IntroductionPhotothermal therapy (PTT) faces limitations due to tumor microenvironment (TME) heterogeneity and single-modality constraints, including hypoxia, redox imbalance, and uneven heat distribution, which compromise therapeutic durability. Integrating nanozyme catalysis with PTT presents a promising strategy to amplify oxidative stress, yet achieving a balance among catalytic efficiency, photothermal performance, biocompatibility, and stability remains challenging.MethodsHerein, we developed an epigallocatechin gallate (EGCG)-modified palladium-based nanozyme (EGCG-PdZyme) for the precision treatment of esophageal cancer. This multifunctional platform was engineered to integrate catalase-like oxygen generation, peroxidase-like reactive oxygen species (ROS) production, and near-infrared photothermal conversion capabilities.ResultsWhile EGCG modification slightly attenuated the intrinsic catalytic activity and peak photothermal temperature, it established an optimized thermo-catalytic synergy. Sustained mild hyperthermia amplified oxidative stress, effectively offsetting the reduced catalytic output and minimizing thermal damage to peritumoral tissues. Mechanistically, persistent photothermal heating boosted enzymatic ROS generation within the TME, initiating a self-amplifying therapeutic cascade. Furthermore, EGCG functionalization significantly enhanced colloidal stability and biosafety, enabling effective tumor ablation with negligible systemic toxicity.DiscussionThis study demonstrates a paradigm shift from maximizing isolated parameters toward achieving a dynamic equilibrium between catalytic functionality and biological compatibility. By integrating TME modulation with controlled photothermal amplification, the EGCG-PdZyme platform offers a viable strategy for clinically translatable precision oncotherapy.

The xenotransplantation of human cells into porcine hosts holds immense potential in the fields of regenerative medicine and organ transplantation. However, the low survival rate of human-derived cells within porcine remains a critical bottleneck constraining the application of xenotransplantation. Whether porcine cells exert negative effect on human cell growth is not studied. Here, we established an in vitro direct co-culture model of human and porcine mesenchymal stem cells (hMSCs and pMSCs) to investigate the competitive relationship between human and porcine-derived cells. The results demonstrated that the proliferation capability of hMSCs in the co-culture system was significantly suppressed compared to those cultured in isolation. Moreover, an increasing number of pMSCs exhibited enhanced inhibition of hMSC proliferation. Notably, results from transwell assays and treatment with porcine-conditioned medium indicated that the inhibition of hMSCs by pMSCs was not mediated through soluble cytokines. To elucidate the underlying molecular mechanisms, RNA sequencing analysis was performed and the result revealed that direct co-culture significantly downregulated the expression of proliferation-related genes in hMSCs, including CYP1B1, SLC7A11, TFAP2C, and PSAT1. Concurrently, the co-culture paradigm disrupted endoplasmic reticulum function and multiple amino acid transport processes within hMSCs, while activating the NF-κB signaling pathway, thereby achieving negative regulation of hMSC proliferation. Collectively, our primary study characterized the competitive interactions between hMSCs and pMSCs and uncovered possible underlying mechanisms which provided new experimental foundations for improving human cell survival in porcine hosts to advance xenotransplantation application.

In brief: The preimplantation embryonic development progress is accompanied by dynamic changes of transcriptome, and many transcription factors have been found to responsible for this progress. This study shows that SP1 is necessary for proper zygotic genome activation and morula-to-blastocyst transition by regulating transcriptome establishment and histone modifications in mouse preimplantation embryos. Abstract: Specificity protein 1 (SP1) is the most active member of the specificity protein and Krüppel-like factor (Sp/KLF) family and is widely expressed across all mammalian cell types. However, more detailed studies on the role of SP1 in preimplantation embryonic development are needed. Here, we analyzed the role of SP1 in the development of preimplantation mouse embryos through supplementation with a small molecular inhibitor (plicamycin) and microinjection of Sp1 siRNA. We found that SP1 was indispensable for zygotic genome activation (ZGA) and the morula-to-blastocyst transition. Plicamycin supplementation arrested embryo development at the 2-cell stage and resulted in aberrant RNA polymerase II preconfiguration. Sp1 expression knockdown by Sp1 siRNA microinjection caused most embryos to arrest at the morula stage, and the expression of NANOG, POU5F1, and CDX2 significantly decreased. Both plicamycin supplementation and Sp1 siRNA microinjection decreased embryonic H3K4me3 levels and increased H3K9me3 levels. Moreover, when Sp1 was overexpressed, the embryos were arrested at the 2-cell stage, the H3K4me3 level increased, and the H3K9me3 level decreased. In conclusion, our findings demonstrate that SP1 is crucial for mouse preimplantation embryonic development through the regulation of gene expression and histone modifications.

