Circ_ZFR contributes to the paclitaxel resistance and progression of non-small cell lung cancer by upregulating KPNA4 through sponging miR-195-5p.

BACKGROUND: A growing body of evidence has demonstrated the vital roles of circular RNAs (circRNAs) in cancer progression and drug resistance. We intended to explore the roles and mechanisms of circ_ZFR in the paclitaxel (PTX) resistance and progression of non-small cell lung cancer (NSCLC). METHODS: Two NSCLC cell lines A549 and H460 were used in this study. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was conducted to measure the levels of circ_ZFR, ZFR, miR-195-5p and karyopherin subunit alpha 4 (KPNA4) mRNA. RNase R assay was used to analyze the characteristic of circ_ZFR. MTT assay was carried out to assess PTX resistance and cell proliferation. Flow cytometry analysis was utilized to analyze cell cycle and apoptosis. Transwell assay was used to examine cell migration and invasion. Western blot assay was conducted to measure the protein levels of Ki67, Twist1, E-cadherin and KPNA4. Dual-luciferase reporter assay was adopted to verify the combination between miR-195-5p and circ_ZFR or KPNA4. Murine xenograft model assay was used to investigate the effect of circ_ZFR on PTX resistance of NSCLC in vivo. RESULTS: Circ_ZFR level was enhanced in PTX-resistant NSCLC tissues and cells. Knockdown of circ_ZFR suppressed PTX resistance, cell cycle process, proliferation, migration and invasion and induced apoptosis in PTX-resistant NSCLC cells. For mechanism analysis, circ_ZFR knockdown markedly downregulated the expression of KPNA4 by sponging miR-195-5p, thereby promoting PTX sensitivity and suppressing cell progression in PTX-resistant NSCLC cells. In addition, circ_ZFR silencing enhanced PTX sensitivity of NSCLC in vivo. CONCLUSION: Circ_ZFR knockdown played a positive role in overcoming PTX resistance of NSCLC via regulating miR-195-5p/KPNA4 axis, which might provide a possible circRNA-targeted therapy for NSCLC.

O- glycosylation can regulate the proliferation and migration of human retinal microvascular endothelial cells through ZFR in high glucose condition.

PURPOSE: Angiogenesis is an essential part of the diabetes retinopathy (DR) process, and Zinc Finger RNA Binding Protein (ZFR) is important for vascularization to occur. However, the function and regulation of ZFR in DR development are not well understood. We hypothesized that high glucose condition could result in ZFR up-regulation in human retinal microvascular endothelial cells (HRMECs), thus contributing to disease progression, and O-glycosylation may be participated in this regulation. METHODS: Retinas were harvested from streptozotocin (STZ)-induced rat model of diabetes. Human umbilical vein endothelial cells (HUVECs) and HRMECs cultured in high glucose concentration, and retinal tissues were detected for ZFR expression. We examined the role of ZFR on vasculogenic processes including proliferation and migration in the cell model of DR. The effect of O-glycosylation modification on ZFR was further assessed in HRMECs. RESULTS: Expression of ZFR was up-regulated in high glucose condition both in vitro and in vivo. ZFR induced proliferation and migration of HRMECs. Inhibition of O-glycosylation modification attenuated the expression of ZFR. CONCLUSION: ZFR plays an important role in the pathogenesis of DR, and its mechanism may through the modification of O-glycosylation. Our research suggests that ZFR may be used as a potential prognostic marker or potential therapeutic target for DR.

ZFR promotes cell proliferation and tumor development in colorectal and liver cancers.

