Elaboration and validation of a prognostic signature associated with disulfidoptosis in lung adenocarcinoma, consolidated with integration of single-cell RNA sequencing and bulk RNA sequencing techniques.

Background: Lung adenocarcinoma (LUAD), the predominant subtype of non-small cell lung cancer (NSCLC), remains a pervasive global public health concern. Disulfidoptosis, a nascent form of regulated cell death (RCD), presents an emerging field of inquiry. Currently, investigations into disulfidoptosis are in their initial stages. Our undertaking sought to integrate single-cell RNA sequencing (scRNA-seq) in conjunction with traditional bulk RNA sequencing (bulk RNA-seq) methodologies, with the objective of delineating genes associated with disulfidoptosis and subsequently prognosticating the clinical outcomes of LUAD patients. Methods: Initially, we conducted an in-depth examination of the cellular composition disparities existing between LUAD and normal samples using scRNA-seq data sourced from GSE149655. Simultaneously, we scrutinized the expression patterns of disulfidoptosis-associated gene sets across diverse cell types. Subsequently, leveraging the bulk RNA-seq data, we formulated disulfidoptosis-related prognostic risk signatures (DRPS) employing LASSO-Cox regression. This was accomplished by focusing on genes implicated in disulfidoptosis that exhibited differential expression within endothelial cells (ECs). Sequentially, the robustness and precision of the DRPS model were rigorously verified through both internal and external validation datasets. In parallel, we executed single-cell trajectory analysis to delve into the differentiation dynamics of ECs. Concluding our study, we undertook a comprehensive investigation encompassing various facets. These included comparative assessments of enrichment pathways, clinicopathological parameters, immune cell abundance, immune response-associated genes, impacts of immunotherapy, and drug predictions among distinct risk cohorts. Results: The scrutiny of scRNA-seq data underscored discernible disparities in cellular composition between LUAD and normal samples. Furthermore, disulfidoptosis-associated genes exhibited marked discrepancies within endothelial cells (ECs). Consequently, we formulated the Disulfidoptosis-Related Prognostic Signature (DRPS) to facilitate prognostic prediction. The prognostic nomogram based on the risk score effectively demonstrated DRPS's robust capacity to prognosticate survival outcomes. This assertion was corroborated by rigorous assessments utilizing both internal and external validation sets, thus affirming the commendable predictive accuracy and enduring stability of DRPS. Functional enrichment analysis shed light on the significant correlation of DRPS with pathways intrinsic to the cell cycle. Subsequent analysis unveiled correlations between DRPS and gene mutations characteristic of LUAD, as well as indications of an immunosuppressive status. Through drug prediction, we explored potential therapeutic agents for low-risk patients. Concluding our investigation, qRT-PCR experiments confirmed the heightened expression levels of EPHX1, LDHA, SHC1, MYO6, and TLE1 in lung cancer cell lines.

Fibrinogen beta chain may be a potential predict biomarker for pre-eclampsia: A preliminary study.

OBJECTIVE: There are no approaches for the early detection of pre-eclampsia (PE). Using parallel reaction monitoring proteomics, we investigated 79 maternal serum protein changes before PE onset and its predictive capability. METHODS: We conducted a nested case-control study with 60 PE patients and 60 normotensive pregnant women matched for age and gestational week. These differentially expressed proteins were quantified using the data-dependent acquisition (DDA) combined parallel response monitoring (PRM) approach, and a PE prediction model was developed using the least absolute shrinkage and selection operator (LASSO) regression. We further examined the link between these biomarkers and PE using bioinformatics. ELISA assay was used to investigate the expression and clinical significance of the critical variables. RESULTS: Among the 79 analyzed proteins, we identified 11 serum proteins with significantly abnormal expression. Fibrinogen beta chain (FGB) was likely connected with the progression of PE due to the positive correlation between their levels and those of hypertension and proteinuria. In addition, an early prediction model for PE with an AUC of 92% was developed using LASSO regression. CONCLUSION: Our research employs predictive algorithms and screens for relevant variables, which could result in a potential approach to enhancing PE prediction.

A Novel Circular RNA CircBRAP May Be Used as an Early Predictor of Preeclampsia and Its Potential Mechanism.