Despite recent advancements in diagnostic techniques and infertility treatments, the precise underlying cause of infertility remains elusive in numerous cases. Elevated immune cell levels in the reproductive tract frequently result in decreased sperm motility and a diminished likelihood of successful embryo implantation. This study aimed to investigate the mechanisms of NETs induction in vitro by sperm or embryos and the effects of DNase I on NETs. NETs stimulated by mouse sperm and embryos were visualized and analyzed using confocal microscopy. The formation and quantification of NETs were studied using inhibitors and PicoGreen. Sperm motility was assessed using computer-aided sperm analysis. Our findings indicated that mouse sperm can activate PMNs by inducing NETs formation, which consisted of DNA with citrullinated histone H3 (citH3) and myeloperoxidase (MPO). The pathways underlying sperm-triggered NETs involve NADPH oxidase, ERK1/2, and p38 MAPK signaling pathways. Furthermore, NETs reduced sperm motility and significantly decreased the success rates of in vitro fertilization (IVF). Treatment with DNase I effectively degraded NETs formation and mitigated these effects mentioned above. Interestingly, it was observed for the first time in vitro that mouse embryos were directly ensnared by NETs, suggesting a potential association with embryonic implantation process. This study presents the first demonstration in a mouse model of the molecular mechanisms underlying sperm-induced NET formation through multiple signaling pathways, as well as the physical entanglement of spermatozoa and embryos within these extracellular structures. These findings may offer novel strategies for managing infertility- related conditions.

The maturation of oocytes has an important impact on the subsequent development of the embryo. However, during the in vitro maturation (IVM) of oocytes, oocytes are separated from the follicular environment, resulting in a low maturation rate of oocytes in vitro. In order to improve maturation rate of IVM of porcine oocytes, this study was conducted to investigate using sodium alginate (SA) to encapsulate porcine mural granulosa cells (MGs) to develop an SA three-dimensional (3D) co-culture system for IVM of porcine oocytes. And, gene expression, reactive oxygen species (ROS), ATP level, mitochondrial membrane potential, parthenogenetic activation development results of cultured oocytes, and as well as ROS and glutathione (GSH) levels in cumulus granulosa cells (CGs) were detected. Our results showed that the maturation rate of the SA 3D co-culture group was 85.41%, that of the negative control (NC) group was 79.24%, and that of the MGs co-culture group was 81.62%. In SA 3D co-culture group, mitochondrial membrane potential level of oocytes was 1.6, ROS level was 19 and the ATP level was 1.7. While in NC group, mitochondrial membrane potential level of oocytes was 1.2, the ROS level was 52, and the ATP level was 0.4. The ROS level in the CGs of SA 3D co-culture group decreased by 1.5 times, and the glutathione content increased by 2.3 times. In the SA 3D co-culture group, GDF9 gene expression level was 2.0, and BMP15 gene expression level was 1.2. While in NC group, GDF9 gene expression level was 0.7, and BMP15 gene expression level was 0.6. The blastocyst rate in the SA 3D co-culture group was 41.4%, and that in the NC group was 36.6%. In conclusion, encapsulating MGs in SA gel and co-culturing them with porcine oocytes in 3D during IVM can improve the developmental potential of oocytes. This result will provide an important reference for improving the methods of in vitro maturation of oocytes.

