circEPB41L2 blocks the progression and metastasis in non-small cell lung cancer by promoting TRIP12-triggered PTBP1 ubiquitylation.

The metastasis of non-small cell lung cancer (NSCLC) is the leading death cause of NSCLC patients, which requires new biomarkers for precise diagnosis and treatment. Circular RNAs (circRNAs), the novel noncoding RNA, participate in the progression of various cancers as microRNA or protein sponges. We revealed the mechanism by which circEPB41L2 (hsa_circ_0077837) blocks the aerobic glycolysis, progression and metastasis of NSCLC through modulating protein metabolism of PTBP1 by the E3 ubiquitin ligase TRIP12. With ribosomal RNA-depleted RNA seq, 57 upregulated and 327 downregulated circRNAs were identified in LUAD tissues. circEPB41L2 was selected due to its dramatically reduced levels in NSCLC tissues and NSCLC cells. Interestingly, circEPB41L2 blocked glucose uptake, lactate production, NSCLC cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, acting as a scaffold, circEPB41L2 bound to the RRM1 domain of the PTBP1 and the E3 ubiquitin ligase TRIP12 to promote TRIP12-mediated PTBP1 polyubiquitylation and degradation, which could be reversed by the HECT domain mutation of TRIP12 and circEPB41L2 depletion. As a result, circEPB41L2-induced PTBP1 inhibition led to PTBP1-induced PKM2 and Vimentin activation but PKM1 and E-cadherin inactivation. These findings highlight the circEPB41L2-dependent mechanism that modulates the "Warburg Effect" and EMT to inhibit NSCLC development and metastasis, offering an inhibitory target for NSCLC treatment.

N6-Methyladenosine Modification of lncCCKAR-5 Regulates Autophagy in Human Umbilical Cord Mesenchymal Stem Cells by Destabilizing LMNA and Inhibits Diabetic Wound Healing.

Long noncoding RNAs are pivotal contributors to the development of human diseases. However, their significance in the context of diabetic wound healing regulated by human umbilical cord mesenchymal stem cells (hUCMSCs) remains unclear. This study sheds light on the involvement of lncCCKAR5 in this process. We found that hUCMSCs exposed to high glucose conditions exhibited a significant downregulation of lncCCKAR5 expression, and lncCCKAR5 played a critical role in modulating autophagy, thus inhibiting apoptosis in hUCMSCs. In addition, the reduction of lncCCKAR5 in cells exposed to high glucose effectively thwarted cellular senescence and facilitated filopodium formation. Mechanistically, lncCCKAR5 served as a scaffold that facilitated the interaction between MKRN2 and LMNA, a key regulator of cytoskeletal function and autophagy. The lncCCKAR5/LMNA/MKRN2 complex played a pivotal role in promoting the ubiquitin-mediated degradation of LMNA, with this effect being further augmented by N6-adenosine methylation of lncCCKAR5. Consequently, our findings underscore the critical role of lncCCKAR5 in regulating the autophagic process in hUCMSCs, particularly through protein ubiquitination and degradation. This intricate regulatory network presents a promising avenue for potential therapeutic interventions in the context of diabetic wound healing involving hUCMSCs.

Long-read sequencing reveals the landscape of aberrant alternative splicing and novel therapeutic target in colorectal cancer.

BACKGROUND: Alternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC). METHODS: Here, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4-5 exclusion was introduced to inhibit neoplasm growth. RESULTS: A total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients' prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4-5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4-5 exclusion have the potential to restrain the CRC growth. CONCLUSIONS: This data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Time-dependent band position difference between vibrational sum and difference frequency generation: a phenomenon originating from dispersion in the visible pulse.

Vibrational spectroscopy is significant for identifying chemical specification. Here, the spectral band frequencies corresponding to the same molecular vibration in sum frequency generation (SFG) and difference frequency generation (DFG) spectra present delay-dependent deviation. Through numerical analysis of time resolved SFG and DFG spectra with a frequency marker in the incident IR pulse, the frequency ambiguity was not caused by any structure and dynamic variation on the surface, but from the dispersion in the incident visible pulse. Our results provide a helpful method to correct the vibrational frequency deviation and improve the assignment accuracy for SFG and DFG spectroscopies.

exRNAdisease: An extracellular RNA transcriptome atlas in human diseases.