Colorectal cancer (CRC) and liver cancer are the second and fourth leading causes of cancer-related deaths in the whole world, respectively, and each year over 1.6 million people die from these diseases. To identify driver genes in CRC and liver cancer, we have performed Sleeping Beauty transposon mutagenesis screens in mouse models. Zinc finger RNA binding protein, ZFR, was one of the novel candidate cancer genes identified in these forward genetic screens. Consistent with this discovery, a pan-cancer analysis of sequencing results of thousands of human cancer genomes demonstrated that ZFR is a potential potent oncogene. In this study, we aimed to investigate ZFR's roles in both types of cancer and found that overexpression of ZFR in CRC and liver cancer cells led to accelerated tumor development. Consistently, knockdown of ZFR resulted in significantly decelerated tumor development. ZFR overexpression also promoted tumor development of immortalized mouse liver cells. ZFR overexpression and shRNA knockdown led to accelerated and decelerated cell proliferation, respectively, indicating that ZFR promotes tumor development mainly by regulating cell proliferation. To identify ZFR's targets in transcription, we performed whole transcriptome sequencing using ZFR small interfering RNAs in a primary human colon cell line. All potential target genes were validated by real time PCR. FAM49B was a tumor suppressor candidate for ZFR targets. When we knocked down the expression of FAM49B in CRC and liver cancer cells, we observed significantly accelerated cell proliferation, consistent with the results with ZFR overexpression. The results presented here demonstrate the oncogenic role of ZFR in both CRC and liver cancer, providing a potential drug target for both cancers' treatment. We also identified ZFR's potential transcriptional targets, and further investigations on those targets, especially FAM49B, will help us understand more about the important role of ZFR in digestive system cancers.

MiR-141-3p functions as a tumor suppressor through directly targeting ZFR in non-small cell lung cancer.

MicroRNAs are important regulators in the development and progression of non-small cell lung cancer (NSCLC). MiR-141-3p has been reported to function as a suppressor or oncogene in several tumors, but the clinical significance and crucial biological functions of miR-141-3p in NSCLC remains largely unclear. In this study, expression levels of miR-141-3p in tissue samples were measured by quantitative real-time polymerase chain reaction. Kaplan-Meier survival analysis and Cox regression assay were performed to evaluate the prognostic value of miR-141-3p. Cell experiments, including CCK-8, colony formation and transwell assays were carried out to explore its functional role. Luciferase reporter assay was used to confirm its target gene. The results showed that miR-141-3p was significantly down-regulated in NSCLC tissues compared with adjacent normal tissues. The decreased miR-141-3p expression was associated with advanced TNM stage and lymph-node metastasis. Patients with low miR-141-3p expression had poor overall survival compared with those with high expression. Down-regulation of miR-141-3p was demonstrated to be an independent prognostic factor for NSCLC. Moreover, ZFR was confirmed as a target gene of miR-141-3p. Meta-analysis based on Oncomine database showed ZFR was significantly up-regulated in human NSCLC tissues. The in vitro experiments showed that restoration of ZFR rescued the miR-141-3p-mediated inhibitory effects on cell proliferation, migration and invasion in NSCLC cells. In conclusions, miR-141-3p might be a prognostic tumor suppressor involved in the NSCLC progression.

Enzyme dimension of the ribosomal protein S4 across plant and animal kingdoms.

BACKGROUND: The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO). METHODS: Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells. RESULTS: Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B, human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic kidney cells, presumably due to the extraribosomal enzyme trait reported. CONCLUSIONS: The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases in the extraribosomal milieu. GENERAL SIGNIFICANCE: The enzyme nature of S4 is relevant toward understanding not only the origin of ribosomal proteins, but also processes in cell biology and diseases.

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.

BACKGROUND: It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease. METHODS: Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis. RESULTS: The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable. CONCLUSION: The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. SIGNIFICANCE: Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.

Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.

STUDY QUESTION: Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI? SUMMARY ANSWER: A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI. WHAT IS KNOWN ALREADY: Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis. STUDY DESIGN, SIZE, DURATION: We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed. MAIN RESULTS AND THE ROLE OF CHANCE: Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study and no functional experiments were performed. WIDER IMPLICATIONS OF THE FINDINGS: The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation. STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Novel requirements for HAP2-mediated gamete fusion in Tetrahymena.

