Impact of Repeated Infantile Exposure to Surgery and Anesthesia on Gut Microbiota and Anxiety Behaviors at Age 6-9.

(1) Background: Preclinical as well as population studies have connected general anesthesia and surgery with a higher risk of abnormal cognitive development, including emotional development. Gut microbiota dysbiosis in neonatal rodents during the perioperative period has been reported, however, the relevance of this to human children who undergo multiple anesthesia for surgeries is unknown. Given the emerging role of altered gut microbes in propagating anxiety and depression, we sought to study whether repeated infantile exposures to surgery and anesthesia affect gut microbiota and anxiety behaviors later in life. (2) Methods: This is a retrospectively matched cohort study comparing 22 pediatric patients of less than 3 years of age with multiple exposures (≥3) to anesthesia for surgeries and 22 healthy controls with no history of exposure to anesthesia. The parent report version of the Spence Children's Anxiety Scale (SCAS-P) was applied to evaluate anxiety in children aged between 6 and 9 years old. Additionally, the gut microbiota profiles of the two groups were compared using 16S rRNA gene sequencing. (3) Results: In behavioral tests, the p-SCAS score of obsessive compulsive disorder and social phobia were significantly higher in children with repeated anesthesia exposure relative to the controls. There were no significant differences between the two groups with respect to panic attacks and agoraphobia, separation anxiety disorder, physical injury fears, generalized anxiety disorder, and the total SCAS-P scores. In the control group, 3 children out of 22 were found to have moderately elevated scores, but none of them had abnormally elevated scores. In the multiple-exposure group, 5 children out of 22 obtained moderately elevated scores, while 2 scored as abnormally elevated. However, no statistically significant differences were detected in the number of children with elevated and abnormally elevated scores. The data show that repeated anesthesia and surgical exposures in children led to long-lasting severe gut microbiota dysbiosis. (4) Conclusions: In this preliminary study, our findings demonstrated that early repeated exposures to anesthesia and surgical predisposes children to anxiety as well as long-term gut microbiota dysbiosis. We should confirm these findings in a larger data population size and with detailed analysis. However, the authors cannot confirm an association between the dysbiosis and anxiety.

T cell proliferation-related genes: Predicting prognosis, identifying the cold and hot tumors, and guiding treatment in clear cell renal cell carcinoma.

Background: Immunotherapy has become a new direction of current research because the effect of traditional radiotherapy and chemotherapy on clear cell renal cell carcinoma (ccRCC) is not satisfactory. T cell proliferation-related genes (TRGs) play a pivotal role in tumor progression by regulating the proliferation, activity, and function of immune cells. The purpose of our study is to construct and verify a prognostic model based on TRGs and to identify tumor subtypes that may guide treatment through comprehensive bioinformatics analyses. Methods: RNA sequencing data, clinical information, and somatic mutation data of ccRCC are obtained from The Cancer Genome Atlas (TCGA) database. We identified the prognosis-related TRGs which were differentially expressed between normal and tumor tissues. After dividing the patients into a train set and a test set according to proportion 1:1 randomly, the least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis were performed to construct a risk-stratified model. Its prediction performance was verified. Then, Gene Set Enrichment Analysis (GSEA), principal component analysis (PCA), tumor microenvironment (TME) analysis, and the half-maximal inhibitory concentration (IC50) prediction were performed between the different groups of patients. To further discuss the immunotherapy between hot and cold tumors, we divided all patients into two clusters based on TRGs through unsupervised learning. Analyzing the gene mutation and calculating the tumor mutation burden (TMB), we further explored the relationship between somatic mutations and grouping or clustering. Results: Risk-stratified model and nomogram predict the prognosis of ccRCC patients accurately. Functional enrichment analyses suggested that TRGs mainly focused on the biological pathways related to tumor progression and immune response. Different tumor microenvironment, drug resistance, and TMB can be distinguished clearly according to both risk stratification and tumor subtype clustering. Conclusion: In this study, a new stratification model of ccRCC based on TRGs was established, which can accurately predict the prognosis of patients. IC50 prediction may guide the application of anti-tumor drugs. The distinction between hot and cold tumors provides a reference for clinical immunotherapy.

METTL3-mediated N6-methyladenosine modification of DUSP5 mRNA promotes gallbladder-cancer progression.

N6-methyladenosine (m6A) RNA methylation and its associated methyltransferase METTL3 play an important role in tumorigenesis of a series of tumors. However, dysregulation of METTL3 in gallbladder cancer (GBC) remains obscure. Here, we showed that upregulated METTL3 level predicted poor prognosis and correlated with increased lymphatic metastasis and high TNM stage. Functionally, we found that METTL3 could promote cell proliferation, invasion, and migration of GBC-SD and NOZ cells. Mechanistically, we revealed the METTL3-mediated m6A-modification profile in GBC cells and identified DUSP5 as the downstream gene of METTL3. METTL3 promoted the degradation of DUSP5 mRNA in a YTHDF2-dependent manner. Rescue assays showed that downregulation of DUSP5 could attenuate the knockdown METTL3-mediated inhibition of cell proliferation, invasion, and migration of GBC-SD and NOZ cells. Thus, our finding shows that elevated METTL3 expression contributes to tumor aggression in GBC, suggesting that METTL3 is a possible prognostic predictor and therapeutic target against GBC.

