ABSTRACT
Temporal lobe epilepsy (TLE) is characterized as an impaired ability of learning and memory with periodic and unpredictable seizures. Status epilepticus (SE) is one of the main causes of TLE. Neuroinflammation and oxidative stress are directly involved in epileptogenesis and neurodegeneration, promoting chronic epilepsy and cognitive deficit. Previous studies have shown that ursolic acid (UA) represses inflammation and oxidative stress, contributing to neuroprotection. Herein, we demonstrated that UA treatment alleviated seizure behavior and cognitive impairment induced by epilepsy. Moreover, UA treatment rescued hippocampal neuronal damage, aberrant neurogenesis, and ectopic migration, which are commonly accompanied by epilepsy occurrence. Our study also demonstrated that UA treatment remarkably suppressed the SE-induced neuroinflammation, evidenced by activated microglial cells and decreased inflammation factors, including TNF-α and IL-1ß. Likewise, the expression levels of oxidative stress damage markers and oxidative phosphorylation (OXPHOS) enzyme complexes of mitochondria were also remarkably downregulated following the UA treatment, suggesting that UA suppressed the damage caused by the high oxidative stress and the defect mitochondrial function induced by SE. Furthermore, UA treatment attenuated GABAergic interneuron loss. In summary, our study clarified the notable anti-seizure and neuroprotective properties of UA in pilocarpine-induced epileptic rats, which is mainly achieved by abilities of anti-inflammation and anti-oxidation. Our study indicates the potential advantage of UA application in ameliorating epileptic sequelae.
ABSTRACT
BACKGROUND: AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells. METHODS: A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, ß-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity. RESULTS: Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner. CONCLUSIONS: In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.
Subject(s)
Colorectal Neoplasms , MicroRNAs , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins/genetics , Chromosomal Instability , Colorectal Neoplasms/genetics , DNA Damage , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , Humans , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Transcription Factors/geneticsABSTRACT
Competitive endogenous messenger RNA (ceRNA) affects transcription of other RNA molecules by competitively binding common microRNAs. Previous studies have shown that TP53INP1 functions as a suppressor in tumor metastasis. Our study elucidated StarD13 messenger RNA as a ceRNA in regulating migration and invasion of breast cancer cells. MicroRNA-125b was identified to induce metastasis of MCF-7 cells and bind with both StarD13 3'UTR and TP53INP1 3'UTR. Therefore, a ceRNA interaction between StarD13 and TP53INP1 mediated by competitively binding to miR-125b was indicated. Importantly, a microRNA-125b binding site at 4546-4560 nt on StarD13 was verified more vital for this ceRNA interaction. Indirectly regulation of SPARC in inducing metastasis of breast cancer cells by StarD13 via competitively binding with TP53INP1 was further confirmed. In conclusion, our findings demonstrate a ceRNA regulatory network which could give a better understanding of metastatic mechanisms of breast cancer.
Subject(s)
3' Untranslated Regions , Breast Neoplasms/pathology , Carrier Proteins/metabolism , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins/metabolism , MicroRNAs/genetics , Neoplasm Invasiveness , Tumor Suppressor Proteins/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Carrier Proteins/genetics , Cell Proliferation , Female , GTPase-Activating Proteins , Heat-Shock Proteins/genetics , Humans , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Wound HealingABSTRACT
To investigate the effects of CYP4Z1 3'UTR in migration of breast cancer cells, a series of assays were used to confirm that overexpression of CYP4Z1 3'UTR could suppress the capacity of migration and adhesion of MCF-7 and MDA-MB-231 cells. EMT (Epithelial-mesenchymal transition)-related proteins were regulated by CYP4Z1 3'UTR. Mesenchyma markers like Vimentin, MMP-2, and MMP-9 were down-regulated, while the expression of E-cadherin was up-regulated with CYP4Z1 3'UTR overexpression. Notably, luciferase reporter and qRT-PCR assays were applied to verify that CYP4Z1 3'UTR was the potential target of miR-9. In addition, our results showed that CYP4Z1 3'UTR repressed the expression of E-cadherin in a miRNA-dependent manner. Combining with our previous study, we have discovered the underlying link between CYP4Z1 and E-cadherin. Therefore, those preliminary data suggest that CYP4Z1 3'UTR could inhibit the migration and EMT of breast cancer cells via acting as a ceRNA for E-cadherin.
Subject(s)
3' Untranslated Regions/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Movement/genetics , Cytochrome P450 Family 4/genetics , Antigens, CD , Cadherins , Cell Adhesion , Cell Line, Tumor , Cytochrome P450 Family 4/metabolism , Epithelial-Mesenchymal Transition/genetics , Female , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Small Interfering/metabolism , TransfectionABSTRACT
Competing endogenous RNAs (ceRNAs) network has been correlated with the initiation and development of cancer. Here, we identify CDH5, HOXD1, and HOXD10 as putative STARD13 ceRNAs and they display concordant patterns with STARD13 in different metastatic potential breast cancer cell lines and tissues. Notably, 3'UTRs of these genes suppress breast cancer metastasis via inhibiting epithelial-mesenchymal transition (EMT) in vitro and in vivo, which are activated through the crosstalk between STARD13 and its ceRNAs in 3'UTR- and miRNA-dependent manners. In addition, Kaplan-Meier survival analysis reveals that mRNA level of STARD13 and its ceRNAs is remarkably associated with survival of breast cancer patients. These results suggest that 3'UTRs of CDH5, HOXD1, and HOXD10 inhibit breast cancer metastasis via serving as STARD13 ceRNAs.