Extracellular RNAs (exRNAs) are types of RNAs that present outside of the cells with highly stable features. exRNAs have been shown to play important roles in aiding intercellular communication and regulating cellular processes. In clinical settings, exRNAs have the potential to serve as biomarkers indicating the presence of a disease.Recently, the high-throughput technologies such as RNA sequencing (RNAseq) and microarray have generated abundant exRNA expression data about various diseases. Thus, it is highly desirable to have a comprehensive database for disease-related exRNA transcriptome data detected in diverse body fluids. Here, a total of 88 previously published exRNA transcriptome datasets were collected. We developed a web-accessible database, exRNADisease, which is designed as a central repository for exRNAs from body fluids in human diseases and healthy controls. In the current version, a total of 18,265 samples were collected, which includes 10 body fluids and 78 human disease types. Six different types of RNAs were included (messenger RNAs, circular RNAs, long noncoding RNAs, microRNAs, transfer RNA fragments and PIWI-interacting RNA). Literature is also included to provide the robust evidence about the relationship between exRNAs and diseases. A user-friendly interface was constructed to conveniently search and browse disease-related exRNAs. exRNADisease will become a useful platform to provide genome-wide view of exRNA deregulation in human diseases and help to identify novel biomarkers. Availability and implementation: exRNAdisease can be freely accessible at http://www.origin-gene.cn/database/exRNAdisease/.

Macrophage migration inhibitory factor (MIF) acetylation protects neurons from ischemic injury.

Ischemia-induced neuronal death leads to serious lifelong neurological deficits in ischemic stroke patients. Histone deacetylase 6 (HDAC6) is a promising target for neuroprotection in many neurological disorders, including ischemic stroke. However, the mechanism by which HDAC6 inhibition protects neurons after ischemic stroke remains unclear. Here, we discovered that genetic ablation or pharmacological inhibition of HDAC6 reduced brain injury after ischemic stroke by increasing macrophage migration inhibitory factor (MIF) acetylation. Mass spectrum analysis and biochemical results revealed that HDAC6 inhibitor or aspirin treatment promoted MIF acetylation on the K78 residue. MIF K78 acetylation suppressed the interaction between MIF and AIF, which impaired MIF translocation to the nucleus in ischemic cortical neurons. Moreover, neuronal DNA fragmentation and neuronal death were impaired in the cortex after ischemia in MIF K78Q mutant mice. Our results indicate that the neuroprotective effect of HDAC6 inhibition and aspirin treatment results from MIF K78 acetylation; thus, MIF K78 acetylation may be a therapeutic target for ischemic stroke and other neurological diseases.

eQTL Highlights the Potential Role of Negative Control of Innate Immunity in Kawasaki Disease.

PURPOSE: Kawasaki disease (KD) is an acute systemic vasculitis mainly found in the medium-sized arteries, especially the coronary arteries. Immune system is involved in the pathogenesis of acute KD in children, but the functional differences in the immune system between healthy children and KD patients remain unclear. PATIENTS AND METHODS: A total of 190 KD patients and 119 healthy controls were recruited for the next-generation sequencing of 512 targeted genes from 4 immune-related pathways. Subsequently, the peripheral blood mononuclear cells (PBMCs) were isolated. RNA sequencing of the LPS treated PBMCs from additional 20 KD patients and 20 healthy controls was used to examine the differentially expressed genes (DEGs). Then, an expression quantitative trait locus (eQTL) analysis combined with previously analyzed RNA data were used to examine the DEGs. Finally, the serum levels of 13 cytokines were detected before and after LPS treatment in 40 samples to confirm the findings from eQTL analysis. RESULTS: A total of 319 significant eQTL were found, and both eQTL analysis and RNA sequencing showed some DEGs were involved in the connective tissue disorders and inflammatory diseases. DEGs that function to negatively regulate immunity were closely related to the pathogenesis of KD. In addition, the serum levels of IL-10 (an inflammatory and immunosuppressive factor) and SCD25 (an important immunosuppressant) reduced significantly in the KD patients. CONCLUSION: Our study shows the expression of factors responsible for the negative control of innate immunity is altered, which plays an important role in the etiology of KD.