Preeclampsia (PE), a pregnancy-related multisystem syndrome, is one of the leading causes of maternal and fetal mortality worldwide. The aim of this study was to combine the plasma protein soluble Fms-related tyrosine kinase 1 (sFLT1) levels with uterine artery Doppler ultrasound findings and CircBRAP levels during the first trimester to predict the occurrence of preeclampsia and to explore the potential mechanism by which CircBRAP functions in preeclampsia. Here, we used qRT-PCR to investigate the expression of CircBRAP in forty-nine pairs of plasma specimens and placental tissues from preeclampsia patients and control subjects. The uterine artery (UtA) pulsatility index (PI) was measured using four-dimensional color Doppler ultrasound, and the sFLT1 levels were evaluated by human immunoassay. Exogenous upregulation or downregulation of CircBRAP expression in TEV-1 trophoblast cells was performed to investigate the role of CircBRAP in cell biological behavior. Mechanistically, luciferase reporter, RNA immunoprecipitation (RIP), and biotin-coupled RNA pull-down assays were conducted to verify the relationship between CircBRAP and sFLT1 in TEV-1 cells. The results showed that the predictive power was strengthened when the plasma sFLT1 and CircBRAP levels were combined with the UtA-PI to predict preeclampsia occurrence. Our study also revealed that CircBRAP may regulate miR-106b and the HIF-2α axis to modulate the proliferation, invasion, and apoptosis of TEV-1 trophoblast cells. In summary, placenta-derived CircBRAP in plasma may be a novel biomarker for preeclampsia that, together with plasma sFLT1 levels and uterine artery Doppler ultrasound findings, can more effectively predict preeclampsia, and CircBRAP may play a potential role in preeclampsia.

circRNA N6-methyladenosine methylation in preeclampsia and the potential role of N6-methyladenosine-modified circPAPPA2 in trophoblast invasion.

Here, we performed N6-methyladenosine (m6A) RNA sequencing to determine the circRNA m6A methylation changes in the placentas during the pathogenesis of preeclampsia (PE). We verified the expression of the circRNA circPAPPA2 using quantitative reverse transcription-PCR. An invasion assay was carried out to identify the role of circPAPPA2 in the development of PE. Mechanistically, we investigated the cause of the altered m6A modification of circPAPPA2 through overexpression and knockdown cell experiments, RNA immunoprecipitation, fluorescence in situ hybridization and RNA stability experiments. We found that increases in m6A-modified circRNAs are prevalent in PE placentas and that the main changes in methylation occur in the 3'UTR and near the start codon, implicating the involvement of these changes in PE development. We also found that the levels of circPAPPA2 are decreased but that m6A modification is augmented. Furthermore, we discovered that methyltransferase­like 14 (METTL14) increases the level of circPAPPA2 m6A methylation and that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) maintains circPAPPA2 stability. Decreases in IGF2BP3 levels lead to declines in circPAPPA2 levels. In summary, we provide a new vision and strategy for the study of PE pathology and report that placental circRNA m6A modification appears to be an important regulatory mechanism.

Plasma SerpinA5 in conjunction with uterine artery pulsatility index and clinical risk factor for the early prediction of preeclampsia.

OBJECT: This study aimed to combine plasma protein SerpinA5 with uterine artery doppler ultrasound and clinical risk factor during the first trimester for prediction of preeclampsia. METHODS AND MATERIALS: This study was a nested cohort study and was divided into the screening set and developing set. The plasma was collected during the first trimester (11+0-13+6 weeks), at the same time, UtA-PI was detected and recorded with four-dimensional color Doppler ultrasound. These pregnancies were followed up until after delivery. The plasma proteins were examined using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and enzyme linked immunosorbent assay (ELISA). Placental samples preserved after delivery were analysed by immunohistochemistry. Clinical risk factors were obtained from medical records or antenatal questionnaires. Upregulation or downregulation of SerpinA5 expression in TEV-1 cells was performed to investigate the role of SerpinA5 in trophoblasts invasion. RESULTS: We demonstrated that SerpinA5 levels were greater not only in preeclampsia placental tissue but also in plasma (both p<0.05), and we found that SerpinA5 may interfere with trophoblastic cell invasion by inhibiting MSP. SerpinA5 may be a potential predictor of preeclampsia. What is more, the sensitivity and specificity of predictive power were strengthened when plasma SerpinA5 was combined with UtA-PI and pre-pregnancy BMI & family history of PE for prediction of preeclampsia. CONCLUSION: These findings showed that placenta-derived plasma SerpinA5 may be a novel biomarker for preeclampsia, which together with uterine artery Doppler ultrasound and clinical risk factor can more effectively predict preeclampsia.

Interaction between retinoic acid and FGF/ERK signals are involved in Dexamethasone-induced abnormal myogenesis during embryonic development.

