Clostridium butyricum Alleviates DEHP Plasticizer-Induced Learning and Memory Impairment in Mice via Gut-Brain Axis.

Di-(2-ethylhexyl) phthalate (DEHP) plasticizer, a well-known environmental and food pollutant, has neurotoxicity. However, it is unknown whether DEHP leads to learning and memory impairment through gut-brain axis and whether Clostridium butyricum can alleviate this impairment. Here, C57BL/6 mice were exposed to DEHP and treated with C. butyricum. Learning and memory abilities were evaluated through the Morris water maze. The levels of synaptic proteins, inflammatory cytokines, and 5-hydroxytryptamine (5-HT) were detected by immunohistochemistry or ELISA. Gut microbiota were analyzed through 16S rRNA sequencing. C. butyricum alleviated DEHP-induced learning and memory impairment and restored synaptic proteins. It significantly relieved DEHP-induced inflammation and recovered 5-HT levels. C. butyricum recovered the richness of the gut microbiota decreased by DEHP, with the Bifidobacterium genus increasing the most. Overall, C. butyricum alleviated DEHP-induced learning and memory impairment due to reduced inflammation and increased 5-HT secretion, which was partly attributed to the recovery of gut microbiota.

Screening of Tnfaip1-Interacting Proteins in Zebrafish Embryonic cDNA Libraries Using a Yeast Two-Hybrid System.

TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1's biological functions.

MiR-93 alleviates DEHP plasticizer-induced neurotoxicity by negatively regulating TNFAIP1 and inhibiting ubiquitin-mediated degradation of CK2ß.

Di-(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used in various products, such as plastic packaging in food industries. As an environmental endocrine disruptor, it induces adverse effects on brain development and function. However, the molecular mechanisms by which DEHP induces learning and memory impairment remain poorly understood. Herein, we found that DEHP impaired learning and memory in pubertal C57BL/6 mice, decreased the number of neurons, downregulated miR-93 and the ß subunit of casein kinase 2 (CK2ß), upregulated tumor necrosis factor-induced protein 1 (TNFAIP1), and inhibited Akt/CREB pathway in mouse hippocampi. Co-immunoprecipitation and western blotting assays revealed that TNFAIP1 interacted with CK2ß and promoted its degradation by ubiquitination. Bioinformatics analysis showed a miR-93 binding site in the 3'-untranslated region of Tnfaip1. A dual-luciferase reporter assay revealed that miR-93 targeted TNFAIP1 and negatively regulated its expression. MiR-93 overexpression prevented DEHP-induced neurotoxicity by downregulating TNFAIP1 and then activating CK2/Akt/CREB pathway. These data indicate that DEHP upregulates TNFAIP1 expression by downregulating miR-93, thus promoting ubiquitin-mediated degradation of CK2ß, subsequently inhibiting Akt/CREB pathway, and finally inducing learning and memory impairment. Therefore, miR-93 can relieve DEHP-induced neurotoxicity and may be used as a potential molecular target for prevention and treatment of related neurological disorders.

Epigenetically modified AP-2α by DNA methyltransferase facilitates glioma immune evasion by upregulating PD-L1 expression.

Programmed death-ligand 1 (PD-L1) ensures that tumor cells escape T-cell-mediated tumor immune surveillance. However, gliomas are characteristic of the low immune response and high-resistance therapy, it is necessary to understand molecular regulatory mechanisms in glioblastoma, especially the limited regulation of PD-L1 expression. Herein, we show that low expression of AP-2α is correlated with high expression of PD-L1 in high-grade glioma tissues. AP-2α binds directly to the promoter of the CD274 gene, not only inhibits the transcriptional activity of PD-L1 but enhances endocytosis and degradation of PD-L1 proteins. Overexpression of AP-2α in gliomas enhances CD8+ T cell-mediated proliferation, effector cytokine secretion, and cytotoxicity in vitro. Tfap2a could increase the cytotoxic effect of Cd8+ T cells in CT26, B16F10, and GL261 tumor-immune models, improve anti-tumor immunity, and promote the efficacy of anti-PD-1 therapy. Finally, the EZH2/H3K27Me3/DNMT1 complex mediates the methylation modification of AP-2α gene and maintains low expression of AP-2α in gliomas. 5-Aza-dC (Decitabine) treatment combines with anti-PD-1 immunotherapy to efficiently suppress the progression of GL261 gliomas. Overall, these data support a mechanism of epigenetic modification of AP-2α that contributes to tumor immune evasion, and reactivation of AP-2α synergizes with anti-PD-1 antibodies to increase antitumor efficacy, which may be a broadly applicable strategy in solid tumors.