Prediction for Intravenous Immunoglobulin Resistance Combining Genetic Risk Loci Identified From Next Generation Sequencing and Laboratory Data in Kawasaki Disease.

Background: Kawasaki disease (KD) is the most common cause of acquired heart disease. A proportion of patients were resistant to intravenous immunoglobulin (IVIG), the primary treatment of KD, and the mechanism of IVIG resistance remains unclear. The accuracy of current models predictive of IVIG resistance is insufficient and doesn't meet the clinical expectations. Objectives: To develop a scoring model predicting IVIG resistance of patients with KD. Methods: We recruited 330 KD patients (50 IVIG non-responders, 280 IVIG responders) and 105 healthy children to explore the susceptibility loci of IVIG resistance in Kawasaki disease. A next generation sequencing technology that focused on 4 immune-related pathways and 472 single nucleotide polymorphisms (SNPs) was performed. An R package SNPassoc was used to identify the risk loci, and student's t-test was used to identify risk factors associated with IVIG resistance. A random forest-based scoring model of IVIG resistance was built based on the identified specific SNP loci with the laboratory data. Results: A total of 544 significant risk loci were found associated with IVIG resistance, including 27 previous published SNPs. Laboratory test variables, including erythrocyte sedimentation rate (ESR), platelet (PLT), and C reactive protein, were found significantly different between IVIG responders and non-responders. A scoring model was built using the top 9 SNPs and clinical features achieving an area under the ROC curve of 0.974. Conclusions: It is the first study that focused on immune system in KD using high-throughput sequencing technology. Our findings provided a prediction of the IVIG resistance by integrating the genotype and clinical variables. It also suggested a new perspective on the pathogenesis of IVIG resistance.

The Platelet microRNA Profile of Kawasaki Disease: Identification of Novel Diagnostic Biomarkers.

Challenging diagnosis and unknown etiology of Kawasaki disease (KD) increase the coronary artery lesions incidence. microRNAs (miRNAs) are the most promising biomarkers because of their stability in peripheral blood and noninvasive measurement procedure, whose potential utility have been proved in cancers. To explore the utility of differentially expressed (DE) miRNAs as early diagnostic markers, 44 patients (25 incomplete KD and 19 complete KD) and 31 febrile controls were recruited for small RNA sequencing. From all the 1922 expressed miRNA, 210 DE miRNAs were found between KD and febrile control groups. Though platelet miRNA profiles of complete KD incomplete KD were much similar through cluster analysis, the DE miRNAs were not identical. Eight DE miRNAs were validated by real-time quantitative PCR (qRT-PCR) in complete or incomplete KD groups using a normalizer, miR-126-3p, which was identified by geNorm and NormFinder tools. The expression level of miRNAs continuous changed over time was observed and the function analysis showed the potential role of miRNAs as therapeutic biomarkers. Additionally, the prediction model for KD showed a sensitivity of 78.8% and a specificity of 71.4%, respectively. This study used small RNA sequencing to identify miRNA biomarkers KD diagnosis based on a large sample size. Our findings shine a light on the understanding of molecular pathogenesis of KD and may improve the accuracy of KD diagnosis and prognosis in clinical.

Translation and functional roles of circular RNAs in human cancer.

