Lnc- <i>HZ14</i> promotes LLPS-mediated SPHK1 aggrephagy degradation to suppress trophoblast cell proliferation in unexplained recurrent miscarriage

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-<i>HZ14</i>, which is highly expressed in unexplained RM vs healthy control (HC) villous tissues, is associated with RM using a new RM case-control group (<i>n</i> = 50). In trophoblast cellular assays, lnc-<i>HZ14</i> suppresses trophoblast cell proliferation by specifically downregulating SPHK1 (sphingosine kinase 1) protein levels. In terms of mechanism, lnc-<i>HZ14</i> 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-<i>HZ14</i> also promotes autophagy by activating ETV4-mediated transcription of <i>ATG101</i> and <i>PPP1R15A/GADD34</i>. 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-<i>HZ14</i> 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-<i>HZ14</i>, 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.<b>Abberviations</b>: 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.