Disturbed sleep is closely associated with an increased risk of metabolic diseases. However, the underlying mechanisms of circadian clock genes linking sleep and lipid profile abnormalities have not been fully elucidated. This study aimed to explore the important role of the circadian clock in regulating impaired cholesterol metabolism at an early stage of sleep deprivation (SD). Sleep disturbance was conducted using an SD instrument. Our results showed that SD increased the serum cholesterol levels. Concentrations of serum leptin and resistin were much lower after SD, but other metabolic hormone concentrations (adiponectin, glucagon, insulin, thyroxine, norepinephrine, and epinephrine) were unchanged before and after SD. Warning signs of cardiovascular diseases [decreased high density lipoprotein (HDL)-cholesterol and increased corticosterone and 8-hydroxyguanosine levels] and hepatic cholestasis (elevated total bile acids and bilirubin levels) were observed after SD. Cholesterol accumulation was also observed in the liver after SD. The expression levels of HMGCR, the critical enzyme for cholesterol synthesis, remained unchanged in the liver. However, the expression levels of liver CYP7A1, the enzyme responsible for the conversion of cholesterol into bile acids, significantly reduced after SD. Furthermore, expression of NR1D1, a circadian oscillator and transcriptional regulator of CYP7A1, strikingly decreased after SD. Moreover, NR1D1 deficiency decreased liver CYP7A1 levels, and SD could exacerbate the reduction of CYP7A1 expression in NR1D1-/- mouse livers. Additionally, NR1D1 deficiency could further increase serum cholesterol levels under SD. These results suggest that sleep disturbance can induce increased serum cholesterol levels and liver cholesterol accumulation by NR1D1 mediated CYP7A1 inhibition.

Emerging evidence suggests that long non-coding RNA (lncRNA) plays a critical role in human disease progression. Recently, a novel lncRNA ST8SIA6-AS1 was shown as an important driver in various cancer types. Nevertheless, its contribution to lung adenocarcinoma (LUAD) remains undocumented. Herein, we found that ST8SIA6-AS1 was frequently overexpressed in LUAD cell lines, tissues, and plasma. Depletion of ST8SIA6-AS1 significantly inhibited LUAD cell proliferation and invasion in vitro and tumor growth in vivo. In term of mechanism, ST8SIA6-AS1 was transcriptionally repressed by tumor suppressor p53, and ST8SIA6-AS1 was mainly located in the cytoplasm and could abundantly sponge miR-125a-3p to increase nicotinamide N-methyltransferase (NNMT) expression, thereby facilitating LUAD malignant progression. Clinically, high ST8SIA6-AS1 was positively correlated with larger tumor size, lymph node metastasis, and later TNM stage. Moreover, ST8SIA6-AS1 was identified as an excellent indicator for MM diagnosis and prognosis. Collectively, our data demonstrate that ST8SIA6-AS1 is a carcinogenic lncRNA in LUAD, and targeting the axis of ST8SIA6-AS1/miR-125a-3p/NNMT may be a promising treatment for LUAD patients.

How the human brain differs from those of non-human primates is largely unknown and the complex drivers underlying such differences at the genomic level remain unclear. In this study, we selected 243 brain-related genes, based on Gene Ontology, and identified 184,113 DNaseI hypersensitive sites (DHSs) within their regulatory regions. To performed comprehensive evolutionary analyses, we set strict filtering criteria for alignment quality and filtered 39,132 DHSs for inclusion in the investigation and found that 2,397 (~6%) exhibited evidence of accelerated evolution (aceDHSs), which was a much higher proportion that DHSs genome-wide. Target genes predicted to be regulated by brain-aceDHSs were functionally enriched for brain development and exhibited differential expression between human and chimpanzee. Alignments indicated 61 potential human-specific transcription factor binding sites in brain-aceDHSs, including for CTCF, FOXH1, and FOXQ1. Furthermore, based on GWAS, Hi-C, and eQTL data, 16 GWAS SNPs, and 82 eQTL SNPs were in brain-aceDHSs that regulate genes related to brain development or disease. Among these brain-aceDHSs, we confirmed that one enhanced the expression of GPR133, using CRISPR-Cas9 and western blotting. The GPR133 gene is associated with glioblastoma, indicating that SNPs within DHSs could be related to brain disorders. These findings suggest that brain-related gene regulatory regions are under adaptive evolution and contribute to the differential expression profiles among primates, providing new insights into the genetic basis of brain phenotypes or disorders between humans and other primates.