CRISPR/Cas9-Mediated Knockout of tnfaip1 in Zebrafish Plays a Role in Early Development.

TNF α-induced protein 1 (TNFAIP1) was first identified in human umbilical vein endothelial cells and can be induced by tumor necrosis factor α (TNFα). Early studies have found that TNFAIP1 is involved in the development of many tumors and is closely associated with the neurological disorder Alzheimer's disease. However, little is known about the expression pattern of TNFAIP1 under physiological conditions and its function during embryonic development. In this study, we used zebrafish as a model to illustrate the early developmental expression pattern of tnfaip1 and its role in early development. First, we examined the expression pattern of tnfaip1 during early zebrafish development using quantitative real-time PCR and whole mount in situ hybridization and found that tnfaip1 was highly expressed in early embryonic development and, subsequently, expression became localized to anterior embryonic structures. To investigate the function of tnfaip1 during early development, we constructed a model of a stably inherited tnfaip1 mutant using the CRISPR/Cas9 system. Tnfaip1 mutant embryos showed significant developmental delays as well as microcephaly and microphthalmia. At the same time, we found decreased expression of the neuronal marker genes tuba1b, neurod1, and ccnd1 in tnfaip1 mutants. Analysis of transcriptome sequencing data revealed altered expression of the embryonic development related genes dhx40, hspa13, tnfrsf19, nppa, lrp2b, hspb9, clul1, zbtb47a, cryba1a, and adgrg4a in the tnfaip1 mutants. These findings suggest an important role for tnfaip1 in the early development of zebrafish.

GCEN: An Easy-to-Use Toolkit for Gene Co-Expression Network Analysis and lncRNAs Annotation.

Gene co-expression network analysis has been widely used in gene function annotation, especially for long noncoding RNAs (lncRNAs). However, there is a lack of effective cross-platform analysis tools. For biologists to easily build a gene co-expression network and to predict gene function, we developed GCEN, a cross-platform command-line toolkit developed with C++. It is an efficient and easy-to-use solution that will allow everyone to perform gene co-expression network analysis without the requirement of sophisticated programming skills, especially in cases of RNA-Seq research and lncRNAs function annotation. Because of its modular design, GCEN can be easily integrated into other pipelines.

Evaluation of pentaerythritol-based and trimethylolpropane-based cationic lipidic materials for gene delivery.

The chemical and physical structure of cationic liposomes pays an important effect on their gene transfection efficiency. Investigation on the structure-function relationship of cationic liposomes will guide the design of novel cationic liposomes with high transfection efficiency and biosafety. In this paper, two novel series of lipids based on the backbone of pentaerythritol and trimethylolpropane were discovered, and their gene transfection efficiencies were assayed in vitro. The four lipids 8c, 9c, 14b, and 15b, exhibited much better transfection efficiency in the HEK293 cell lines compared with Lipo2000, lipid 9c also showed good transfection efficiency in the SW480 cell lines. And the structure-efficiency relationship revealed that a hydroxyethyl polar head group boosted transfer potency in trimethylolpropane-type lipids, but reduced in pentaerythritol-type lipids.

Polydatin protects neuronal cells from hydrogen peroxide damage by activating CREB/Ngb signaling.