Despite the common application in pregnancy at clinical practice, it remains ambiguous whether dexamethasone (Dex) exposure can affect embryonic myogenesis. In this study, firstly we showed that 10-6 M Dex (Cheng et al., 2016; 2017) treatment resulted in abnormal myogenesis in chicken embryos. Secondly, we demonstrated that 10-6 M Dex-induced abnormality of myogenesis resulted from aberrant cell proliferation, as well as from alteration of the differentiation process from the early stage of somitogenesis up to the late stage of myogenesis. The above-mentioned results caused by Dex exposure might be due to the aberrant gene expressions of somite formation (Raldh2, Fgf8, Wnt3a, ß-catenin, Slug, Paraxis, N-cadherin) and differentiation (Pax3, MyoD, Wnt3a, Msx1, Shh). Thirdly, RNA sequencing implied the statistically significant differential gene expressions in regulating the myofibril and systemic development, as well as a dramatical alteration of retinoic acid (RA) signaling during somite development in the chicken embryos exposed to Dex. The subsequent validation experiments verified that Dex treatment indeed led to a metabolic change of RA signaling, which was up-regulated and principally mediated by FGF-ERK signaling revealed by means of the combination of chicken embryos and in vitro C2C12 cells. These findings highlight that 10-6 M Dex exposure enhances the risk of abnormal myogenesis through interfering with RA signaling during development.

circCRAMP1L is a novel biomarker of preeclampsia risk and may play a role in preeclampsia pathogenesis via regulation of the MSP/RON axis in trophoblasts.

BACKGROUND: Preeclampsia is a severe disease in pregnant women, which is primarily managed by early screening and prevention. Circular RNAs (circRNAs) have increasingly been shown to be important biological regulators involved in numerous diseases. Further, increasing evidence has demonstrated that circRNAs can be used as diagnostic biomarkers. This study was conducted to evaluate the potential of circCRAMP1L, previously identified to be downregulated in preeclampsia, as a novel biomarker for predicting the development of preeclampsia. METHODS: We measured the expression of circCRAMP1L, which is reportedly relevant to trophoblast physiology, in plasma samples from 64 patients with preeclampsia and 64 age-, gestational age-, and body mass index-matched healthy pregnant women by qRT-PCR. MTT proliferation and transwell invasion assays revealed the biological role of circCRAMP1L in preeclampsia pathogenesis. RNA immunoprecipitation and dual-luciferase reporter assays clarified the mechanism underlying the biological function of circCRAMP1L in TEV-1 cells. RESULTS: circCRAMP1L circulating levels were significantly lower in patients with preeclampsia (2.66 ± 0.82, △Ct value) than in healthy pregnant women (3.95 ± 0.67, △Ct value, p <  0.001). The area under the receiver operating characteristic curve for circCRAMP1L was 0.813. Univariate and multivariate analyses identified circCRAMP1L as an independent predictor of preeclampsia. Furthermore, when circCRAMP1L was utilised in combination with its target protein macrophage stimulating protein (MSP), the predictive performance increased, with an area under the receiver operating characteristic curve of 0.928 (95% CI 0.882-0.974), 80.0% sensitivity, and 80.0% specificity. The in vitro results indicated that circCRAMP1L regulates cell proliferation, and invasion via MSP and RON proteins. We investigated the molecular mechanisms of these effects. In vitro, relative to the control group, circCRAMP1L overexpression significantly enhanced cell proliferation; furthermore, trophoblast cell invasion increased proportionally with circCRAMP1L expression. RNA immunoprecipitation and luciferase reporter gene illustrated that circCRAMP1L participated in regulation of trophoblast cell by regulating MSP. CONCLUSION: Reduced plasma levels of circCRAMP1L may be associated with an increased risk of preeclampsia, and circCRAMP1L may be a novel biomarker of preeclampsia risk.

CircSFXN1 regulates the behaviour of trophoblasts and likely mediates preeclampsia.

Preeclampsia (PE) is the second most common complication that threatens the health of pregnant women and their foetuses; however, the underlying mechanisms are poorly understood. Circular RNAs (circRNAs) are involved in various human diseases, and an increasing number of studies have revealed the vital role of circRNAs in PE. Here, we investigated the biological function of circRNA-SFXN1 (CircSFXN1) in PE and the associated molecular mechanisms. Microarray data analysis revealed that CircSFXN1 was highly expressed in PE placentas compared to control placentas; this finding was confirmed by qRT-PCR. In vitro, CircSFXN1 overexpression significantly inhibited the invasion of TEV-1 trophoblasts and blocked the angiogenesis of human umbilical vein endothelial cells (HUVECs), while CircSFXN1 knockdown promoted trophoblast invasion and stimulated HUVEC angiogenesis. For in vivo evaluation, pregnant Sprague-Dawley rats were randomly selected for tail vein injection with sFLT1-expressing adenovirus, which resulted in elevated blood pressure and increased proteinuria; si-CircSFXN1 reversed these increases. Mechanistic analyses via RNA-protein pulldown, RNA immunoprecipitation (RIP) and dual-luciferase reporter assays showed that CircSFXN1 recruits sFLT1 and modulates the biological behaviour of trophoblasts by binding sFLT1. In summary, we identified a novel circRNA that regulates tumorigenic activities, suggesting a new pathway governing CircSFXN1/sFLT1-mediated regulation of trophoblast behaviour.