Background: Recently, RNA-binding proteins (RBPs) were reported to interact with target mRNA to regulate gene posttranscriptional expression, and RBP-mediated RNA modification can regulate the expression and function of proto-oncogenes and tumor suppressor genes. We systematically analyzed the expression of RBPs in pancreatic adenocarcinoma (PAAD) and constructed an RBP-associated prognostic risk model. Methods: Gene expression data of normal pancreatic samples as well as PAAD samples were downloaded from TCGA-PAAD and GTEx databases. Wilcoxon test and univariate Cox analysis were, respectively, applied to screen differential expression RBPs (DE-RBPs) and prognostic-associated RBPs (pRBPs). Functional enrichment was analyzed by GO, KEGG, and GSEA. Protein-protein interaction (PPI) network was constructed by STRING online database. Modeling RBPs were selected by multivariate Cox analysis. Kaplan-Meier survival and Cox analysis were applied to evaluate the effects of risk score on the overall survival of PAAD patients. ROC curves and validation cohort were applied to verify the accuracy of the model. Nomogram was applied for predicting 1-, 3-, and 5-year overall survival (OS) of PAAD patients. At last, modeling RBPs were further analyzed to explore their differential expression, prognostic value, as well as enrichment pathways in PAAD. Results: RBPs (453) were differentially expressed in normal and tumor samples, besides, 28 of which were prognostic associated. DE-RBPs (453) are functionally associated with ribosome, ribonuclease, spliceosome, etc. Eight RBPs (PABPC1, PRPF6, OAS1, RBM5, LSM12, IPO7, FXR1, and RBM6) were identified to construct a prognostic risk model. Higher risk score not only predicted poor prognosis but also was an independent poor prognostic indicator, which was verified by ROC curves and validation cohort. Eight modeling RBPs were confirmed to be significantly differentially expressed between normal and tumor samples from RNA and protein level. Besides, all of eight RBPs were related with overall survival of PAAD patients. Conclusions: We successfully constructed an RBP-associated prognostic risk model in PAAD, which has a potential clinical application prospect.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Background: Breast cancer (BRCA) is a life-threatening malignancy in women with an unsatisfactory prognosis. The purpose of this study was to explore the prognostic biomarkers and a risk signature based on ferroptosis-related RNA-binding proteins (FR-RBPs). Methods: FR-RBPs were identified using Spearman correlation analysis. Differentially expressed genes (DEGs) were identified by the "limma" R package. The univariate Cox and multivariate Cox analyses were executed to determine the prognostic genes. The risk signature was constructed and verified with the training set, testing set, and validation set. Mutation analysis, immune checkpoint expression analysis in high- and low-risk groups, and correlation between risk signature and chemotherapeutic agents were conducted using the "maftools" package, "ggplot2" package, and the CellMiner database respectively. The Human Protein Atlas (HPA) database was employed to confirm protein expression trends of prognostic genes in BRCA and normal tissues. The expression of prognostic genes in cell lines was verified by Real-time quantitative polymerase chain reaction (RT-qPCR). Kaplan-meier (KM) plotter database analysis was applied to predict the correlation between the expression levels of signature genes and survival statuses. Results: Five prognostic genes (GSPT2, RNASE1, TIPARP, TSEN54, and SAMD4A) to construct an FR-RBPs-related risk signature were identified and the risk signature was validated by the International Cancer Genome Consortium (ICGC) cohort. Univariate and multivariate Cox regression analysis demonstrated the risk score was a robust independent prognostic factor in overall survival prediction. The Tumor Mutational Burden (TMB) analysis implied that the high- and low-risk groups responded differently to immunotherapy. Drug sensitivity analysis suggested that the risk signature may serve as a chemosensitivity predictor. The results of GSEA suggested that five prognostic genes might be related to DNA replication and the immune-related pathways. RT-qPCR results demonstrated that the expression trends of prognostic genes in cell lines were consistent with the results from public databases. KM plotter database analysis suggested that high expression levels of GSPT2, RNASE1, and SAMD4A contributed to poor prognoses. Conclusion: In conclusion, this study identified the FR-RBPs-related prognostic genes and developed an FR-RBPs-related risk signature for the prognosis of BRCA, which will be of great significance in developing new therapeutic targets and prognostic molecular biomarkers for BRCA.