As the first acetylated nucleoside to be discovered, N-acetyltransferase 10 (NAT10)-catalyzed RNA N4-acetylcytidine (ac4C) modification is involved in the occurrence of various diseases. However, the roles of RNA ac4C in preimplantation embryo development still need more detailed studies. Here, we analyzed the role of RNA ac4C in preimplanted embryonic development in mice through Nat10 siRNA microinjection and growing oocyte stage-specific Nat10 knockout (Zp3-Nat10lox/lox). We found that NAT10 was indispensable for both the morula-to-blastocyst transition and zygotic genome activation (ZGA). Nat10 knockdown by Nat10 siRNA microinjection caused most embryos to arrest at the morula stage, and the expression levels of NANOG and CDX2 were significantly decreased. Moreover, the mRNA stability of Nanog was also significantly decreased in morulae after Nat10 knockdown. Zp3-Nat10lox/lox female mice were completely sterile, and the embryos from Zp3-Nat10lox/lox females were arrested at the 2-cell stage. Both the degradation of maternal mRNA and ZGA were deficient in 2-cell embryos from Zp3-Nat10lox/lox females. In conclusion, our findings demonstrate that NAT10 is crucial for both ZGA and the morula-to-blastocyst transition processes during mouse preimplantation embryonic development.

The Myc family is essential for cell proliferation, differentiation, and metabolism, particularly in embryonic development and stem cell functions. However, the specific role of Myc in porcine early embryonic development is not fully understood. This study observed high Myc expression during the four-cell stage of porcine embryos. Inhibition of Myc using 10058-F4 impaired embryonic development, disrupted energy metabolism, and increased DNA methylation. Mechanistically, these effects were dependent on α-KG, a TCA cycle intermediate and cofactor for TET demethylation enzymes. Sequencing analysis of four-cell embryos post-Myc inhibition revealed downregulation of key metabolic enzymes related to α-KG, such as CS, IDH2, leading to reduced α-KG levels. Supplementation with α-Ketoglutarate (α-KG) mitigated the negative effects of Myc inhibition, including lower blastocyst rates, decreased ATP levels, and increased 5 mC levels. In conclusion, Myc regulates the expression of key metabolic enzymes during the four-cell stage, influencing early embryonic metabolism and epigenetic reprogramming.

Research question: What is the role and mechanism of action of transcription factor AP-2 gamma (TFAP2C) in porcine early embryo development? Design: TFAP2C siRNA were injected into porcine oocytes, which subsequently underwent IVF. Different stages of embryos were collected for RNA sequencing, quantitative polymerase chain reaction, immunofluorescence staining to explore the affects in gene expression and epigenetic modification. Porcine fetal fibroblasts were transfected with siRNA, and cells were collected for chromatin immunoprecipitation and dual luciferase reporter assays. Results: The deficiency of TFAP2C led to disorders in early embryonic development; 1208 genes were downregulated and 792 genes were upregulated in TFAP2C knockdown (TFAP2C-KD) embryos. The expression of epigenetic modification enzymes KDM5B, SETD2 were significantly elevated in the TFAP2C-KD group (P < 0.001). Meanwhile, the modification levels of H3K4me3 and H3K4me2 were significantly decreased (P = 0.0021, P = 0.0029), and H3K36me3 and DNA methylation were significantly increased in TFAP2C-KD group (P = 0.0045, P = 0.0025). DNMT1 was mainly expressed in nuclei in the TFAP2C-KD group (P = 0.0103). In addition, TFAP2C could bind to the promoter region of SETD2, and the mutation of the TFAP2C binding site resulted in increased activity of SETD2 promoter (P < 0.001). Conclusions: The knockdown of TFAP2C affects early embryonic development by regulating histone modification and DNA methylation.