Circular RNAs (circRNAs) are a new class of non-coding RNAs formed by covalently closed loops through backsplicing. Recent methodologies have enabled in-depth characterization of circRNAs for identification and potential functions. CircRNAs play important roles in various biological functions as microRNA sponges, transcriptional regulators and combining with RNA binding proteins. Recent studies indicated that some cytoplasmic circRNAs can be effectively translated into detectable peptides, which enlightened us on the importance of circRNAs in cellular physiology function. Internal Ribosome Entry site (IRES)- and N6-methyladenosines (m6A)-mediated cap-independent translation initiation have been suggested to be potential mechanism for circRNA translation. To date, several translated circRNAs have been uncovered to play pivotal roles in human cancers. In this review, we introduced the properties and functions of circRNAs, and characterized the possible mechanism of translation initiation and complexity of the translation ability of circRNAs. We summarized the emerging functions of circRNA-encoded proteins in human cancer. The works on circRNA translation will open a hidden human proteome, and enhance us to understand the importance of circRNAs in human cancer, which has been poorly explored so far.

[Determination of nine pesticide residues in honey using high performance liquid chromatography-tandem mass spectrometry].

A method is proposed for the simultaneous determination of nine benzimidazole and neonicotinoid pesticides present in honey by employing automatic solid-phase extraction with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A honey sample was dissolved in a phosphate buffer (pH=7.8) followed by ultrasonic extraction. The extracts were then purified through solid-phase extraction (SPE) with hydrophilic-lipophilic balance (HLB) cartridges. Finally, nitrogen was blown on the obtained mixture, and the mixture was subsequently filtered for conducting HPLC-MS/MS analysis. Nine compounds were detected under the multiple reaction monitoring (MRM) mode, and the corresponding quantification was performed by employing the method of internal standards. The nine detected pesticides demonstrated good linearity in the range of 0.002-0.05 mg/L, with the correlation coefficient values (r2) being higher than 0.99. The limits of detection (LODs) (S/N=3) and limits of quantification (LOQs) (S/N=10) were found to be in the ranges of 0.1-1.0 µg/kg and 0.3-2.0 µg/kg, respectively. Furthermore, the results indicated that the recoveries of the nine detected pesticides range from 78.2%-101.2% at three spiked levels of 5.0, 10.0, and 20.0 µg/kg with a relative standard deviation (RSD) range of 1.3%-14.3% (n=6). Hence, the proposed method is rapid and can be employed for accurate determination of pesticide residues in large quantities of honey samples.

The Evolution and Expression Pattern of Human Overlapping lncRNA and Protein-coding Gene Pairs.

Long non-coding RNA overlapping with protein-coding gene (lncRNA-coding pair) is a special type of overlapping genes. Protein-coding overlapping genes have been well studied and increasing attention has been paid to lncRNAs. By studying lncRNA-coding pairs in human genome, we showed that lncRNA-coding pairs were more likely to be generated by overprinting and retaining genes in lncRNA-coding pairs were given higher priority than non-overlapping genes. Besides, the preference of overlapping configurations preserved during evolution was based on the origin of lncRNA-coding pairs. Further investigations showed that lncRNAs promoting the splicing of their embedded protein-coding partners was a unilateral interaction, but the existence of overlapping partners improving the gene expression was bidirectional and the effect was decreased with the increased evolutionary age of genes. Additionally, the expression of lncRNA-coding pairs showed an overall positive correlation and the expression correlation was associated with their overlapping configurations, local genomic environment and evolutionary age of genes. Comparison of the expression correlation of lncRNA-coding pairs between normal and cancer samples found that the lineage-specific pairs including old protein-coding genes may play an important role in tumorigenesis. This work presents a systematically comprehensive understanding of the evolution and the expression pattern of human lncRNA-coding pairs.

Construction of differential mRNA-lncRNA crosstalk networks based on ceRNA hypothesis uncover key roles of lncRNAs implicated in esophageal squamous cell carcinoma.

