Objective To explore the mechanism of lncRNA-BC200 (BC200) targeting the ubiquitination of Beta-site APP cleaving enzyme 1 (BACE1) and regulating the repair of nerve cell injury. Methods Mouse hippocampal neuron cell line HT22 was divided into four groups: control group, oxygen-glucose deprivation/reoxygenation(OGD/R) group, OGD/R+si-NC group and OGD/R+si-BC200 group. In order to further explore the relationship between BC200 and BACE1, HT22 cells were divided into four groups: OGD/R group, OGD/R+si-BC200 group, OGD/R+si-BC200+NC group and OGD/R+si-BC200+ BACE1 group. Twenty male C57BL/6J mice were randomly assigned to the following four groups: control group, middle cerebral artery occlusion (MCAO) group, MCAO+si-BC200 group and MCAO+si-BC200+BACE1 group. The mRNA expression levels of BC200 and BACE1 in cells were measured by real-time quantitative reverse transcription polymerase chain reaction. The expressions of c-caspase-3, B-cell lymphoma 2 (Bcl2), Bcl2 associated X protein(BAX) and BACE1 were detected by western blot, and the apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test. Results Compared with the control group, the activity of HT22 cells in OGD/R group decreased significantly, and the percentage of apoptotic cells increased significantly. Compared with OGD/R+si-NC group, the activity of HT22 cells in OGD/R+si-BC200 group increased significantly, and the percentage of apoptotic cells decreased significantly. Compared with the control group, the expression of BACE1 protein in HT22 cells in OGD/R group was significantly enhanced. Compared with OGD/R+si-NC group, the expression of BACE1 protein in HT22 cells in OGD/R+si-BC200 group decreased significantly. It was observed that after OGD/R treatment, the ubiquitination level of BACE1 decreased significantly and the expression of BACE1 protein increased significantly. After transfection with si-BC200, the ubiquitination level of BACE1 protein increased significantly, while the expression of BACE1 protein decreased significantly. Compared with OGD/R+si-BC200+NC group, the percentage of apoptotic cells, the expression of c-caspase-3 and Bax protein in HT22 cells in OGD/R+si-BC200+BACE1 group increased significantly, and the expression of Bcl2 protein decreased significantly. Compared with the control group, the number of cerebral infarction areas and TUNEL positive cells in MCAO group increased significantly, and the survival number of neurons decreased significantly. Compared with the MCAO group, the number of cerebral infarction areas and TUNEL positive cells in MCAO+si-BC200 group decreased significantly, and the survival number of neurons increased significantly, while the addition of BACE1 reversed the improvement of si-BC200 transfection. Conclusion The combination of BC200 and BACE1 inhibit the ubiquitination of BACE1, and participate in mediating the expression enhancement of BACE1 induced by OGD/R. Specific blocking of BC200/BACE1 axis may be a potential therapeutic target to protect neurons from apoptosis induced by cerebral ischemia/reperfusion.
Animals ; Amyloid Precursor Protein Secretases/genetics* ; RNA, Long Noncoding/physiology* ; Aspartic Acid Endopeptidases/genetics* ; Male ; Neurons/pathology* ; Mice ; Mice, Inbred C57BL ; Apoptosis/genetics* ; Ubiquitination ; Cell Line ; Hippocampus/metabolism* ; bcl-2-Associated X Protein/genetics* ; Caspase 3/genetics* ; Infarction, Middle Cerebral Artery/metabolism*

Alzheimer's disease (AD), a neurodegenerative disorder with complex etiologies, manifests through a cascade of pathological changes before clinical symptoms become apparent. Among these early changes, alterations in the expression of non-coding RNAs (ncRNAs) have emerged as pivotal events. In this study, we focused on the aberrant expression of ncRNAs and revealed that Lamr1-ps1, a pseudogene of the laminin receptor, significantly exacerbates early spatial learning and memory deficits in APP/PS1 mice. Through a combination of bioinformatics prediction and experimental validation, we identified the miR-29c/Bace1 pathway as a potential regulatory mechanism by which Lamr1-ps1 influences AD pathology. Importantly, augmenting the miR-29c-3p levels in mice ameliorated memory deficits, underscoring the therapeutic potential of targeting miR-29c-3p in early AD intervention. This study not only provides new insights into the role of pseudogenes in AD but also consolidates a foundational basis for considering miR-29c as a viable therapeutic target, offering a novel avenue for AD research and treatment strategies.
Animals ; Alzheimer Disease/pathology* ; Pseudogenes/genetics* ; Mice ; Memory Disorders/metabolism* ; MicroRNAs/genetics* ; Disease Models, Animal ; Spatial Learning/physiology* ; Mice, Transgenic ; Presenilin-1/genetics* ; Male ; Amyloid Precursor Protein Secretases/metabolism* ; Mice, Inbred C57BL ; Aspartic Acid Endopeptidases/metabolism*

