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Abnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12 suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12 downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12 (mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174 patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12 upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12 was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3 could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12 and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment.Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type. [ABSTRACT FROM AUTHOR]

Human trophoblast cells are crucial for healthy pregnancy. However, whether the defective homologous recombination (HR) repair of dsDNA break (DSB) in trophoblast cells may induce miscarriage is completely unknown. Moreover, the abundance of BRCA1 (a crucial protein for HR repair), its recruitment to DSB foci, and its epigenetic regulatory mechanisms, are also fully unexplored. In this work, it is identified that a novel lnc‐HZ10, which is highly experssed in villous tissues of recurrent miscarriage (RM) vs their healthy control group, suppresses HR repair of DSB in trophoblast cell. Lnc‐HZ10 and AhR (aryl hydrocarbon receptor) form a positive feedback loop. AhR acts as a transcription factor to promote lnc‐HZ10 transcription. Meanwhile, lnc‐HZ10 also increases AhR levels by suppressing its CUL4B‐mediated ubiquitination degradation. Subsequently, AhR suppresses BRCA1 transcription; and lnc‐HZ10 (mainly 1‐447 nt) interacts with γ‐H2AX; and thus, impairs its interactions with BRCA1. BPDE exposure may trigger this loop to suppress HR repair in trophoblast cells, possibly inducing miscarriage. Knockdown of murine Ahr efficiently recovers HR repair in placental tissues and alleviates miscarriage in a mouse miscarriage model. Therefore, it is suggested that AhR/lnc‐HZ10/BRCA1 axis may be a promising target for alleviation of unexplained miscarriage. [ABSTRACT FROM AUTHOR]

A novel method to prepare low temperature poly-Si thin film on glass substrate is presented based on two-step laser crystallization.

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Dpo4 is a representative model of Y-family DNA polymerase and is therefore one of the most intensively studied DNA polymerase. 6 mA, an epigenetic marker, plays important roles in regulation of various biological processes. However, its effects on DNA replication by Dpo4 is completely unknown. Here, we found that 6 mA and its intermediate Hyp inhibits primer extension by Dpo4, showing an obvious blockage just one nucleotide before 6 mA or Hyp. 6 mA reduces dTTP incorporation efficiency, next-base extension efficiency, binding affinity of DNA to Dpo4, binding affinity of dTTP to Dpo4-DNA complex, the fraction of productive Dpo4 or productive ternary complex, and the burst incorporation rate, explaining the inhibition effects of 6 mA on DNA replication by Dpo4. Hyp is similar to G and dCTP is preferentially incorporated opposite Hyp by Dpo4, resulting in A:T to G:C mutation. Relative to dTTP incorporation opposite unmodified A, Hyp reduces dCTP incorporation efficiency, next-base extension efficiency, the priority in extension beyond correct pair, binding affinity of Dpo4 to DNA, binding of dCTP to Dpo4-DNA complex, and the burst incorporation efficiency, explaining the inhibition effects of Hyp on DNA replication by Dpo4. This work provides insight in the effects of epigenetically modified 6 mA and Hyp on DNA replication by a representative Y-family DNA polymerase Dpo4., (Copyright © 2019 Elsevier Inc. All rights reserved.)

N 6 -methyladenine (6mA), a newly identified epigenetic modification, plays important roles in regulation of various biological processes. However, the effect of 6mA on DNA replication has been little addressed. In this work, we investigated how 6mA affected DNA replication by DNA polymerase of Pseudomonas aeruginosa Phage PaP1 (gp90 exo - ). The presence of 6mA, as well as its intermediate hypoxanthine (Hyp), inhibited DNA replication by gp90 exo - . The 6mA reduced dTTP incorporation efficiency by 10-fold and inhibited next-base extension efficiency by 100-fold. Differently, dCTP was preferentially incorporated opposite Hyp among four dNTPs. Gp90 exo - reduced the extension priority beyond the 6mA:T pair rather than the 6mA:C mispair and preferred to extend beyond Hyp:C rather than the Hyp:T pair. Incorporation of dTTP opposite 6mA and dCTP opposite Hyp showed fast burst phases. The burst rate and burst amplitude were both reduced for 6mA compared with unmodified A. Moreover, the total incorporation efficiency ( k pol / K d,dNTP ) was decreased for dTTP incorporation opposite 6mA and dCTP incorporation opposite Hyp compared with dTTP incorporation opposite A. 6mA reduced the incorporation rate ( k pol ), and Hyp increased the dissociation constant ( K d,dNTP ). However, 6mA or Hyp on template did not affect the binding of DNA polymerase to DNA in binary or ternary complexes. This work provides new insight into the inhibited effects of epigenetic modification of 6mA on DNA replication in PaP1.