The ancestral gamete fusion protein, HAP2, catalyzes sperm-egg fusion in a broad range of taxa dating to the last eukaryotic common ancestor. Remarkably, HAP2 orthologs are structurally related to the class II fusogens of modern-day viruses, and recent studies make clear that these proteins utilize similar mechanisms to achieve membrane merger. To identify factors that may regulate HAP2 activity, we screened mutants of the ciliate Tetrahymena thermophila for behaviors that mimic Δhap2 knockout phenotypes in this species. Using this approach, we identified two new genes, GFU1 and GFU2, whose products are necessary for the formation of membrane pores during fertilization and show that the product of a third gene, namely ZFR1, may be involved in pore maintenance and/or expansion. Finally, we propose a model that explains cooperativity between the fusion machinery on apposed membranes of mating cells and accounts for successful fertilization in T. thermophila's multiple mating type system.

Circ_ZFR affects FABP7 expression to regulate breast cancer progression by acting as a sponge for miR-223-3p.

BACKGROUND: Breast cancer (BC) is a common malignancy in women. Circular RNAs (circRNAs) have been reported to play a key role in the development of BC; however, the effect of circular RNA zinc finger RNA binding protein (circ_ZFR) in BC is unknown. METHODS: Abundances of circ_ZFR, fatty acid binding protein 7 (FABP7), and microRNA-223-3p (miR-223-3p) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The circular structure of circ_ZFR was validated by RNase R treatment. Cell proliferation, migration, invasion, and apoptosis were assessed by colony formation, cell counting kit-8, Transwell, flow cytometry assays, respectively. All protein levels were determined by Western blot. Dual-luciferase reporter assay was used to confirm the relationship between miR-223-3p and circ_ZFR or FABP7. A xenograft model was established to understand the effect of circ_ZFR on BC cell growth in vivo. RESULTS: The expression levels of circ_ZFR and FABP7 were higher in BC tissues and cell lines, whereas miR-223-3p expression was lower. Knockdown of circ_ZFR or FABP7 in BC cells reduced proliferation, migration, invasion, and epithelial mesenchymal transition (EMT), and induced apoptosis in vitro, whereas the opposite effects were observed in circ_ZFR-overexpressed cells. Furthermore, circ_ZFR might act as a sponge for miR-223-3p to regulate FABP7 expression, thereby promoting the progression of BC cells in vitro and in vivo. CONCLUSION: Circ_ZFR might act as a miRNA sponge for miR-223-3p to regulate FABP7, thereby promoting proliferation, migration, invasion, and EMT of BC cells, and inhibiting cell apoptosis.

Circular RNA ZFR promotes cell cycle arrest and apoptosis of colorectal cancer cells via the miR-147a/CACUL1 axis.

Background: Colorectal cancer (CC) is one of the most prevalent malignancies worldwide. Nonetheless, its pathogenicity and molecular mechanisms have not been completely elucidated yet. The potential clinical value of circular RNAs (circRNAs) in tumor diagnosis, treatment, and prognosis has received considerable attention. Methods: Here, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) examined the levels of circular ZFR (circZFR) in CC cells. The expression of circZFR was knocked down in CC cells and cell viability was detected using Cell Counting Kit-8 (CCK-8) and colony formation assays. Cell cycle progression was assessed by flow cytometry and the expression levels of cyclin-associated proteins were detected by western blot analysis. The transferase dUTP nick end labeling (TUNEL) assay was used to detect apoptosis and western blot analysis was used to evaluate the expression levels of apoptosis-associated proteins. Subsequently, the interactions between circZFR and microRNA (miR)-147a and between miR-147a and CDK2 associated cullin domain 1 (CACUL1) were predicted by the Encyclopedia of RNA Interactomes database and verified by luciferase reporter assays. Finally, plasmid transfection, CCK-8, and flow cytometry assays were used to explore the associated mechanism of action. Results: CircZFR was highly expressed in CC cell lines. Interference with its expression inhibited proliferation and induced G1/S cell cycle arrest and apoptosis in CC cells. The expression levels of miR-147a and CACUL1 were decreased and increased, respectively, in CC cells. These data demonstrated that circZFR could target miR-147a and CACUL1 to regulate the cell cycle and apoptosis of CC cells and, ultimately, promote the progression of CC. Conclusions: Knockdown of the expression of circZFR upregulated miR-147a expression and reduced CACUL1 expression levels, thereby inhibiting the proliferation of CC cells and inducing cell cycle arrest and apoptosis.