Increasing evidence has indicated that lncRNAs acting as competing endogenous RNAs (ceRNAs) play crucial roles in tumorigenesis, metastasis and diagnosis of cancer. However, the function of lncRNAs as ceRNAs involved in esophageal squamous cell carcinoma (ESCC) is still largely unknown. In this study, clinical implications of two intrinsic subtypes of ESCC were identified based on expression profiles of lncRNA and mRNA. ESCC subtype-specific differential co-expression networks between mRNAs and lncRNAs were constructed to reveal dynamic changes of their crosstalks mediated by miRNAs during tumorigenesis. Several well-known cancer-associated lncRNAs as the hubs of the two networks were firstly proposed in ESCC. Based on the ceRNA mechanism, we illustrated that the"loss" of miR-186-mediated PVT1-mRNA and miR-26b-mediated LINC00240-mRNA crosstalks were related to the two ESCC subtypes respectively. In addition, crosstalks between LINC00152 and EGFR, LINC00240 and LOX gene family were identified, which were associated with the function of "response to wounding" and "extracellular matrix-receptor interaction". Furthermore, functional cooperation of multiple lncRNAs was discovered in the two differential mRNA-lncRNA crosstalk networks. These together systematically uncovered the roles of lncRNAs as ceRNAs implicated in ESCC.

A genome-wide association study identifies GRK5 and RASGRP1 as type 2 diabetes loci in Chinese Hans.

Substantial progress has been made in identification of type 2 diabetes (T2D) risk loci in the past few years, but our understanding of the genetic basis of T2D in ethnically diverse populations remains limited. We performed a genome-wide association study and a replication study in Chinese Hans comprising 8,569 T2D case subjects and 8,923 control subjects in total, from which 10 single nucleotide polymorphisms were selected for further follow-up in a de novo replication sample of 3,410 T2D case and 3,412 control subjects and an in silico replication sample of 6,952 T2D case and 11,865 control subjects. Besides confirming seven established T2D loci (CDKAL1, CDKN2A/B, KCNQ1, CDC123, GLIS3, HNF1B, and DUSP9) at genome-wide significance, we identified two novel T2D loci, including G-protein-coupled receptor kinase 5 (GRK5) (rs10886471: P = 7.1 × 10(-9)) and RASGRP1 (rs7403531: P = 3.9 × 10(-9)), of which the association signal at GRK5 seems to be specific to East Asians. In nondiabetic individuals, the T2D risk-increasing allele of RASGRP1-rs7403531 was also associated with higher HbA(1c) and lower homeostasis model assessment of ß-cell function (P = 0.03 and 0.0209, respectively), whereas the T2D risk-increasing allele of GRK5-rs10886471 was also associated with higher fasting insulin (P = 0.0169) but not with fasting glucose. Our findings not only provide new insights into the pathophysiology of T2D, but may also shed light on the ethnic differences in T2D susceptibility.

Evolutionary relationships between miRNA genes and their activity.

BACKGROUND: The emergence of vertebrates is characterized by a strong increase in miRNA families. MicroRNAs interact broadly with many transcripts, and the evolution of such a system is intriguing. However, evolutionary questions concerning the origin of miRNA genes and their subsequent evolution remain unexplained. RESULTS: In order to systematically understand the evolutionary relationship between miRNAs gene and their function, we classified human known miRNAs into eight groups based on their evolutionary ages estimated by maximum parsimony method. New miRNA genes with new functional sequences accumulated more dynamically in vertebrates than that observed in Drosophila. Different levels of evolutionary selection were observed over miRNA gene sequences with different time of origin. Most genic miRNAs differ from their host genes in time of origin, there is no particular relationship between the age of a miRNA and the age of its host genes, genic miRNAs are mostly younger than the corresponding host genes. MicroRNAs originated over different time-scales are often predicted/verified to target the same or overlapping sets of genes, opening the possibility of substantial functional redundancy among miRNAs of different ages. Higher degree of tissue specificity and lower expression level was found in young miRNAs. CONCLUSIONS: Our data showed that compared with protein coding genes, miRNA genes are more dynamic in terms of emergence and decay. Evolution patterns are quite different between miRNAs of different ages. MicroRNAs activity is under tight control with well-regulated expression increased and targeting decreased over time. Our work calls attention to the study of miRNA activity with a consideration of their origin time.