Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.

Pyrimidinergic receptor P2Y6 expression is elevated in lung adenocarcinoma and is associated with poor prognosis.

BACKGROUD: Previous in vitro studies have indicated that pyrimidinergic receptor P2Y6 (P2RY6, P2Y6 receptor) may function as a cancer-promoting factor in lung adenocarcinoma (LUAD). However, the prognostic significance of P2RY6 expression in LUAD has not been investigated. OBJECTIVE: This study aimed to assess the impact of P2RY6 expression on the survival of patients with LUAD. METHODS: First, we assessed P2RY6 mRNA and protein expression in LUAD and non-cancerous lung tissues using the online bioinformatics analysis tool GEPIA, fresh LUAD tissues, and LUAD tissue microarrays (TMAs). Second, we investigated the correlation between P2RY6 expression and clinicopathological parameters of LUAD patients based on data from The Cancer Genome Atlas (TCGA) database and TMAs. Finally, we analyzed the prognostic significance of P2RY6 expression in LUAD using the online survival analysis tool Kaplan-Meier Plotter and data from TMAs. RESULTS: We demonstrated that P2RY6 mRNA and protein expression levels in LUAD tissues were significantly higher than those in non-cancerous lung tissues. The expression of P2RY6 in LUAD was positively correlated with poor differentiation, more lymph node metastasis, and more advanced clinical stage. Higher P2RY6 expression level was correlated with shorter survival of the LUAD patients. Univariate and multivariate Cox regression analyses indicated that higher P2RY6 tumor expression was an independent unfavorable prognostic factor for LUAD patients. CONCLUSIONS: P2RY6 expression was elevated in LUAD and correlated with poor prognosis.

Overexpression of PELP1 in Lung Adenocarcinoma Promoted E2 Induced Proliferation, Migration and Invasion of the Tumor Cells and Predicted a Worse Outcome of the Patients.

The expression of Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) has been reported to be dysregulated in non-small cell lung carcinoma, especially in lung adenocarcinoma (LUAD). Therefore, we aimed to investigate the functional and prognostic roles of PELP1 in LUAD in this study. We first immunolocalized PELP1 in 76 cases of LUAD and 17 non-pathological or tumorous lung (NTL) tissue specimens and correlated the findings with the clinicopathological parameters of the patients. We then performed in vitro analysis including MTT, flow cytometry, wound healing, and transwell assays in order to further explore the biological roles of PELP1 in 17-ß-estradiol (E2) induced cell proliferation, migration, and invasion of LUAD cells. We subsequently evaluated the prognostic significance of PELP1 in LUAD patients using the online survival analysis tool Kaplan-Meier Plotter. The status of PELP1 immunoreactivity in LUAD was significantly higher than that in the NTL tissues and significantly positively correlated with less differentiated features of carcinoma cells, positive lymph node metastasis, higher clinical stage as well as the status of ERα, ERß, and PCNA. In vitro study did reveal that E2 promoted cell proliferation and migration and elevated PELP1 protein level in PELP1-high A549 and H1975 cells but not in PELP1-low H-1299 cells. Knock down of PELP1 significantly attenuated E2 induced cell proliferation, colony formation, cell cycle progress as well as migration and invasion of A549 and H1975 cells. Kaplan-Meier Plotter revealed that LUAD cases harboring higher PELP1 expression had significantly shorter overall survival. In summary, PELP1 played a pivotal role in the estrogen-induced aggressive transformation of LUAD and could represent adverse clinical outcome of the LUAD patients.

Estradiol-Induced MMP-9 Expression via PELP1-Mediated Membrane-Initiated Signaling in ERα-Positive Breast Cancer Cells.

Proline-, glutamic acid-, leucine-rich protein 1 (PELP1) is a novel estrogen receptor (ER) coregulator, demonstrated distinctive characters from other ERα coregulators, and has been suggested to be involved in metastasis of several cancers. In ERα-positive breast cancer, PELP1 overexpression enhanced ruffles and filopodium-like structure stimulated by estradiol (E2) through extranuclear cell signaling transduction hereby increased cell motility. However, whether PELP1 is also involved in extracellular matrix remodeling of ERα-positive breast cancer cells is still unknown. In this study, we investigated the role of PELP1 in E2-induced MMP-9 expression and the underlined mechanism. The results demonstrated the following: E2-induced ERα-positive MCF-7 breast cancer cell MMP-9 mRNA and protein expression in a rapid response and concentration-dependent manner. Knocked down PELP1 significantly suppressed E2-induced MMP-9 expression. E2-bovine serum albumin (BSA), a large molecular membrane-impenetrable conjugate of E2, can also upregulate MMP-9 protein expression in MCF-7, and the action of E2-BSA can be abolished by PI3K inhibitor LY294002; treating MCF-7 simultaneously with PELP1-shRNA and LY294002 did not show synergetic inhibitory effect on E2-BSA-induced MMP-9 expression. Our results indicated that estrogen-induced MMP-9 expression in ER-positive breast cancer cells may be through PELP1-mediated PI3K/Akt signaling pathway.

Kdm6a overexpression improves the development of cloned mouse embryos.

Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

RNAi-mediated knockdown of Parp1 does not improve the development of female cloned mouse embryos.

Somatic cell nuclear transfer is an important technique for life science research, but its efficiency is still extremely low, and most genes that are important during early development, such as X chromosome-linked genes, are not appropriately expressed during this process. Poly (ADP-ribose) polymerase (PARP) is an enzyme that transfers ADP ribose clusters to target proteins. PARP family members such as PARP1 participate in cellular signalling pathways through poly (ADP-ribosylation) (PARylation), which ultimately promotes changes in chromatin structure, gene expression, and the localization and activity of proteins that mediate signalling responses. PARP1 is associated with X chromosome inactivation (Xi). Here, we showed that abnormal Xi occurs in somatic cell nuclear transfer (NT) blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. Parp1 expression was higher in female NT blastocysts than that in intracytoplasmic sperm injection (ICSI) embryos but not in male NT blastocysts. After knocking down Parp1 expression, both the pre-rRNA 47S and X-inactivation-specific transcript (Xist) levels increased. Moreover, the expression of genes on the inactivated X chromosome, such as Magea6 and Msn, were also increased in the NT embryos. However, the development of Parp1si NT embryos was impaired, although total RNA sequencing showed that overall gene expression between the Parp1si NT blastocysts and the control was similar. Our findings demonstrate that increases in the expression of several genes on the X chromosome and of rRNA primary products in NT blastocysts with disrupted Parp1 expression are insufficient to rescue the impaired development of female cloned mouse embryos and could even exacerbate the associated developmental deficiencies.

Methyl-CpG-Binding Protein 2 Improves the Development of Mouse Somatic Cell Nuclear Transfer Embryos.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis.

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8-cell stage (named as a2 PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4-cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4-cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post-implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP-a4 PgESCs which derived from GFP-4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5-day fetus totally derived from GFP-a4 PgESCs with germline contribution by 8-cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a4 PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.