Long noncoding RNAs (lncRNAs) are implicated in a number of regulatory functions in eukaryotic genomes. In humans, KCNQ1OT1 is a 91 kb imprinted lncRNA that inhibits multiple surrounding genes in cis. Among them, CDKN1C is closely related to KCNQ1OT1 and is involved in multiple epigenetic disorders. Here, we found that pigs also had a relatively conserved paternal allele expressing KCNQ1OT1 and had a shorter 5 ' end (similar to 27 kb) compared to human KCNQ1OT1. Knockdown of KCNQ1OT1 using antisense oligonucleotides (ASO) showed that upregulation of CDKN1C expression in pigs. However, porcine KCNQ1OT1 did not affect the DNA methylation status of the CpG islands in the promoters of KCNQ1OT1 and CDKN1C. Inhibition of DNA methyltransferase using Decitabine treatment resulted in a significant increase in both KCNQ1OT1 and CDKN1C expression, suggesting that the regulation between KCNQ1OT1 and CDKN1C may not be dependent on RNA interference. Further use of chromosome conformation capture and reverse transcription-associated trap detection in the region where CDKN1C was located revealed that KCNQ1OT1 bound to the CDKN1C promoter and affected chromosome folding. Phenotypically, inhibition of KCNQ1OT1 at the cumulus-oocyte complex promoted cumulus cell transformation, and to upregulated the expression of ALPL at the early stage of osteogenic differentiation of porcine bone marrow mesenchymal stem cells. Our results confirm that the expression of KCNQ1OT1 imprinting in pigs as well as porcine KCNQ1OT1 regulates the expression of CDKN1C through direct promoter binding and chromatin folding alteration. And this regulatory mechanism played an important role in cell differentiation.

Oocyte senescence alters the shape and function, thereby weakening the fertilization potential. Nicotinamide mononucleotide (NMN) reverses age-related dysfunctions in various organs. Studies had shown long-term administration of NMN reduced the physiological decline associated in aged mice and reversed the aging of the ovaries. However, the protective effect of NMN on aged porcine oocytes is still unclear. In this study, we investigated the effects of NMN on aging porcine oocytes and subsequent embryonic development. We established a model of senescence of porcine oocytes after ovulation by extending the culture time in vitro. NMN supplementation significantly reduced reactive oxygen species (ROS) levels in senescence oocytes and increased the mRNA levels of antioxidant genes SOD1 and Cat. The mitochondrial membrane potential of aged oocytes treated with NMN was increased compared with that of untreated oocytes. In addition, the mRNA level of apoptosis-related gene Bax was significantly decreased in senescence oocytes treated with NMN, while the mRNA level of anti-apoptosis-related gene BCL-2 was significantly increased. Furthermore, NMN supplementation enhanced the subsequent development ability of senescent oocytes during in vitro aging. Compared with untreated senescent oocytes, the blastocyst formation rate and pluripotent genes of senescent oocytes treated with NMN were significantly increased. Taken together, these results suggest that NMN is beneficial for delaying the aging process in porcine oocytes.

Epigenetic modifications play critical roles during somatic cell nuclear transfer (SCNT) embryo development. Whether RNA N6-methyladenosine (m6A) affects the developmental competency of SCNT embryos remains unclear. Here, we showed that porcine bone marrow mesenchymal stem cells (pBMSCs) presented higher RNA m6A levels than those of porcine embryonic fibroblasts (pEFs). SCNT embryos derived from pBMSCs had higher RNA m6A levels, cleavage, and blastocyst rates than those from pEFs. Compared with pEFs, the promoter region of METTL14 presented a hypomethylation status in pBMSCs. Mechanistically, DNA methylation regulated METTL14 expression by affecting the accessibility of transcription factor SP1 binding, highlighting the role of the DNA methylation/SP1/METTL14 pathway in donor cells. Inhibiting the DNA methylation level in donor cells increased the RNA m6A level and improved the development efficiency of SCNT embryos. Overexpression of METTL14 significantly increased the RNA m6A level in donor cells and the development efficiency of SCNT embryos, whereas knockdown of METTL14 suggested the opposite result. Moreover, we revealed that RNA m6A-regulated TOP2B mRNA stability, translation level, and DNA damage during SCNT embryo development. Collectively, our results highlight the crosstalk between RNA m6A and DNA methylation, and the crucial role of RNA m6A during nuclear reprogramming in SCNT embryo development.