The study aims to investigate the impacts of prolyl oligopeptidase (POP) on the replication of foot-and-mouth disease virus (FMDV) in BHK-21 cells. Firstly, the effects of FMDV replication on POP expression in BHK-21 cells were analyzed by Western blotting and Real-time reverse transcription polymerase chain reaction (RT-qPCR). Secondly, a eukaryotic expression plasmid for POP was constructed, and the effects of POP overexpression on the replication of two different serotypes of FMDV were assessed by Western blotting, RT-qPCR, and virus titer assays. Thirdly, specific small interfering RNAs (siRNAs) targeting POP were synthesized, and their efficiency in interfering with endogenous POP expression was identified by RT-qPCR. The impacts of downregulating endogenous POP expression on FMDV replication were further evaluated by Western blotting, RT-qPCR, and virus titer assays. The results indicated that FMDV infection did not significantly affect POP expression in BHK-21 cells. Overexpression of POP dose-dependently enhanced the replication of both FMDV/O and FMDV/A serotypes. Conversely, siRNA-mediated downregulation of endogenous POP expression markedly suppressed FMDV/O replication. This study is the first to demonstrated that the role of the host POP protein in promoting FMDV replication in BHK-21 cells, thereby providing a critical theoretical foundation and potential molecular targets for developing efficient candidate cell strains for foot-and-mouth disease inactivated vaccines.
Foot-and-Mouth Disease Virus/genetics* ; Virus Replication/genetics* ; Prolyl Oligopeptidases ; Serine Endopeptidases/physiology* ; Animals ; Cell Line ; RNA, Small Interfering/genetics* ; Foot-and-Mouth Disease/virology* ; Cricetinae

Acute Kidney Injury ; etiology ; Betacoronavirus ; Coronavirus Infections ; complications ; Humans ; Kidney ; enzymology ; Pandemics ; Peptidyl-Dipeptidase A ; physiology ; Pneumonia, Viral ; complications ; Sequence Analysis, RNA ; methods ; Serine Endopeptidases ; physiology ; Single-Cell Analysis ; methods

OBJECTIVETo investigate the effects of YOD1 overexpression on the proliferation and migration of human oral keratinocytes (HOKs), and to clarify whether the mechanisms involve transforming growth factor-β (TGF-β) signaling.

METHODSHOKs were transfected with the plasmid pEGFP-N3-YOD1 containing YOD1. The mRNA levels of YOD1 and TGF-β were determined by qPCR. The protein expressions of YOD1, TGF-β, Smad2/3, Smad4, and phospho-Smad2/3 were determined by western blotting. Cell proliferation and migration were evaluated by Cell Counting Kit-8 assay and wound healing assay, respectively.

RESULTSThe mRNA and protein levels of YOD1 were higher in HOKs transfected with YOD1. YOD1 overexpression significantly enhanced the migration of HOKs. The mRNA and protein levels of TGF-β3 were increased by YOD1 overexpression. HOKs transfected with YOD1 exhibited increased phospho-Smad2/3 levels.

CONCLUSIONYOD1 overexpression enhances cell migration by promoting TGF-β3 signaling which may play an important role in lip and palate formation. YOD1 mutation may contribute to aberrant TGF-β3 signaling associated with decreased cell migration resulting in NSCLP.

Cell Movement ; physiology ; Cell Proliferation ; Cells, Cultured ; Endopeptidases ; genetics ; metabolism ; Humans ; Keratinocytes ; physiology ; Signal Transduction ; physiology ; Smad Proteins ; genetics ; metabolism ; Thiolester Hydrolases ; genetics ; metabolism ; Transforming Growth Factor beta3 ; genetics ; metabolism

BACKGROUNDTwo recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD).

METHODSThe anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition.

RESULTSInhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis.

CONCLUSIONSInhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD.