Oxidative stress­induced neuronal cell death contributes significantly to the physiological processes of a number of neurological disorders. Polydatin (PD) has been reported to protect against Alzheimer's disease (AD), ischemic stroke and traumatic brain injury. However, the underlying neuroprotective mechanisms remain to be elucidated. The current study suggested that PD activates AKT/cAMP response element­binding protein (CREB) signaling and induces neuroglobin (Ngb) to protect neuronal cells from hydrogen peroxide (H2O2) in vitro. PD inhibited the H2O2­induced neuronal cell death of primary mouse cortical neurons and N2a cells. Functional studies showed that PD attenuated H2O2­induced mitochondrial dysfunction and mitochondrial reactive oxygen species production. Mechanistically, PD was verified to induce the phosphorylation of AKT and CREB and increase the protein level of Ngb. The luciferase assay results showed that Ngb transcriptional activity was activated by CREB, especially after PD treatment. It was further indicated that PD increased the transcription of Ngb by enhancing the binding of CREB to the promoter region of Ngb. Finally, Ngb knockdown largely attenuated the neuroprotective role of PD against H2O2. The results indicated that PD protected neuronal cells from H2O2 by activating CREB/Ngb signaling in neuronal cells, indicating that PD has a neuroprotective effect against neurodegenerative diseases.

ADNP prompts the cisplatin-resistance of bladder cancer via TGF-ß-mediated epithelial-mesenchymal transition (EMT) pathway.

Activity-dependent neuroprotective protein (ADNP) is vital for embryonic development and brain formation. Besides, the upregulated expression of ADNP enhances tumorigenesis in some human tumors like bladder cancer (BC). However, the potential roles of ADNP in drug resistance and the related mechanisms in BC is unknown. We performed this study to elucidate the influence of ADNP in the chemoresistance of BC and tried to explore the underlying molecular mechanism. The expressions of ADNP in BC from progression and non-progression patient specimens were measured by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). In vitro experiments including colony formation, cell counting kit-8 (CCK-8), wound healing, and in vivo tumorigenesis assay were performed to explore the effects of ADNP on chemoresistance of BC. The impacts of ADNP on TGF-ß/Smad signaling pathways were explored by western blot. Our results showed that the expression of ADNP mRNA and protein were significantly upregulated in BC tissues of the patients who suffered tumor-progression via RT-PCR and western blot. Cox regression survival analysis revealed that patients with high ADNP expression closely linked to shorter tumor-free survival. ADNP downregulation in BC showed more sensitive to cisplatin in vivo, while ADNP overexpression showed the opposite results. Additionally, we confirmed that ADNP promoted cell migration and EMT, thereby inducing cisplatin resistance, which may be related to TGF-ß / Smad signaling pathway.

DDX21 interacts with nuclear AGO2 and regulates the alternative splicing of SMN2.

AGO2 is the only member of mammalian Ago protein family that possesses the catalytic activity and plays a central role in gene silencing. Recently researches reported that multiple gene silencing factors, including AGO2, function in the nuclei. The molecular mechanisms of the gene silencing factors functioning in nuclei are conducive to comprehend the roles of gene silencing in pretranslational regulation of gene expression. Here, we report that AGO2 interacts with DDX21 indirectly in an RNA-dependent manner by Co-IP and GST-Pulldown assays and the 2 proteins present nuclei foci in the immunofluorescence experiments. We found that DDX21 up-regulated the protein level of AGO2 and participated in target gene, SNM2, alternative splicing involved in AGO2 by the indirect interaction with AGO2, which produced different transcripts of SMN2 in discrepant expression level. This study laid important experiment foundation for the further analysis of the nuclear functions of gene silencing components.

Heterotopic Cesarean Scar Pregnancy: An Analysis of 20 Cases Following in vitro Fertilization-Embryo Transfer.

We report a series of 20 cases of heterotopic cesarean scar pregnancy in this study. The results show that transvaginal sonography offers highly accurate diagnoses of heterotopic cesarean scar pregnancy during the first trimester. Careful exclusion of cesarean scar pregnancy is of great clinical importance for patients with a history of cesarean section after in vitro fertilization-embryo transfer, even when an intrauterine pregnancy has been detected. We recommend single embryo transfer for patients with a history of cesarean section. Expectant management may provide the opportunity for a live cesarean scar pregnancy to develop, albeit at high risks of placenta accreta and hemorrhage, and this needs further verification in the future.

TNFAIP1 Is Upregulated in APP/PS1 Mice and Promotes Apoptosis in SH-SY5Y Cells by Binding to RhoB.