NLGN3 promotes neuroblastoma cell proliferation and growth through activating PI3K/AKT pathway.

Neuroblastoma is the most common extracranial solid tumor of childhood, previous studies show synaptic protein neuroligin-3 (NLGN3) promotes glioma proliferation and growth, However, no investigation about the role of NLGN3 in neuroblastoma was reported. Here, we found NGLGN3 was significantly upregulated in neuroblastoma cells and tissues, its overexpression significantly promoted neuroblastoma cell proliferation and growth determined by MTT analysis, colony formation assay, cell cycle progression analysis, BrdU incorporation assay and animal model, while its knockdown inhibited cell proliferation and growth. Then we found NLGN3 could increase the phosphorylation level of AKT and the transcription activity of FOXO family, suggesting NLGN3 activated PI3K/AKT pathway, inhibition of PI3K/AKT pathway in NLGN3 overexpressing cells inhibited cell proliferation, confirming NLGN3 promoted neuroblastoma proliferation through activating PI3K/AKT pathway. In summary, we found NLGN3 promoted neuroblastoma cell proliferation and growth through activating PI3K/AKT pathway and providing a new target for neuroblastoma therapy.

GDNF family receptor alpha 2 promotes neuroblastoma cell proliferation by interacting with PTEN.

Neuroblastoma is a childhood tumor, and high-stage neuroblastoma has a poor prognosis. The regulatory mechanisms for neuroblastoma progression are poorly understood. In present study, we found that GDNF family receptor alpha 2 (GFRA2) was upregulated in neuroblastoma cells and tissues, and its overexpression promoted neuroblastoma cell proliferation, as revealed using colony formation, soft agar growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays Tumor suppressor phosphatase and tensin homolog (PTEN) is an inhibitor of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) pathway that interacts with GFRA2. A luciferase activity assay showed GFRA2 inhibits the transcriptional activity of the forkhead box O (FOXO) family proteins, which suggested that GFRA2 activated the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway in GFRA2 overexpressing cells decreased cell proliferation, confirming that GFRA2 promoted neuroblastoma cell proliferation by activating the PI3K/AKT pathway. In summary, cell proliferation via the GFRA2-PTEN-PI3K/AKT axis may represent new target to develop treatments for neuroblastoma.

The relationship between amniotic fluid miRNAs and congenital obstructive nephropathy.

Exosomes are small membrane vesicles with size of 30-100 nm, which were found in bodily fluids including amniotic fluid and saliva. The biological materials in exosomes, such as proteins and RNA, can be used as novel potential biomarkers for diagnostic assays. The purpose of this study was to assess whether exosomal microRNAs (miRNAs) could be used as biomarkers to prenatally diagnose congenital hydronephrosis and to evaluate fetal kidney function. Transmission electron microscopy (TEM), flow cytometry (FACS), and western-blot were applied to identify exosomes in the amniotic fluid from fetuses with congenital hydronephrosis and healthy controls. Exosomal miRNA was extracted according to the manufacturer's protocol and used for microarray. The differentially expressed miRNAs were selected for further study. The miRNA targets were analyzed to assess their possible function in the pathophysiology of obstructive nephropathy, and the miRNA array results were confirmed by qPCR. Amniotic fluid exosomes were identified based on CD24 and CD9 expression. The has-miR-942, has-miR-4289, has-miRPlus-A1073, and has-miR-195-3p were up-regulated in amniotic fluid exosomes from fetuses with congenital hydronephrosis comparing with those in healthy controls, and 35 had reduced expression levels. These results were confirmed by using qPCR. After integrating the miRNAs targets predicted via three databases and subjecting those target genes to KEGG pathway analysis, we found that the target genes of hsa-miR-300 and hsa-miR-299-5p were determined to be part of the Wnt signaling pathway. In addition, DVL2, PP2R5A, SRFP2, and SIAH1 predicted as target genes of has-miR-300 and has-miR-299-5p are informative for further exploration of congenital hydronephrosis pathologies. The reduced expression of hsa-miR-300 and hsa-miR-299-5p in the amniotic fluid of congenital hydronephrosis could be a biomarker for kidney fibrosis associated with congenital obstructive nephropathy.

miR-1303 promotes the proliferation of neuroblastoma cell SH-SY5Y by targeting GSK3ß and SFRP1.

Neuroblastoma (NB) is one of the most common solid tumors in children, many microRNAs regulate progression and development of NB. Here, we found miR-1303 was upregulated in NB cells and tissues, miR-1303 overexpression promoted the proliferation of SH-SY5Y NB cell investigated by MTT assay, colony formation assay and anchorage-independent growth ability assay, while miR-1303 knockdown reduced this effect. mechanism analysis suggested glycogen synthase kinase 3 beta (GSK3ß) and secreted frizzled-related protein 1 (SFRP1) were the target of miR-1303, luciferase assay revealed miR-1303 directly bound to the 3'UTR of GSK3ß and SFRP1. miR-1303 increased expression of MYC and CyclinD1, and decreased the expression of p21 and p27, and further demonstrated miR-1303 promotes NB proliferation. Moreover, there was a negative correlation between miR-1303 expression and GSK3ß and SFRP1 expression in NB tissues, confirming GSK3ß and SFRP1 were the targets of miR-1303 in NB tissues. Collectively, our findings suggested miR-1303 promotes NB proliferation by targeting GSK3ß and SFRP1, and might be a target for NB therapy.