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

Background: The complex formed by disulfiram (DSF) and copper (Cu) is safe and effective for the prevention and treatment of triple-negative breast cancer (TNBC). Although previous studies have shown that DSF/Cu induces ferroptosis, the mechanism remains unclear. Methods: The mitochondrial morphology of TNBC treated with DSF/Cu was observed by transmission microscopy, and intracellular levels of iron, lipid reactive oxygen species (ROS), malondialdehyde, and glutathione were evaluated to detect the presence of ferroptosis. Target genes for the DSF/Cu-activated ferroptosis signaling pathway were examined by transcriptome sequencing analysis. Expression of the target gene, HOMX1, was detected by qRT-PCR, immunofluorescence and western blot. Results: The mitochondria of TNBC cells were significantly atrophied following treatment with DSF/Cu for 24 h. Addition of DSF/Cu supplement resulted in significant up-regulation of intracellular iron, lipid ROS and malondialdehyde levels, and significant down-regulation of glutathione levels, all of which are important markers of ferroptosis. Transcriptome analysis confirmed that DSF/Cu activated the ferroptosis signaling pathway and up-regulated several ferroptosis target genes associated with redox regulation, especially heme oxygenase-1 (HMOX-1). Inhibition of ferroptosis by addition of the ROS scavenger N-acetyl-L-cysteine (NAC) significantly increased the viability of DSF/Cu-treated TNBC cells. Conclusions: These results show that DSF/Cu increases lipid peroxidation and causes a sharp increase in HMOX1 activity, thereby inducing TNBC cell death through ferroptosis. DSF/Cu is a promising therapeutic drug for TNBC and could lead to ferroptosis-mediated therapeutic strategies for human cancer.

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naive-like conversion, based on a naive medium (NM). The results showed successful conversion of the primed bovine iPSCs to naive-like state within 3-4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naive-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naive-like iPSCs. We further analyzed the naive markers and differentiation po-tential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naive-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naive-like bovine iPSCs, and improve the conversion efficiency.(c) 2022 Elsevier Inc. All rights reserved.

Abstract Background The transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited. Results In this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species. Conclusions Our results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

Epigenetic mechanisms play an important role in oogenesis and early embryo development in mammals. Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy. Recent studies suggest that DMSO detrimentally affects porcine embryonic development, yet the mechanism of the process in parthenogenetically activated porcine embryos has not been reported. In this study, we found that treatment of embryos with 1.5% DMSO significantly decreased the cleavage and blastocyst rates, total cell number of blastocysts and the anti-apoptotic gene BCL-2 transcription level; however, the percentage of apoptotic cells and the expression levels of the pro-apoptotic gene BAX were not changed. Treatment with DMSO significantly decreased the expression levels of DNMT1 , DNMT3a , DNMT3b , TET1 , TET2 , TET3 , KMT2C , MLL2 and SETD3 in most of the stages of embryonic development and increased 5-mC signals, while the staining intensity for 5-hmC had no change in porcine preimplantation embryos from 2-cell to the blastocyst stages. Meanwhile, DMSO decreased the level of H3K4me3 during the development of parthenogenetically activated porcine embryos. After treatment with DMSO, expression levels of the pluripotency-related genes POU5F1 and NANOG decreased significantly (P <0.01), whereas the imprinted gene H19 did not change (P >0.05). In conclusion, these results suggest that DMSO can affect genome-wide DNA methylation and histone modification by regulating the expression of epigenetic modification enzymes, and DMSO also influences the expression level of pluripotent genes. These dysregulations lead to defects in embryonic development.