Alzheimer's disease (AD) poses a significant threat to human life and health. The intraneuronal accumulation of ß-amyloid (Aß) plaques in the brains of AD patients results in neuronal cell death, which is a key factor that triggers multiple changes in the pathogenesis of AD. The inhibition of Aß-induced neuronal cell death may potentially help in the intervention and treatment of AD. Our previous study reported that tumor necrosis factor α-induced protein 1 (TNFAIP1) is induced by and promotes Aß25-35-induced neurotoxicity in mouse neuronal cells, but the roles and regulatory mechanisms of TNFAIP1 are still largely unknown. In this study, our experimental results show that TNFAIP1 and p-TNFAIP1 (phosphorylation of TNFAIP1 at Ser280) are overexpressed in the neurons of the cortex and hippocampus in the brains of APP/PS1 mice, and the transcription factor NF-κB is involved in the Aß-induced upregulation of TNFAIP1. Moreover, our results suggest that TNFAIP1 contributes to the Aß-induced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential (∆Ψm), and neuronal cell death in human SH-SY5Y cells. We further revealed that Aß increases the binding of TNFAIP1 to RhoB, and knockdown of RhoB attenuates the TNFAIP1-induced apoptosis of human SH-SY5Y cells. These data suggest that TNFAIP1 is closely associated with AD pathogenesis, and overexpression of TNFAIP1 in the neurons of the brains of AD patients plays a role in apoptosis, at least in part, via RhoB signaling.

Corrigendum: Purification and Identification of miRNA Target Sites in Genome Using DNA Affinity Precipitation.

[This corrects the article DOI: 10.3389/fgene.2019.00778.].

Reference intervals of gestational sac, yolk sac, embryonic length, embryonic heart rate at 6-10 weeks after in vitro fertilization-embryo transfer.

BACKGROUND: Accurately determining the normal range of early pregnancy markers can help to predict adverse pregnancy outcomes. The variance in ovulation days leads to uncertain accuracy of reference intervals for natural pregnancies. While the gestational age (GA) is accurate estimation during in vitro fertilization-embryo transfer (IVF-ET). Thus, the objective of this research is to construct reference intervals for gestational sac diameter (GSD), yolk sac diameter (YSD), embryonic length (or crown-rump length, CRL) and embryonic heart rate (HR) at 6-10 gestational weeks (GW) after IVF-ET. METHODS: From January 2010 to December 2016, 30,416 eligible singleton pregnancies were retrospectively recruited. All included participants had full records of early ultrasound measurements and phenotypically normal live neonates after 37 GW, with birth weights > the 5th percentile for gestational age. The curve-fitting method was used to screen the optimal models to predict GSD, CRL, YSD and HR based on gestational days (GD) and GW. Additionally, the percentile method was used to calculate the 5th, 50th, and 95th percentiles. RESULTS: There were significant associations among GSD, CRL, YSD, HR and GD and GW, the models were GSD = - 29.180 + 1.070 GD (coefficient of determination [R2] = 0.796), CRL = - 11.960 - 0.147 GD + 0.011 GD2 (R2 = 0.976), YSD = - 2.304 + 0.184 GD - 0.011 GD2 (R2 = 0.500), HR = - 350.410 + 15.398 GD - 0.112 GD2 (R2 = 0.911); and GSD = - 29.180 + 7.492 GW (R2 = 0.796), CRL = - 11.960 - 1.028 GW + 0.535 GW2 (R2 = 0.976), YSD = - 2.304 + 1.288 GW - 0.054 GW2 (R2 = 0.500), HR = - 350.410 + 107.788 GW - 5.488 GW2 (R2 = 0.911), (p < 0.001). CONCLUSIONS: Reference intervals for GSD, YSD, HR and CRL at 6-10 gestational weeks after IVF-ET were established.

TNFAIP1 Mediates Formaldehyde-Induced Neurotoxicity by Inhibiting the Akt/CREB Pathway in N2a Cells.