Despite considerable progress has been made in the understanding of the genetics and molecular biology of renal cell carcinoma (RCC), therapeutic options of patients with papillary renal cell carcinoma (PRCC) are limited. Immunotherapy based on immune checkpoint inhibitors (ICIs) has become a hot point in researching new drug for tumor and been tested in a number of human clinical trials. In this study, an immune-related gene prognostic index (IRGPI) was developed and provided a comprehensive and systematic analysis of distinct phenotypic and molecular portraits in the recognition, surveillance, and prognosis of PRCC. The reliability of the IRGPI was evaluated using independent datasets from GEO database and the expression levels of the genes in the IRGPI detected by real-time PCR. Collectively, the currently established IRGPI could be used as a potential biomarker to evaluate the response and efficacy of immunotherapy in PRCC.

The sturgeon (Acipenseriformes) is an important farmed species because of its economical value. However, neither gene transfer nor gene editing techniques have been established in sturgeon for molecular breeding and gene functional study until now. In this study, we accomplished gene transfer and gene editing in sterlet (Acipenser ruthenus), which has the shortest sexual maturation period of sturgeons. The plasmid encoding enhanced green fluorescent protein (EGFP) was transferred into the embryos of sterlet at injection concentration of 100 ng/μL, under which condition high survival rate and gene transfer rate could be achieved. Subsequently, exogenous EGFP was efficiently disrupted by transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease/guide RNA (gRNA), with injection concentrations of 300 ng/μL TALENs, or 100 ng/μL Cas9 nuclease and 30 ng/μL gRNA, respectively, under which condition high survival rate and gene mutation rate could be achieved. Finally, the endogenous gene no tail in sterlet was successfully mutated by Cas9 nuclease/gRNA. We observed the CRISPR-induced no tail mutation, at a high efficiency with the mutant P0 embryos displaying the expected phenotype of bent spine and twisted tail.

To determine the relationship between glioma and muscle aging and to predict prognosis by screening for co-expressed genes, this study examined the relationship between glioma and sarcopenia. The study identified eight co-downregulated miRNAs, three co-upregulated miRNAs, and seven genes associated with overall glioma survival, namely, KRAS, IFNB1, ALCAM, ERBB2, STAT3, FOSL1, and EN2. With a multi-factor Cox regression model incorporating FOSL1 and EN2, we obtained ROC curves of 0.702 and 0.709, respectively, suggesting that glioma prognosis can be predicted by FOSL1 and EN2, which are differentially expressed in both cancer and aged muscle. FOSL1 and EN2 were analyzed using Gene Set Enrichment Analysis to identify possible functional pathways. RT-qPCR and a dual-luciferase reporter gene system verified that hsa-miR-33a targets FOSL1 and EN2. We found that hsa-mir-33a co-targeting FOSL1 and EN2 has a good predictive value for glioblastoma and skeletal muscle reduction.

RNA stability plays an important role in gene expression. Here, using 3' end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3'UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3'-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5' splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity.

Multiple myeloma (MM) is a malignant hematopoietic disease that is usually incurable. RNA-binding proteins (RBPs) are involved in the development of many tumors, but their prognostic significance has not been systematically described in MM. Here, we developed a prognostic signature based on eight RBP-related genes to distinguish MM cohorts with different prognoses.