The study of preimplantation development is of great significance to reproductive biology and regenerative medicine. With the development of high-throughput deep sequencing technology, it has been found that lncRNAs play a very important role in the regulation of embryonic development. In this study, key lncRNAs that regulate embryonic development were screened by analyzing the expression pattern of lncRNAs in porcine in vivo fertilization (IVV) embryos. By knocking down lncRNA expression in in vitro fertilization (IVF) embryos, we investigated its function and mechanism of regulating embryonic development. The results showed that the expression pattern of lncRNA was consistent with the time of gene activation. The lncRNAs were highly expressed in the 4-cell to blastocyst stage but barely expressed in the oocytes and 2-cell stage. So we speculated this part of lncRNAs may regulate gene expression. The lncRNA LOC102165808 (named lncT because the gene near this lncRNA is TFAP2C) was one of them. The knockdown (KD) of lncT inhibited embryonic development, resulting in decreased H3K4me3, H3K4me2, and H3K9me3, and increased DNA methylation. Meanwhile, RNAseq showed SIN3A was the top decreased gene in lncT-KD embryos. There was a severe blastocyst formation defect in SIN3A-KD embryos. Both lncT and SIN3A could affect NANOG and induce more cell apoptosis. In conclusion, the knockdown of lncT inhibits embryonic development by regulating H3K4me3, H3K4me2, DNA methylation, pluripotency gene, and apoptosis, and SIN3A is one of the downstream genes of lncT in regulating embryonic development.

Because of the lack of specific molecular targeted therapies, triple-negative breast cancer (TNBC) has high tumour recurrence and metastasis rates. It is urgent to develop novel chemotherapeutic strategies to improve patient survival. DNA damaging agents have been shown to sensitize cancer to genotoxic chemotherapies. We first found that 6-thioguanine (6-TG) can activate the NF-кB signalling pathway. Our results showed that NF-кB signalling was reduced when cells were treated with 6-TG/disulfiram (DSF)/Cu. DSF/Cu enhanced the 6-TG-mediated inhibition of proliferation. 6-TG/DSF/Cu inhibited cell cycle progression, causing cell cycle arrest in the S phase and G2/M phase. Moreover, the combined effect of 6-TG and DSF/Cu induced apoptosis, and either agent alone was able to induce apoptosis. The accumulation of γH2A indicated that DSF/Cu increased the DNA damage induced by 6-TG. Combined treatment with 6-TG and DSF/Cu synergistically reduced the levels of both phosphorylated and total ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), suggesting that DSF/Cu promoted 6-TG-induced DNA damage by suppressing ATR protein kinases, therefore enhancing cell apoptosis. In conclusion, we demonstrate that the combination of 6-TG and DSF/Cu exerted a significant synergistic antitumour effect on human TNBC in vitro and in vivo by enhancing DNA damage and disrupting DNA damage checkpoints. We propose that this combination therapy could be a novel strategy for the treatment of TNBC.

Maturation of oocytes and early embryo development are regulated precisely by numerous factors at transcriptional and posttranslational levels through precise mechanisms. N6-methyladenosine (m6A) is the most common modification in mRNA which regulates RNA metabolism and gene expression. However, the role of RNA m6A on porcine oocyte maturation and early embryogenesis is largely unknown. Here, we found that oocytes treated with cycloleucine (CL), an RNA m6A inhibitor, express impaired cumulus expansion, increased production of reactive oxygen species (ROS) in the mitochondria, and delayed maturation of oocytes by disrupting spindle organization and chromosome alignment. Also, CL halted the development of embryos at the 4-cell stage and resulted in low-quality blastocysts. Furthermore, CL treatment decreased the RNA m6A, H3K4me3, and H3K9me3 levels, but increased the acetylation level of H4K16 during parthenogenetic embryonic development in pigs. Single-cell RNA-seq (scRNA-seq) analysis further revealed that CL treatment dramatically up-regulated the expression of metabolism-related genes (SLC16A1, and MAIG3 etc.) and maternal related genes, including BTG4, WEE2, and BMP15 among others, at the blastocyst stage. Taken together, inhibition of RNA m6A by CL impaired meiosis of oocytes and early embryonic development of porcine via RNA m6A methylation, histone modifications, and altering the expression of metabolism-related genes in blastocysts.