Formaldehyde (FA) is a common air pollutant. Exposure to exogenous FA can cause damage to the nervous system, such as learning and memory impairment, balance dysfunction, and sleep disorders. Excessive production of endogenous FA also causes memory impairment and is thought to be associated with Alzheimer's disease (AD). Tumor necrosis factor alpha-induced protein 1 (TNFAIP1) plays a crucial role in neurodevelopment and neurological diseases. However, the role of TNFAIP1 in FA-induced neurotoxicity is unclear. Herein, using a mouse neuroblastoma cell line (N2a cells), we explored the mechanism of TNFAIP1 in FA-induced neurotoxicity, the involvement of the Akt/CREB signaling pathway, and how the expression of TNFAIP1 is regulated by FA. We found that exposure to 100 µM or 200 µM FA for 24 h led to decreased cell viability, increased cell apoptosis and neurite retraction, increased reactive oxygen species (ROS) levels, upregulated protein expression of TNFAIP1 and decreased the levels of phosphorylated Akt and CREB in the Akt/CREB pathway. Knockdown of TNFAIP1 using a TNFAIP1 small interfering RNA (siRNA) expression vector prevented FA from inhibiting the Akt/CREB pathway, thus reducing cell apoptosis and restoring cell viability and neurite outgrowth. Clearance of ROS by vitamin E (Vit E) repressed the FA-mediated upregulation of TNFAIP1 expression. These results suggest that FA increases the expression of TNFAIP1 by inducing oxidative stress and that upregulated TNFAIP1 then inhibits the Akt/CREB pathway, consequently leading to cell apoptosis and neurite retraction. Therefore, TNFAIP1 is a potential target for alleviating FA-induced neurotoxicity and related neurological disorders.

Tumor necrosis factor α-induced protein 1 as a novel tumor suppressor through selective downregulation of CSNK2B blocks nuclear factor-κB activation in hepatocellular carcinoma.

BACKGROUND: Tumor necrosis factor α-induced protein 1 (TNFAIP1) is frequently downregulated in cancer cell lines and promotes cancer cell apoptosis. However, its role, clinical significance and molecular mechanisms in hepatocellular carcinoma (HCC) are unknown. METHODS: The expression of TNFAIP1 in HCC tumor tissues and cell lines was measured by Western blot and immunohistochemistry. The effects of TNFAIP1 on HCC proliferation, apoptosis, metastasis, angiogenesis and tumor formation were evaluated by Cell Counting Kit-8 (CCK8), Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL), transwell, tube formation assay in vitro and nude mice experiments in vivo. The interaction between TNFAIP1 and CSNK2B was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), Co-immunoprecipitation and Western blot. The mechanism of how TNFAIP1 regulated nuclear factor-kappaB (NF-κB) pathway was analyzed by dual-luciferase reporter, immunofluorescence, quantitative Real-time polymerase chain reaction (RT-qPCR) and Western blot. FINDINGS: The TNFAIP1 expression is significantly decreased in HCC tissues and cell lines, and negatively correlated with the increased HCC histological grade. Overexpression of TNFAIP1 inhibits HCC cell proliferation, metastasis, angiogenesis and promotes cancer cell apoptosis both in vitro and in vivo, whereas the knockdown of TNFAIP1 in HCC cell displays opposite effects. Mechanistically, TNFAIP1 interacts with CSNK2B and promotes its ubiquitin-mediated degradation with Cul3, causing attenuation of CSNK2B-dependent NF-κB trans-activation in HCC cell. Moreover, the enforced expression of CSNK2B counteracts the inhibitory effects of TNFAIP1 on HCC cell proliferation, migration, and angiogenesis in vitro and in vivo. INTERPRETATION: Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-κB pathway, implying that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC.

