduct or containing neutravidin to mimic HBV polymerase, were used in our study; it remains unclear if the HBV polymerase removal processes are the same in mouse and human cells. Conclusions: Collectively, our data suggest that the HBV life cycle is blocked post entry and likely before the repair stage in mouse cells, which yields critical insights that will aid in the construction of a mouse model with inbred susceptibility to HBV infection. Lay summary: Hepatitis B virus (HBV) is only known to infect humans and chimpanzees in nature. Mouse models are often used in modeling disease pathogenesis and preclinical research to assess the efficacy and safety of interventions before they are then tested in human participants. However, because mice are not susceptible to HBV infection it is difficult to accurately model human infection (and test potential treatments) in mouse models. Herein, we have shown that mice are able to perform a key step in the HBV life cycle, tightening the net around the possible reason why HBV can not efficiently infect and replicate in mice.

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein.

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication.

Small-Molecule Inhibitors Targeting FEN1 for Cancer Therapy.

DNA damage repair plays a key role in maintaining genomic stability and integrity. Flap endonuclease 1 (FEN1) is a core protein in the base excision repair (BER) pathway and participates in Okazaki fragment maturation during DNA replication. Several studies have implicated FEN1 in the regulation of other DNA repair pathways, including homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Abnormal expression or mutation of FEN1 in cells can cause a series of pathological responses, leading to various diseases, including cancers. Moreover, overexpression of FEN1 contributes to drug resistance in several types of cancers. All this supports the hypothesis that FEN1 could be a therapeutic target for cancer treatment. Targeting FEN1 has been verified as an effective strategy in mono or combined treatment of cancer. Small-molecule compounds targeting FEN1 have also been developed and detected in cancer regression. In this review, we summarize the recent development of small-molecule inhibitors targeting FEN1 in recent years, thereby expanding their therapeutic potential and application.

miR-4324 inhibits ovarian cancer progression by targeting FEN1.

BACKGROUND: Ovarian cancer is one of the most lethal malignancies, with a 1.9% mortality rate worldwide. The dysregulation of the FEN1 gene and miR-4324 has been associated with cancer progression. However, the relationship between miR-4324 and-FEN1 requires further investigation. METHODS: miR-4324 and FEN1 expressions in ovarian cancer tissues and cell lines were measured via RT-qPCR. The interaction between miR-4324 and FEN1 was assessed using luciferase and RNA pull-down assays. The effects of miR-4324 and FEN1 on cell proliferation, adhesion and apoptosis were determined by CCK-8, BrdU, colony formation, cell adhesion, Caspase-3 and western blot assays in ovarian cancer cell lines CaOV3 and OVCAR3, respectively. RESULTS: The results showed that miR-4324 expression was significantly decreased and FEN1 expression was enhanced in ovarian cancer tissues and cell lines. miR-4324 inhibitor promoted cell proliferation, adhesion and migration, and prevented apoptosis. Furthermore, the downregulation of FEN1 inhibited ovarian cancer cell growth and increased apoptosis. miR-4324 inhibited FEN1 expression and repressed ovarian cancer progression. CONCLUSION: Our study found that miR-4324 inhibited FEN1 expression, suppressed cell growth, and increased apoptosis in ovarian cancer cells. Therefore, we identified miR-4324 and FEN1 as potential therapeutic targets for ovarian cancer treatment.

Label-free and homogeneous detection of flap endonuclease 1 by ligation-promoted hyperbranched rolling circle amplification platform.

The structure-specific endonuclease FEN1 participates in various genome maintenance pathways in eukaryotes and is associated with different human diseases. Herein, we demonstrate label-free and homogeneous detection of FEN1 based on ligation-promoted hyperbranched rolling circle amplification (HRCA). This assay can be performed isothermally with the involvement of primers 1 and 2 and a circular DNA substrate with a 5'-flap. When FEN1 is present, it cleaves 5'-flap of circular DNA substrate to obtain a circular padlock probe with the assistance of Taq DNA ligase. The circular padlock probe can serve as a template to initiate HRCA in the presence of primers 1 and 2 and Vent (exo-) DNA polymerase. The obtained dsDNA fragments can produce an enhanced fluorescence signal with SYBR Green I as indicator. This method displays good specificity and high sensitivity, and it can be employed to screen FEN1 inhibitors and quantitatively detect FEN1 activity in human cancer cells, with potential applications in early diagnosis and drug discovery.

FEN1 Status and Its Correlation with Clinicopathologic Characteristic in Colorectal Cancer.

OBJECTIVE: The goal of this study was to investigate the status of FEN1 in colorectal cancer (CRC) and determine the potential correlation between FEN1 expression level and clinicopathological parameters in CRC patients. METHODS: Expression of FEN1 in CRC tissue on tissue microarray was detected using immunohistochemistry (IHC). The relationship between FEN1 expression status and clinicopathologic characteristics of CRC was analyzed by the Chi-square test. The survival data of TCGA Colon Cancer (COAD) were obtained from ucsc xena browser (https://xenabrowser.net/). Patients were separated into higher and lower expression groups by median FEN1 expression. The association with prognosis of CRC patients was determined by Kaplan-Meier survival analysis with Log-rank test. RESULTS: FEN1expression level and cellular localization had wide variability among different individuals; we classified the staining results into four types: both positive in nucleus and cytoplasm, both negative in nucleus and cytoplasm, only positive in the nucleus, only positive in the cytoplasm. Moreover, FEN1 expression status only correlated with patient's metastasis status, and the patients in the NLCL group showed more risk of cancer cell metastasis. CONCLUSION: Our results indicate that FEN1 expression level and cellular localization had wide variability in CRC and is not a promising biomarker in CRC.

Identification of Flap Endonuclease 1 With Diagnostic and Prognostic Value in Breast Cancer.

OBJECTIVE: This study aims to identify the potential value of flap endonuclease 1 (FEN1) as a diagnostic and prognostic marker for breast cancer (BC). METHODS: ELISA was used to measure serum FEN1 levels and ECLIA for CA153 and CEA levels. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic value. Oncomine and UALCAN databases were used to analyze the differences in FEN1 mRNA and protein expressions. Kaplan-Meier Plotter database was then used to assess the prognostic value. RESULTS: Bioinformatics analysis showed that the FEN1 mRNA and protein levels were significantly higher in BC tissues than in normal tissues. FEN1 was detected in culture medium of BC cell lines and serum FEN1 concentrations were significantly increased in BC patients than in cancer-free individuals. Besides, FEN1 exhibited higher diagnostic accuracy (AUC values>0.800) than CA153 and CEA for distinguishing BC patients, especially early BC, from the healthy and benign groups, or individually. Additionally, serum FEN1 levels were significantly associated with the stage (P=0.001) and lymph invasion (P=0.016), and serum FEN1 levels were increased with the development of BC. Furthermore, serum FEN1 levels were significantly decreased in post-operative patients than in pre-operative patients (P=0.016). Based on the Kaplan-Meier Plotter database, the survival analysis indicated that FEN1 overexpression was associated with poor prognoses for overall survival (OS), relapse-free survival (RFS), and distant metastasis-free survival (DMFS) in BC patients. CONCLUSION: FEN1 might be a novel diagnostic and prognostic marker for BC.