Identifying causal regulatory variants and their target genes from the majority of non-coding disease-associated genetic loci is the main challenge in post-Genome-Wide Association Studies (GWAS) functional studies. Although chromosome conformation capture (3C) and its derivative technologies have been successfully applied to nominate putative causal genes for non-coding variants, many GWAS target genes have not been identified yet. This study generated a high-resolution contact map from epithelial ovarian cancer (EOC) cells with two H3K27ac-HiChIP libraries and analyzed the underlying gene networks for 15 risk loci identified from the largest EOC GWAS. By combinatory analysis of 4,021 fine-mapped credible variants of EOC GWAS and high-resolution contact map, we obtained 162 target genes that mainly enriched in cancer related pathways. Compared with GTEx eQTL genes in ovarian tissue and annotated proximal genes, 132 HiChIP targets were first identified for EOC causal variants. More than half of the credible variants (CVs) involved interactions that were over 185 kb in distance, indicating that long-range transcriptional regulation is an important mechanism for the function of GWAS variants in EOC. We also found that many HiChIP gene targets showed significantly differential expressions between normal ovarian and EOC tumor samples. We validated one of these targets by manipulating the rs9303542 located region with CRISPR-Cas9 deletion and dCas9-VP64 activation experiments and found altered expression of HOXB7 and HOXB8 at 17q21.32. This study presents a systematic analysis to identify putative target genes for causal variants of EOC, providing an in-depth investigation of the mechanisms of non-coding regulatory variants in the etiology and pathogenesis of ovarian cancer.

Long non-coding RNA (LncRNAs) are newly highlighted key factors controlling brown adipogenesis and development, but their regulatory effect to white adipocyte is still merely understood. Deciphering their underlying mechanism could be a novel way to discovering potential targets of obesity. Therefore, we conducted a whole transcriptome analysis in white adipose tissue from obese patients for the first time. Six obese patients and five control subjects were selected for microarray assay. Differentially expressed coding genes (DEGs), targets of lncRNAs, and alternatively spliced genes in obesity group were systematically compared in a functional framework based on a global gene regulatory network. It was demonstrated that all the three kinds of transcripts were enriched in pathways related to glucose metabolism while only DEGs showed closer proximity to neuro-endocrine-immune system. Thus, a lncRNA-regulated core network was constructed by a stepwise strategy using DEGs as seed nodes. From the core network, we identified a decreased lncRNA, uc001kfc.1, as potential cis-regulator for phosphatase and tensin homolog (PTEN) to enhance insulin sensitivity of white adipocytes in obese patients. We further validated the down-regulation of uc001kfc.1 and PTEN in an independent testing sample set enrolling 22 subjects via qRT-PCR. Although whether the decreased uc001kfc.1 correlated with low risk of diabetes deserved to be examined in an expanded cohort with long-term follow-up visit, the present study highlighted the potential of lncRNA regulating glucose homeostasis in human adipose tissue from a global perspective. With further improvement, such network-based analyzing protocol proposed in this study could be applied to interpreting function of more lncRNAs from other whole transcriptome data.

Epigenetic modifications play an important role in central nervous system disorders. As a widespread posttranscriptional RNA modification, the role of the m5C modification in cerebral ischemia-reperfusion injury (IRI) remains poorly defined. Here, we successfully constructed a neuronal oxygen-glucose deprivation/reoxygenation (OGD/R) model and obtained an overview of the transcriptome-wide m5C profiles using RNA-BS-seq. We discovered that the distribution of neuronal m5C modifications was highly conserved, significantly enriched in CG-rich regions and concentrated in the mRNA translation initiation regions. After OGD/R, modification level of m5C increased, whereas the number of methylated mRNA genes decreased. The amount of overlap of m5C sites with the binding sites of most RNA-binding proteins increased significantly, except for that of the RBM3-binding protein. Moreover, hypermethylated genes in neurons were significantly enriched in pathological processes, and the hub hypermethylated genes RPL8 and RPS9 identified by the protein-protein interaction network were significantly related to cerebral injury. Furthermore, the upregulated transcripts with hypermethylated modification were enriched in the processes involved in response to stress and regulation of apoptosis, and these processes were not identified in hypomethylated transcripts. In final, we verified that OGD/R induced neuronal apoptosis in vitro using TUNEL and western blot assays. Our study identified novel m5C mRNAs associated with ischemia-reperfusion in neurons, providing valuable perspectives for future studies on the role of the RNA methylation in cerebral IRI.