Vasoconstrictor antagonism improves functional and structural vascular alterations and liver damage in rats with early NAFLD.

BACKGROUND & AIMS: Intrahepatic vascular resistance is increased in early non-alcoholic fatty liver disease (NAFLD), potentially leading to tissue hypoxia and triggering disease progression. Hepatic vascular hyperreactivity to vasoconstrictors has been identified as an underlying mechanism. This study investigates vasoconstrictive agonism and antagonism in 2 models of early NAFLD and in non-alcoholic steatohepatitis (NASH). METHODS: The effects of endothelin-1 (ET-1), angiotensin II (ATII) and thromboxane A2 (TxA2) agonism and antagonism were studied by in situ ex vivo liver perfusion and preventive/therapeutic treatment experiments in a methionine-choline-deficient diet model of steatosis. Furthermore, important results were validated in Zucker fatty rats after 4 or 8 weeks of high-fat high-fructose diet feeding. In vivo systemic and portal pressures, ex vivo transhepatic pressure gradients (THPG) and transaminase levels were measured. Liver tissue was harvested for structural and mRNA analysis. RESULTS: The THPG and consequent portal pressure were significantly increased in both models of steatosis and in NASH. ET-1, ATII and TxA2 increased the THPG even further. Bosentan (ET-1 receptor antagonist), valsartan (ATII receptor blocker) and celecoxib (COX-2 inhibitor) attenuated or even normalised the increased THPG in steatosis. Simultaneously, bosentan and valsartan treatment improved transaminase levels. Moreover, bosentan was able to mitigate the degree of steatosis and restored the disrupted microvascular structure. Finally, beneficial vascular effects of bosentan endured in NASH. CONCLUSIONS: Antagonism of vasoconstrictive mediators improves intrahepatic vascular function. Both ET-1 and ATII antagonists showed additional benefit and bosentan even mitigated steatosis and structural liver damage. In conclusion, vasoconstrictive antagonism is a potentially promising therapeutic option for the treatment of early NAFLD. LAY SUMMARY: In non-alcoholic fatty liver disease (NAFLD), hepatic blood flow is impaired and the blood pressure in the liver blood vessels is increased as a result of an increased response of the liver vasculature to vasoconstrictors. Using drugs to block the constriction of the intrahepatic vasculature, the resistance of the liver blood vessels decreases and the increased portal pressure is reduced. Moreover, blocking the vasoconstrictive endothelin-1 pathway restored parenchymal architecture and reduced disease severity.

An Update on the Roles of circRNA-ZFR in Human Malignant Tumors.

CircRNAs (circular RNAs) are single-stranded RNAs that form covalently closed loops and function as important regulatory elements of the genome through multiple mechanisms. Increasing evidence had indicated that circRNAs, which might serve as either oncogenes or tumor suppressors, played vital roles in the pathophysiology of human diseases, especially in tumorigenesis and progression. CircRNA-ZFR (circular RNA zinc finger RNA binding protein) is a circular RNA that had attracted much attention in recent years. It has been found that circRNA-ZFR was abnormally expressed in a variety of malignant tumors, and its dysregulated expression was closely related to tumor stage, cancer metastasis and patients' prognosis. Recent studies had shown that aberrantly expressed circRNA-ZFR could regulate the malignant biological behaviors of tumors through various mechanisms; further exploration of circRNA-ZFR expression in tumors and its regulation on malignant biological behaviors such as tumor proliferation, invasion and drug resistance will provide new ideas for clinical tumors diagnosis and treatment.

