Activation of HAND2-FGFR signaling pathway by lncRNA HAND2-AS1 in adenomyosis†.

Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2 expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2 promoter and the first exon was also monitored in these samples. Our results revealed that HAND2 expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2 in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2 and HAND2-AS1, and the first exon of HAND2 gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1 expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2 in response to HAND2-AS1 silencing, FGF9 expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1 exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1 is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.

Hydrogen Sulfide Exerted a Pro-Angiogenic Role by Promoting the Phosphorylation of VEGFR2 at Tyr797 and Ser799 Sites in Hypoxia-Reoxygenation Injury.

The protective effects of hydrogen sulfide (H2S) against ischemic brain injury and its role in promoting angiogenesis have been established. However, the specific mechanism underlying these effects remains unclear. This study is designed to investigate the regulatory impact and mechanism of H2S on VEGFR2 phosphorylation. Following expression and purification, the recombinant His-VEGFR2 protein was subjected to LC-PRM/MS analysis to identify the phosphorylation sites of VEGFR2 upon NaHS treatment. Adenovirus infection was used to transfect primary rat brain artery endothelial cells (BAECs) with the Ad-VEGFR2WT, Ad-VEGFR2Y797F, and Ad-VEGFR2S799A plasmids. The expression of VEGFR2 and recombinant Flag-VEGFR2, along with Akt phosphorylation, cell proliferation, and LDH levels, was assessed. The migratory capacity and tube-forming potential of BAECs were assessed using wound healing, transwell, and tube formation assays. NaHS notably enhanced the phosphorylation of VEGFR2 at Tyr797 and Ser799 sites. These phosphorylation sites were identified as crucial for mediating the protective effects of NaHS against hypoxia-reoxygenation (H/R) injury. NaHS significantly enhanced the Akt phosphorylation, migratory capacity, and tube formation of BAECs and upregulated the expression of VEGFR2 and recombinant proteins. These findings suggest that Tyr797 and Ser799 sites of VEGFR2 serve as crucial mediators of H2S-induced pro-angiogenic effects and protection against H/R injury.

Regulation of compound leaf development in mungbean (Vigna radiata L.) by CUP-SHAPED COTYLEDON/NO APICAL MERISTEM (CUC/NAM) gene.

MAIN CONCLUSION: The results provide a significant verification of functional redundancy and diversity of CUC/NAM genes in legumes. The CUP-SHAPED COTYLEDON/NO APICAL MERISTEM (CUC/NAM) orthologs play key roles for plant organ boundary formation and organ development. Here, we performed a forward screen of the gamma irradiation mutagenesis population in mungbean and characterised a mutant, reduced rachis and fused leaflets (rrf1), which gave rise to the formation of compound leaves with reduced rachis and fused leaflets. Map-based cloning revealed that RRF1 encoded a CUC/NAM protein in mungbean. Phylogenetic analysis indicated that legume CUC1/CUC2 genes were classified as belonging to two subclades, and there are different copies of CUC1/CUC2 genes in legumes. Transcriptomic analysis showed that expression levels of a set of developmental regulators, including class I KNOTTED-LIKE HOMEOBOXI (KNOXI) gene and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) gene, were altered in rrf1 mutants compared to the wild-type plants. Furthermore, rrf1 genetically interacted with heptafoliate leaflets1 (hel1), a mutant displaying a seven-leaflet compound leaf, to regulate leaf development in mungbean. Our results suggest functional redundancy and diversity of two subclades of CUC1/CUC2 genes in legumes, following the duplication of an ancestral gene.

The Causal Effect of Urate Level on Female Infertility: A Mendelian Randomization Study.

BACKGROUND/OBJECTIVE: This study aimed to investigate the causal relationship between urate level and female infertility using Mendelian randomization (MR) analysis. METHODS: To identify instrumental variables, we selected independent genetic loci associated with serum urate levels in individuals of European ancestry, utilizing data from large-scale genome-wide association studies (GWAS). The GWAS dataset included information on serum urate levels from 288,649 CKDGen participants. Female infertility data, including different etiologic classifications, consisted of 13,142 female infertility patients and 107,564 controls. We employed four MR methods, namely inverse variance weighted (IVW), MR-Egger, weighted median, and weighted model, to investigate the causal relationship between urate levels and female infertility. The Cochran Q-test was used to assess heterogeneity among single nucleotide polymorphisms (SNPs), and the MR-Egger intercept test was employed to evaluate the presence of horizontal pleiotropy. Additionally, a "leave-one-out" sensitivity analysis was conducted to examine the influence of individual SNPs on the MR study. RESULTS: The IVW analysis demonstrated that elevated serum urate levels increased the risk of female infertility (odds ratio [OR] = 1.18, 95% confidence interval [CI]: 1.07-1.33). Furthermore, serum urate levels were found to be associated with infertility due to cervical, vaginal, or other unknown causes (OR = 1.16, 95% CI: 1.06-1.26), also confirmed by other methods. Heterogeneity among instrumental variables was assessed using Cochran's Q-test (p < 0.05), so a random-effects IVW approach was employed in the effects model. The MR-Egger intercept test indicated no presence of horizontal pleiotropy. A "leave-one-out" sensitivity analysis was conducted, demonstrating that no individual SNP had a substantial impact on the overall findings. CONCLUSIONS: In the European population, the urate level is significantly and causally associated with an increased risk of female infertility.