Long non-coding RNAs (lncRNAs) emerge as critical regulators across a wide variety of biological functions in living organisms. However, to date, no systematic characterization of lncRNAs has been investigated in the ectoparasitic mite Varroa destructor, the most severe biotic threat to honey bees worldwide. Here, we performed an initial genome-wide identification of lncRNAs in V. destructor via high-throughput sequencing technology and reported, for the first time, the transcriptomic landscape of lncRNAs in the devastating parasite. By means of a lncRNA identification pipeline, 6,645 novel lncRNA transcripts, encoded by 3,897 gene loci, were identified, including 2,066 sense lncRNAs, 2,772 lincRNAs, and 1,807 lncNATs. Compared with protein-coding mRNAs, V. destructor lncRNAs are shorter in terms of full length, as well as of the ORF length, contain less exons, and express at lower level. GO term and KEGG pathway enrichment analyses of the lncRNA target genes demonstrated that these predicted lncRNAs may be potentially responsible for the regulatory functions of cellular and biological progresses in the reproductive phase of V. destructor. To our knowledge, this is the first catalog of lncRNA profile in the parasitiformes species, providing a valuable resource for genetic and genomic studies. Understanding the characteristics and features of lncRNAs in V. destructor would promote sustainable parasite control.

Objective: Currently available evidence regarding the association between collagen type XII α1 (COL12A1) polymorphism and risk of anterior cruciate ligament rupture (ACLR) remains elusive. The aim of our present study was to assess the association between COL12A1 rs970547 polymorphism and ACLR risk. Methods: Five online databases, namely, PubMed, EMBASE, ISI Web of Science, CENTRAL, and CNKI, were searched from their inception data up to December 2020 to identify relative observational studies. The methodological quality of each individual study was evaluated using the Newcastle-Ottawa Scale (NOS). The "model-free approach" was employed to estimate the magnitude of effect of COL12A1 rs970547 polymorphism on ACLR, and the association was expressed using odds ratio (OR) and its associated 95% confidence interval (95% CI). Subgroup analysis was performed by ethnicity and sex of included subjects. Results: Eight studies involving 1,477 subjects with ACLR and 100,439 healthy controls were finally included in our study. The methodological quality of included studies was deemed moderate to high based on NOS scores. The "model-free" approach suggested no genotype differences between ACLR and healthy control for the rs970547 polymorphism, but we still used the allele model to present the combined data. Under the random-effect model, there was no significant difference in the frequency of effecting allele between ACLR and control (OR: 0.91, 95% CI 0.77, 1.08; p = 0.28). Stratified analysis by sex and ethnicity also showed no difference in allele frequency. Conclusion: The findings of this current meta-analysis suggested that rs970547 was not associated with ACLR risk in male, female, and the overall population among Asians or Caucasians.

It is now clear that major malignancies are heterogeneous diseases associated with diverse molecular properties and clinical outcomes, posing a great challenge for more individualized therapy. In the last decade, cancer molecular subtyping studies were mostly based on transcriptomic profiles, ignoring heterogeneity at other (epi-)genetic levels of gene regulation. Integrating multiple types of (epi)genomic data generates a more comprehensive landscape of biological processes, providing an opportunity to better dissect cancer heterogeneity. Here, we propose sparse canonical correlation analysis for cancer classification (SCCA-CC), which projects each type of single-omics data onto a unified space for data fusion, followed by clustering and classification analysis. Without loss of generality, as case studies, we integrated two types of omics data, mRNA and miRNA profiles, for molecular classification of ovarian cancer (n = 462), and breast cancer (n = 451). The two types of omics data were projected onto a unified space using SCCA, followed by data fusion to identify cancer subtypes. The subtypes we identified recapitulated subtypes previously recognized by other groups (all P- values < 0.001), but display more significant clinical associations. Especially in ovarian cancer, the four subtypes we identified were significantly associated with overall survival, while the taxonomy previously established by TCGA did not (P- values: 0.039 vs. 0.12). The multi-omics classifiers we established can not only classify individual types of data but also demonstrated higher accuracies on the fused data. Compared with iCluster, SCCA-CC demonstrated its superiority by identifying subtypes of higher coherence, clinical relevance, and time efficiency. In conclusion, we developed an integrated bioinformatic framework SCCA-CC for cancer molecular subtyping. Using two case studies in breast and ovarian cancer, we demonstrated its effectiveness in identifying biologically meaningful and clinically relevant subtypes. SCCA-CC presented a unique advantage in its ability to classify both single-omics data and multi-omics data, which significantly extends the applicability to various data types, and making more efficient use of published omics resources.