Unbiased Identification of trans Regulators of ADAR and A-to-I RNA Editing.

Adenosine-to-inosine RNA editing is catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes that deaminate adenosine to inosine. Although many RNA editing sites are known, few trans regulators have been identified. We perform BioID followed by mass spectrometry to identify trans regulators of ADAR1 and ADAR2 in HeLa and M17 neuroblastoma cells. We identify known and novel ADAR-interacting proteins. Using ENCODE data, we validate and characterize a subset of the novel interactors as global or site-specific RNA editing regulators. Our set of novel trans regulators includes all four members of the DZF-domain-containing family of proteins: ILF3, ILF2, STRBP, and ZFR. We show that these proteins interact with each ADAR and modulate RNA editing levels. We find ILF3 is a broadly influential negative regulator of editing. This work demonstrates the broad roles that RNA binding proteins play in regulating editing levels, and establishes DZF-domain-containing proteins as a group of highly influential RNA editing regulators.

Circular RNA ZFR accelerates non-small cell lung cancer progression by acting as a miR-101-3p sponge to enhance CUL4B expression.

BACKGROUND: Non-small-cell lung carcinoma (NSCLC) is the most common type of lung cancer. Circular RNA ZFR (circZFR) is an identified circular RNA (circRNA) that is correlated with cancer progression. However, the role of circZFR in NSCLC remains unknown. In the present study, we aimed to investigate the function of circZFR in NSCLC and the underlying mechanism. METHODS: The expression patterns of circZFR were determined using qRT-PCR in NSCLC samples and cell lines. The subcellular distribution of circZFR in NSCLC cells was analyzed by fluorescence in situ hybridization (FISH). Cell proliferation was examined utilizing the CCK-8 assay. Cell migration and invasion were evaluated using the Transwell assay. We used the bioinformatics software TargetScan and miRanda to predict circRNA-miRNA and miRNA-mRNA interactions. RESULTS: Our results showed that circZFR expressions were significantly upregulated in NSCLC tissues and cell lines. Knockdown of circZFR significantly inhibited the cell proliferation, migration and invasion of NSCLC cells in vitro. Furthermore, we demonstrated that circZFR acted as a sponge to absorb microRNA-101-3p (miR-101-3p) and promoted cullin 4B (CUL4B) expression. CONCLUSIONS: Collectively, our results demonstrated that circZFR exhibited a carcinogenic role by sponging miR-101-3p and regulating CUL4B expression in NSCLC. These findings provided evidence for understanding the role of circZFR in NSCLC tumourigenesis.

Use of zero-frequency resonator for automatically detecting systolic peaks of photoplethysmogram signal.

This work investigates the application of zero-frequency resonator (ZFR) for detecting systolic peaks of photoplethysmogram (PPG) signals. Based on the authors' studies, they propose an automated noise-robust method, which consists of the central difference operation, the ZFR, the mean subtraction and averaging, the peak determination, and the peak rejection/acceptance rule. The method is evaluated using different kinds of PPG signals taken from the standard MIT-BIH polysomnographic database and Complex Systems Laboratory database and the recorded PPG signals at their Biomedical System Lab. The method achieves an average sensitivity (Se) of 99.95%, positive predictivity (Pp) of 99.89%, and overall accuracy (OA) of 99.84% on a total number of 116,673 true peaks. Evaluation results further demonstrate the robustness of the ZFR-based method for noisy PPG signals with a signal-to-noise ratio (SNR) ranging from 30 to 5 dB. The method achieves an average Se = 99.76%, Pp = 99.84%, and OA = 99.60% for noisy PPG signals with a SNR of 5 dB. Various results show that the method yields better detection rates for both noise-free and noisy PPG signals. The method is simple and reliable as compared with the complexity of signal processing techniques and detection performance of the existing detection methods.

Circular RNA-ZFR Inhibited Cell Proliferation and Promoted Apoptosis in Gastric Cancer by Sponging miR-130a/miR-107 and Modulating PTEN.