Subject(s)
Breast Neoplasms/pathology , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Liver Neoplasms/secondary , Lung Neoplasms/secondary , RNA, Long Noncoding/genetics , Tumor Suppressor Proteins/metabolism , 3' Untranslated Regions , Animals , Apoptosis , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Proliferation , Female , GTPase-Activating Proteins , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , MicroRNAs/genetics , Prognosis , RNA, Messenger , Survival Rate , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
Patients with estrogen receptor α (ERα)-positive breast cancer can be treated with endocrine therapy using anti-estrogens such as tamoxifen; nonetheless, patients often develop resistance limiting the success of breast cancer treatment. The potential mechanisms remain elusive. In detail, many miRNAs have been associated with breast cancer tamoxifen resistance, but no studies have addressed the role of miRNA-mediated competitive endogenous RNAs network (ceRNET) in tamoxifen resistance. The ceRNET between CYP4Z1 and pseudogene CYP4Z2P has been revealed to promote breast cancer angiogenesis. However, its function in tamoxifen resistance remains unclear. Here we report CYP4Z1 and CYP4Z2P were downregulated in MCF-7 cells compared with tamoxifen-resistant MCF-7-TamR cells. Enforced upregulation of CYP4Z1- or CYP4Z2P-3'UTR level renders MCF-7 Cells resistant to tamoxifen. We find that overexpression of CYP4Z1- or CYP4Z2P-3'UTR enhances the transcriptional activity of ERα through the activation of ERα phosphorylation. Furthermore, we find that CYP4Z1- and CYP4Z2P-3'UTRs increase ERα activity dependent on cyclin-dependent kinase 3 (CDK3). Reporter gene and western blot assays revealed that CYP4Z1- and CYP4Z2P-3'UTRs act as CDK3 ceRNAs. More importantly, the blocking of CYP4Z1- and CYP4Z2P-3'UTRs reversed tamoxifen resistance in MCF-7-TamR cells. Our data demonstrates that the ceRNET between CYP4Z1 and pseudogene CYP4Z2P acts as a sub-ceRNET to promote CDK3 expression in ER-positive breast cancer and is a potential therapeutic target for treatment of tamoxifen-resistant breast cancer.
Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/genetics , Cytochrome P450 Family 4/genetics , Drug Resistance, Neoplasm/genetics , Pseudogenes , RNA, Neoplasm/metabolism , Tamoxifen/pharmacology , 3' Untranslated Regions , Breast Neoplasms/drug therapy , Down-Regulation , Female , HEK293 Cells , Humans , MCF-7 CellsABSTRACT
Competitive endogenous messenger RNAs (ceRNAs) affect other RNAs transcription through competitively binding common microRNAs (miRNAs). In this study we identified long non-coding RNA (lncRNA) MALAT1 can function as a ceRNA of cell division cycle 42 (cdc42) 3'UTR in inducing migration and invasion of breast cancer cells via miR-1. We found that miR-1 bound both MALAT1 and cdc42 3'UTR directly. Further study showed that MALAT1 induced migration and invasion of breast cancer cells while reduced the level of cdc42. Our results suggest that MALAT1 regulated migration and invasion of breast cancer cells via affecting cdc42 through binding miR-1 competitively.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Long Noncoding/genetics , cdc42 GTP-Binding Protein/genetics , 3' Untranslated Regions , Binding, Competitive , Breast Neoplasms/physiopathology , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Down-Regulation , Epithelial-Mesenchymal Transition/genetics , Female , Humans , MCF-7 Cells , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Invasiveness/physiopathology , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Up-RegulationABSTRACT
CXCR4 is the most common chemokine receptor expressed on tumor cells, and it is closely correlated with cancer cell stemness. This study was carried out to explore whether CXCR4 could function as a competitive endogenous RNA to promote metastasis, proliferation and survival in MCF-7 breast cancer cells. We validated that CXCR4, together with TRAF6 and EGFR, was directly targeted by miR-146a in MCF-7 cells. Overexpression of CXCR4 3'UTR inhibited the activity of miR-146a, thus elevating the expression of CXCR4, TRAF6 and EGFR. These oncoproteins further activated NF-κB pathway and promoted the proliferation, migration, invasion and anti-apoptotic activity of MCF-7 cells. Collectively, our study provided new insights into the function of CXCR4 in breast cancer: it promotes tumor progression as both a protein-coding gene and a non-coding RNA, complicating the mechanism by which oncogenes promote tumor progression.