Osteosarcoma is a common malignant primary bone tumor in adolescents and children. Numerous studies have shown that circRNAs were involved in the proliferation and invasion of various tumors. However, the role of circRNAs in osteosarcoma remains unclear. Here, we aimed to explore the regulatory network among circRNA-miRNA-mRNA in osteosarcoma.

For lung adenocarcinoma (LUAD), patients of different stages have strong heterogeneity, and their overall prognosis varies greatly. Thus, exploration of novel biomarkers to better clarify the characteristics of LUAD is urgent. Multi-omics information of LUAD patients were collected form TCGA. Three independent LUAD cohorts were obtained from gene expression omnibus (GEO). A multi-omics correlation analysis of METTL5 was performed in TCGA dataset. To build a METTL5-associated prognostic score (MAPS). Spathial and random forest methods were first applied for feature selection. Then, LASSO was implemented to develop the model in TCGA cohort. The prognostic value of MAPS was validated in three independent GEO datasets. Finally, functional annotation was conducted using gene set enrichment analysis (GSEA) and the abundances of infiltrated immune cells were estimated by ImmuCellAI algorithm. A total of 901 LUAD patients were included. The expression of METTL5 in LUAD was significantly higher than that in normal lung tissue. And high expression of METTL5 indicated poor prognosis in all different stages (P < 0.001, HR = 1.81). Five genes (RAC1, C11of24, METTL5, RCCD1, and SLC7A5) were used to construct MAPS and MAPS was significantly correlated with poor prognosis (P < 0.001, HR = 2.15). Furthermore, multivariate Cox regression analysis suggested MAPS as an independent prognostic factor. Functional enrichment revealed significant association between MAPS and several immune components and pathways. This study provides insights into the potential significance of METTL5 in LUAD and MAPS can serve as a promising biomarker for LUAD.

It is estimated that around 10-20% of hypospadias are caused by genetic abnormalities worldwide although the spectrum of associated genes does vary across different ethnicities. The prevalence of hypospadias among the Chinese population has been increasing the last couple of decades. However, the pathogenesis underlying the disease and its associated genetic abnormality remains unclear. Here we performed a genetic analysis of 81 children with karyotype 46, XY and the hypospadias phenotype in order to characterize the genetic components that contribute to the development of hypospadias in Chinese patients. 15 candidate genes, including sex determination genes-SOX9, SRY, NR0B1 (DAX1), NR5A1 (SF1), DHH, sex differentiation genes-AR, SRD5A2, MAMLD1, INSL3, and hypospadias-associated genes-FGF8, FGF10, BMP4, BMP7, ATF3, and MID1 were screened by using next generation sequencing. A total of 18 patients were found to have mutations identified by PCR and sequencing, including 11 cases of SRD5A2 genes, 6 cases of AR genes, and 1 case of MID1 gene, respectively. One novel missense mutation p.I817N was discovered in AR gene. Further molecular analysis found that subcellular localization of the ARI 81 7N was the same as that of wild type ARWT in the absence or presence of hormone. But it led to 50% reduction in AR-induced transcriptional activity in the presence of either the synthetic androgen R1881 or the natural ligand dihydrotestosterone. Our results indicate that SRD5A2 and AR genes are two top candidate genes associated with 46, XY hypospadias in Chinese patients. Further epidemiological and genetic analysis are still needed to further clarify the pathogenesis of hypospadias in Han Chinese patients.