ABSTRACT High incidence of recurrent miscarriage (RM, recurrent abnormal early embryo loss) largely limits global human reproduction. However, it is unclear how the pathogenesis greatly restricts its effective clinical treatment. In our previous studies, we have identified a group of novel long non-coding RNAs (lncRNAs), which might regulate the occurrence of RM through unknown biological mechanisms. In this study, we confirm that a novel lncRNA, lnc-HZ14, which is highly expressed in unexplained RM vs healthy control (HC) villous tissues, is associated with RM using a new RM case-control group (n = 50). In trophoblast cellular assays, lnc-HZ14 suppresses trophoblast cell proliferation by specifically downregulating SPHK1 (sphingosine kinase 1) protein levels. In terms of mechanism, lnc-HZ14 upregulates SQSTM1/p62 protein levels, enhances its protein interactions with polyubiquitin-modified SPHK1, promotes the formation of SQSTM1-SPHK1 bodies through liquid-liquid phase separation (LLPS), and accelerates SPHK1 aggrephagy degradation. Meanwhile, lnc-HZ14 also promotes autophagy by activating ETV4-mediated transcription of ATG101 and PPP1R15A/GADD34. The cellular mechanisms are consistent with those in villous tissues of RM patients and in placental tissues of a mouse miscarriage model, excepting that there is no lnc-HZ14 homolog in mouse. As for miscarriage treatment, therapeutic upregulation of SPHK1 by treatment with phorbol 12-myristate 13-acetate (PMA), an SPHK1 agonist recovers mouse placental proliferation and alleviates mouse miscarriage. Collectively, this study shows for the first time the regulatory roles of lnc-HZ14, LLPS, and aggrephagy degradation of SPHK1 in unexplained recurrent miscarriage, uncovering novel pathogenesis and biological mechanisms of unexplained RM and also providing potential targets for treatment against miscarriage. Abberviations: 3-MA: 3-methyladenine; Act D: actinomycin D; ATG101: autophagy related 101; Baf A1: bafilomycin, A1; CCK8: Cell Counting Kit-8; CHX: cycloheximide; CQ: chloroquine; EdU: 5-ethynyl-2’-deoxyuridine; EGFP: enhanced green fluorescent protein; ETV4/PEA3: ETS variant transcription factor 4; FISH: fluorescence in situ hybridization; IP: immunoprecipitation; KRT7: keratin 7; LLPS: liquid-liquid phase separation; lncRNA: long non-coding RNAs; MAP1LC3B/ LC3B: microtubule associated protein 1 light chain 3 beta; MKI67/Ki67: marker of proliferation Ki-67; MTT: methylthiazolyldiphenyl tetrazolium bromide; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; PMA: phorbol 12-myristate 13-acetate; RACE: rapid amplification of cDNA ends; RM: recurrent miscarriage; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TUBB: tubulin beta; Ub: ubiquitin.

Tobacco smoke is an important inducer of airway epithelial cell aging. Punicalagin(PCG) is a natural anti-aging compound. The effect of PCG on tobacco smoke-induced airway epithelial cell senescence is unknown. Our study investigated whether PCG can treat the human bronchial epithelial cell line (BEAS-2B) aging by inhibiting the protease-activated receptor 2 (PAR2)/m- TOR pathway. Bioinformatics techniques were used to analyze the potential biological functions of PAR2. Molecular dynamics evaluated the binding ability of PCG and PAR2. The CCK8 assay was used to detect the cytotoxicity of CSE and PCG. The activity of the PAR2/mTOR pathway and the expression of the characteristic aging markers p16, p21, and SIRT1 are detected by qRT-PCR and Western blotting. Cell senescence was observed by Senescence-associated β-galactosidase (SA-β-gal) staining. The senescence-associated secretory phenotype (SASP): concentrations of interleukin IL-6, IL-8, and TNF- α were detected by ELISA. The GSE57148 bioinformatics analysis dataset showed that PAR2 regulates lung senescence through the mTOR signaling pathway. Molecular dynamics results found that PCG and PAR2 had a strong and stable binding force. CSE induces BEAS-2B cell senescence and activates the PAR2/mTOR pathway. Inhibition of PAR2 mitigated the senescence changes. In addition, PCG's pretreatment can significantly alleviate CSE-induced BEAS-2B cell senescence while inhibiting the PAR2/mTOR pathway. PCG has a therapeutic effect on the senescence of airway epithelial cells.