SNHG16 as the miRNA let-7b-5p sponge facilitates the G2/M and epithelial-mesenchymal transition by regulating CDC25B and HMGA2 expression in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) play crucial roles in hepatocellular carcinoma (HCC). However, the underlying molecular mechanisms of small nucleolar RNA host gene 16 (SNHG16) for regulating the cell cycle and epithelial to mesenchymal transition (EMT) remain elusive. In this study, SNHG16 expression profiles of HCC tissues or cell lines were compared with those of normal tissues or hepatocyte cell line. The effect of SNHG16 knockdown in HCC cell lines was investigated by using in vitro loss-of-function experiments and in vivo nude mouse experiments. The potential molecular regulatory mechanism of SNHG16 in HCC progression was investigated by using mechanistic experiments and rescue assays. The results revealed that SNHG16 was highly expressed in HCC tissues and cell lines, which predicted poor prognosis of HCC patients. On one hand, the downregulation of SNHG16 induced G2/M cell cycle arrest, inducing cell apoptosis and suppression of cell proliferation. On the other hand, it inhibited cell metastasis and EMT progression demonstrated by in vitro loss-of-function cell experiments. Besides, knockdown of SNHG16 increased the sensitivity of HCC cells to cisplatin. For the detailed mechanism, SNHG16 was demonstrated to act as a let-7b-5p sponge in HCC. SNHG16 facilitated the G2/M cell cycle transition by directly acting on the let-7b-5p/CDC25B/CDK1 axis, and promoted cell metastasis and EMT progression by regulating the let-7b-5p/HMGA2 axis in HCC. In addition, the mechanism of SNHG16 for regulating HCC cell proliferation and metastasis was further confirmed in vivo by mouse experiments. Furthermore, these results can provide new insights into HCC treatment and its molecular pathogenesis, which may enlighten the further research of the molecular pathogenesis of HCC.

Knockdown of TNFAIP1 prevents di-(2-ethylhexyl) phthalate-induced neurotoxicity by activating CREB pathway.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer. It has neurotoxicity and exposure to it causes impairment of neurodevelopment, behavior and cognition. However, the molecular mechanisms responsible for the DEHP-induced neurotoxicity are not yet clearly defined. Tumor necrosis factor-induced protein 1 (TNFAIP1) was first discovered in umbilical vein endothelial cells and was further found to be important in the progress of Alzheimer's disease. Herein we explore the mechanism of TNFAIP1 in DEHP-induced neurotoxicity with the involvement of cyclic AMP response elements binding protein (CREB) signaling pathway in a mouse neuroblastoma cell line (N2a cells). We found that exposure to DEHP induced apoptosis and downregulated the expression of brain-derived neurotrophic factor (BDNF), synaptic proteins PSD 95 and synapsin-1 while upregulated the expression of TNFAIP1 and decreased the levels of phosphorylated Akt, CaMK Ⅳ, catalytic subunits of PKA and CREB in CREB signaling pathway. Knockdown of TNFAIP1 using TNFAIP1 small interfering RNA (siRNA) expression vector prevented DEHP from inhibiting CREB pathway, thus reduced apoptosis and restored expression of BDNF, PSD 95 and synapsin-1. Our data indicate that downregulation of TNFAIP1 prevents DEHP-induced neurotoxicity via activating CREB pathway. Therefore, TNFAIP1 is a potential target for relieving the DEHP-induced neurotoxicity and related neurological disorders.

Purification and Identification of miRNA Target Sites in Genome Using DNA Affinity Precipitation.

Combination with genomic DNA is one of the important ways for microRNAs (miRNAs) to perform biological processes. However, because of lack of an experimental method, the identified genomic sites targeted by microRNA were only located in the promoter and enhancer regions. In this study, based on affinity purification of labeled biotin at the 3'-end of miRNAs, we established an efficiently experimental method to screen miRNA binding sequences in the whole genomic regions in vivo. Biotinylated miR-373 was used to test our approach in MCF-7 cells, and then Sanger and next-generation sequencing were used to screen miR-373 binding sequences. Our results demonstrated that the genomic fragments precipitated by miR-373 were located not only in promoter but also in intron, exon, and intergenic. Eleven potentially miR-373 targeting genes were selected for further study, and all of these genes were significantly regulated by miR-373. Furthermore, the targeting sequences located in E-cadherin, cold-shock domain-containing protein C2 (CSDC2), and PDE4D genes could interact with miR-373 in MCF-7 cells rather than HeLa cells, which is consistent with our data that these three genes can be regulated by miR-373 in MCF-7 cells while not in HeLa cells. On the whole, this is an efficient method to identify miRNA targeting sequences in the whole genome.