PURPOSE: This study aimed to probe into the associations among circular RNA ZFR (circ-ZFR), miR-130a/miR-107, and PTEN, and to investigate the regulatory mechanism of circ-ZFR‒miR-130a/miR-107‒PTEN axis in gastric cancer (GC). MATERIALS AND METHODS: GSE89143 microarray data used in the study were acquired from publicly available Gene Expression Omnibus database to identify differentially expressed circular RNAs inGC tissues. The expressions of circ-ZFR, miR-130a, miR-107, and PTEN were examined by real-time reverse transcription polymerase chain reaction, while PTEN protein expression was measured by western blot. The variation of GC cell proliferation and apoptosis was confirmed by cell counting kit-8 assay and flow cytometry analysis. The targeted relationships among circZFR, miR-130a/miR-107, and PTEN were predicted via bioinformatics analysis and demonstrated by dual-luciferase reporter assay and RNA immunoprecipitation assay. The impact of ZFR on gastric tumor was further verified in xenograft mice model experiment. RESULTS: Circ-ZFR and PTEN were low-expressed whereas miR-107 and miR-130a were highexpressed in GC tissues and cells. There existed targeted relationships and interactions between miR-130a/miR-107 and ZFR/PTEN. Circ-ZFR inhibited GC cell propagation, cell cycle and promoted apoptosis by sponging miR-107/miR-130a, while miR-107/miR-130a promoted GC cell propagation and impeded apoptosis through targeting PTEN. Circ-ZFR inhibited cell proliferation and facilitated apoptosis in GC by sponging miR-130a/miR-107 and modulating PTEN. Circ-ZFR curbed GC tumor growth and affected p53 protein expression in vivo. CONCLUSION: Circ-ZFR restrained GC cell proliferation, induced cell cycle arrest and promoted apoptosis by sponging miR-130a/miR-107 and regulating PTEN.

Zinc finger RNA-binding protein promotes non-small-cell carcinoma growth and tumor metastasis by targeting the Notch signaling pathway.

Metastatic non-small-cell lung carcinoma (NSCLC) is typically incurable. The development of anti-metastatic therapies is hampered because the mechanisms regulating metastasis in NSCLC are not well known. Currently, there is no effective treatment for NSCLC once it has progressed to the metastatic stage. Therefore, further elucidation of the molecular mechanisms underlying the metastasis of NSCLC cells is urgently required for improving NSCLC treatment. Here, we report that the zinc finger RNA-binding protein (ZFR) is over-expressed in NSCLC cells and demonstrate that ZFR is a promising therapeutic target in metastatic NSCLC. The use of shRNA to knockdown ZFR impaired cell proliferation in vitro and tumor growth in vivo. Moreover, silencing of ZFR inhibited metastasis almost completely. In contrast, over-expression of ZFR in cells significantly enhanced NSCLC cell growth and metastasis. Finally, ZFR increased the metastatic potential of NSCLC cells in a Notch1-dependent manner. Collectively, our study reveals a critical role of ZFR in NSCLC tumor growth and metastasis, suggesting ZFR as a novel target for the treatment of NSCLC.

Purification and In Vitro Characterization of Zinc Finger Recombinases.

Zinc finger recombinases (ZFRs) are designer site-specific recombinases that have been adapted for a variety of genome editing purposes. Due to their modular nature, ZFRs can be customized for targeted sequence recognition and recombination. There has been substantial research on the in vivo properties and applications of ZFRs; however, in order to fully understand and customize them, it will be necessary to study their properties in vitro. Experiments in vitro can allow us to optimize catalytic activities, improve target specificity, measure and minimize off-target activity, and characterize key steps in the recombination pathway that might be modified to improve performance. Here, we present a straightforward set of protocols for the expression and purification of ZFRs, an assay system for catalytic proficiency in vitro and bandshift assays for detection of sequence-specific DNA interactions.