H2S protects rat cerebral ischemia-reperfusion injury by inhibiting expression and activation of hippocampal ROCK2 at the Thr436 and Ser575 sites.

BACKGROUND: H2S is an endogenous gas signal molecule, which protects cerebral ischemia/reperfusion (I/R) injury by phosphorylating rho-associated coiled coil-containing protein kinase 2 (ROCK2) at Tyr722, and inhibiting ROCK2 protein expression and activities. We previously reported that H2S protected rat neurons from hypoxia/reoxygenation injury in vitro through inhibiting phosphorylation of ROCK2 at Thr436 and Ser575, but it is unclear whether these two sites are involved in protection of H2S against cerebral I/R injury. METHOD: Rats transfected with wild-type and mutant eukaryotic plasmids of ROCK2 in hippocampus were used to establish I/R model by ligating bilateral common carotid artery. Rat behavioral deficit was detected by water maze assay, and ROCK2, lactate dehydrogenase (LDH), nerve-specific enolase (NSE) and reactive oxygen species (ROS) were determined by ELISA. ROCK2 expressions was examined by western-blot assay, and bcl-2 and Bax mRNAs were examined by RT-qPCR. RESULTS: NaHS (4.8mg/kg) significantly inhibited the I/R-increased serum LDH, NSE and ROS in the ROCK2wild-pEGFP-N1-transfected rats, but had no obvious effect in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats; inhibitions of NaHS on the I/R-increased escape latency and the I/R-decreased percentage of target quadrant distance to total distance were markedly attenuated or abolished in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats compared with those in the ROCK2wild-pEGFP-N1-transfected rats; NaHS obviously inhibited the I/R-increased hippocampal ROCK2 and GFP-ROCK2 proteins, Bax mRNA, and ROCK2 activity, as well as the I/R-decreased hippocampal bcl-2 mRNA in the hippocampus of the ROCK2wild-pEGFP-N1-transfected rats, but had no significant effect in the ROCK2T436A-pEGFP-N1- or the ROCK2S575F-pEGFP-N1-transfected rats. CONCLUSION: H2S protects cerebral I/R injury in rats by inhibiting expression and activation of hippocampal ROCK2 via the Thr436 and Ser575 sites.

Regulation of HAND2 Expression by LncRNA HAND2-AS1 in Ovarian Endometriosis Involving DNA Methylation.

HAND2 is a critical mediator of progesterone receptor signaling in endometrium. Silencing of HAND2 expression is associated with female infertility and endometrial cancers. We recently observed that lncRNA HAND2-AS1 and HAND2 are expressed coordinately in human endometrial stromal cells. To investigate involvement of HAND2-AS1 and HAND2 in pathogenesis of endometriosis, we employed immunohistochemistry, in situ hybridization, and quantitative real-time PCR to assess their expression in normal endometrium and the ectopic lesions obtained from patients with ovarian endometriosis. HAND2 promoter methylation was also monitored in these samples. Our results revealed that HAND2 and HAND2-AS1 expression levels were reduced but promoter methylation was enhanced significantly in ectopic endometrium when compared with the normal controls. Fluorescence in situ hybridization showed that HAND-AS1 is predominantly localized in the nuclei of endometrial stromal cells in contrast to the cytoplasmic distribution in epithelial cell compartment. To further investigate regulation of HAND2 expression by HAND2-AS1, HAND2-AS1 was silenced or overexpressed in human endometrial stromal cells. Our studies showed that expression levels of HAND2 and its direct target IL15 were attenuated markedly in HAND2-AS1 silenced cells but enhanced significantly in the overexpressed human endometrial stromal cells. Silencing of HAND2-AS1 also impaired endometrial stromal cell decidualization as indicated by downregulation of decidual biomarkers IGFBP1 and PRL. In addition, HAND2 promoter methylation was also enhanced upon HAND2-AS1 silencing. RNA immunoprecipitation studies further revealed that HAND2-AS1 is capable of binding to DNA methyltransferase DNMT1, indicating that HAND2-AS1 governs HAND2 expression epigenetically involving DNA methylation.

Genetic control of compound leaf development in the mungbean (Vigna radiata L.).

Many studies suggest that there are distinct regulatory processes controlling compound leaf development in different clades of legumes. Loss of function of the LEAFY (LFY) orthologs results in a reduction of leaf complexity to different degrees in inverted repeat-lacking clade (IRLC) and non-IRLC species. To further understand the role of LFY orthologs and the molecular mechanism in compound leaf development in non-IRLC plants, we studied leaf development in unifoliate leaf (un) mutant, a classical mutant of mungbean (Vigna radiata L.), which showed a complete conversion of compound leaves into simple leaves. Our analysis revealed that UN encoded the mungbean LFY ortholog (VrLFY) and played a significant role in leaf development. In situ RNA hybridization results showed that STM-like KNOXI genes were expressed in compound leaf primordia in mungbean. Furthermore, increased leaflet number in heptafoliate leaflets1 (hel1) mutants was demonstrated to depend on the function of VrLFY and KNOXI genes in mungbean. Our results suggested that HEL1 is a key factor coordinating distinct processes in the control of compound leaf development in mungbean and its related non-IRLC legumes.