Adrenocorticotropic Hormone ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Endopeptidases ; metabolism ; Endosomal Sorting Complexes Required for Transport ; antagonists & inhibitors ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; Indenes ; pharmacology ; Mice ; Pyrazines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Ubiquitin Thiolesterase ; antagonists & inhibitors ; metabolism

BACKGROUNDAmyloid β (Aβ) has been established as a key factor for the pathological changes in the brains of patients with Alzheimer's disease (AD), and cellular senescence is closely associated with aging and cognitive impairment. However, it remains blurred whether, in the AD brains, Aβ accelerates the neuronal senescence and whether this senescence, in turn, impairs the cognitive function. This study aimed to explore the expression of senescence-associated genes in the hippocampal tissue from young to aged 5XFAD mice and their age-matched wild type (WT) mice to determine whether senescent neurons are present in the transgenic AD mouse model.

METHODSThe 5XFAD mice and age-matched wild type mice, both raised from 1 to 18 months, were enrolled in the study. The senescence-associated genes in the hippocampus were analyzed and differentially expressed genes (DEGs) were screened by quantitative real-time polymerase chain reaction. Cognitive performance of the mice was evaluated by Y-maze and Morris water maze tests. Oligomeric Aβ (oAβ) (1-42) was applied to culture primary neurons to simulate the in vivo manifestation. Aging-related proteins were detected by Western blotting analysis and immunofluorescence.

RESULTSIn 5XFAD mice, of all the DEGs, the senescence-associated marker p16 was most significantly increased, even at the early age. It was mainly localized in neurons, with a marginal expression in astrocytes (labeled as glutamine synthetase), nil expression in activated microglia (labeled as Iba1), and negatively correlated with the spatial cognitive impairments of 5XFAD mice. oAβ (1-42) induced the production of senescence-related protein p16, but not p53 in vitro, which was in line with the in vivo manifestation.

CONCLUSIONSoAβ-accelerated neuronal senescence may be associated with the cognitive impairment in 5XFAD mice. Senescence-associated marker p16 can serve as an indicator to estimate the cognitive prognosis for AD population.

Alzheimer Disease ; metabolism ; physiopathology ; Amyloid Precursor Protein Secretases ; genetics ; metabolism ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; genetics ; metabolism ; Brain ; metabolism ; physiopathology ; Cells, Cultured ; Cellular Senescence ; genetics ; physiology ; Cognition ; physiology ; Cognition Disorders ; metabolism ; physiopathology ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neurons ; metabolism ; pathology ; Real-Time Polymerase Chain Reaction

OBJECTIVETo observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC).

METHODSSix human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay.

RESULTSRT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05).

CONCLUSIONSHtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

Adolescent ; Adult ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Child ; Down-Regulation ; Genetic Vectors ; High-Temperature Requirement A Serine Peptidase 1 ; Humans ; Lentivirus ; physiology ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; Transfection ; Young Adult

The picornavirus family comprises many small viruses, several of which are important pathogens of humans and livestock. The 3C protease (3Cpro) of different species and genera of picornavirus contains the classic G-X-C-G motif and Cys-His-Asp/Glu catalytic triad. 3Cpro conducts maturation cleavage in the regions of VP2-VP3 and VP3-VP1 in P1, 2A-2B and 2B-2C in P2 and the whole P3. Picornavirus 3Cpro has been shown to have significant substrate preference in Q-G/S/A/V/H/R and E-S/G/R/M as well as species and genera specificity through analyses of the maturation cleavage of picornavirus polyproteins. Innate immune adaptors such as TRIF, MAVS, IRF3, IRF7 and NEMO have various potential cleavage sites in picornavirus 3Cpro (TRIF and NEMO show considerable diversity in their cleavage sites). Useful information will be provided for the development of broad-spectrum antiviral agents as well as evasion mechanisms of the innate immune system against picornavirus 3Cpro through continued research of picornavirus 3Cpro.
Cysteine Endopeptidases ; physiology ; Immunity, Innate ; Picornaviridae ; enzymology ; immunology ; Viral Proteins ; physiology ; Virus Replication

Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P≤0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.
Amino Acid Motifs ; Aminoacyltransferases ; drug effects ; genetics ; Antigens, Bacterial ; drug effects ; Bacterial Adhesion ; drug effects ; Bacterial Proteins ; drug effects ; genetics ; Biofilms ; drug effects ; Cysteine Endopeptidases ; drug effects ; genetics ; Dose-Response Relationship, Drug ; Humans ; Mutation ; genetics ; Nicotine ; administration & dosage ; pharmacology ; Peptidoglycan ; drug effects ; genetics ; Saliva ; physiology ; Streptococcus mutans ; drug effects ; enzymology ; growth & development ; Sucrose ; pharmacology