An integrative platform for detection of RNA 2'-O-methylation reveals its broad distribution on mRNA.

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers.

Comprehensive profiling of EGFR mutation subtypes reveals genomic-clinical associations in non-small-cell lung cancer patients on first-generation EGFR inhibitors.

Common sensitizing mutations in epidermal growth factor receptor (cEGFR), including exon 19 deletions (19-Del) and exon 21 L858R substitution, are associated with high sensitivity to EGFR-TKIs in NSCLC patients. The treatment for NSCLC patients with uncommon EGFR (uEGFR) mutations remains a subject of debate due to heterogeneity in treatment responses. In this manuscript, the targeted next-generation sequencing (NGS) data of a large cohort of EGFR-mutated NSCLC patients was assessed to elucidate genomic profiles of tumors carrying cEGFR or uEGFR mutations. The results showed that NSCLC patients with uEGFR mutations were more likely to harbor co-occurring genetic alterations in the Hippo pathway and a higher TMB compared with cEGFR-positive patients. Smoking-related mutations were found to significantly enriched in uEGFR-positive patients. Subgroup analyses were performed to identify potential prognostic biomarkers in patients harboring various EGFR subtype mutations. L858R-positive patients with co-existing ARID2 mutations had shorter progression-free survival (PFS) than those who were L858R- or 19-Del-positive but ARID2-negative (median: 2.3 vs. 12.0 vs. 8.0 months, P = 0.038). Furthermore, mutational profiles, such as top frequently mutated genes and mutational signatures of patients with various EGFR subtype mutations were significantly different. Our study analyzed the mutational landscape of NSCLC patients harboring cEGFR and uEGFR mutations, revealing specific genomic characteristics associated with uEGFR mutations that might explain the poor prognosis of first-generation EGFR-TKIs.

Mature Red Blood Cells Contain Long DNA Fragments and Could Acquire DNA from Lung Cancer Tissue.

Red blood cells (RBC) are commonly known as cells with no nucleus or mitochondria and are assumed to be a transportation vehicle. This study confirms that RBC contain long DNA fragments inside with stain by both microscope and flow cytometry, which covers most nuclear and mitochondrial genome regions by next-generation sequencing (NGS). Such characteristics demonstrate a significant difference compared with A549 cell line or paired peripheral blood mononuclear cell as nucleated cells. To further explore the characteristics of RNA DNA, DNA from 20 RBC samples is sequenced by NGS. Interestingly, several gaps and multiple regions with copy number variation are observed significantly different between different samples, which could be used to distinguish samples with different health status accurately. Using an in vitro co-culture system, it is shown that RBC could absorb DNA-bearing tumorigenic mutations from cancer cell lines but requires cell-to-cell contact. Finally, based on a small scale clinical trial, it is confirmed that common genetic mutations of cancer tissues could be detected in RBC from patients with early-stage non-small-cell lung cancer. This study highlights a new biological phenomenon involving RBC and its translational potential as a novel liquid biopsy technology platform for early cancer screening and diagnosis of malignancy.

Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

Tumor cells can evade attack by phagocytes by upregulating the self-marker CD47. The mechanisms underlying tumor CD47 upregulation, however, remain unclear. Here, we report that human lung adenocarcinoma CD47 is upregulated by interferon-γ (IFN-γ), the level in the tumor microenvironment of which is markedly increased after tumor metastasis and chemotherapy. The IFN-γ receptor is expressed in various human lung adenocarcinoma tissues regardless of the CD47 protein expression, and lung adenocarcinoma CD47 expression is significantly enhanced following tumor metastasis or chemotherapy treatment. In line with this, CD47 expression in various lung cancer cells is markedly increased by IFN-γ treatment. Mechanistically, IFN-γ promotes CD47 expression by activating interferon regulatory factor-1 (IRF-1), which binds to an IRF-1-binding domain within the CD47 promoter region and increases CD47 transcription. Functionally, IFN-γ-enhanced CD47 expression facilitates human lung cancer cell invasion both in vitro and in vivo, whereas IFN-γ-induced CD47 upregulation and cancer metastasis are blocked by mutating the IRF-1-binding site within the CD47 promoter. Our results reveal IFN-γ-enhanced CD47 expression as a novel mechanism promoting human lung adenocarcinoma progression.

Long Noncoding RNA CTD-2245E15.3 Promotes Anabolic Enzymes ACC1 and PC to Support Non-Small Cell Lung Cancer Growth.

Long noncoding RNAs (lncRNA) have been shown to play critical regulatory roles in the onset and progression of human cancers. However, the functions of a large proportion of lncRNAs are still unexplored. Here we describe a novel lncRNA, CTD-2245E15.3, that promotes lung tumorigenesis by regulating the anabolic enzymes acetyl-CoA carboxylase 1 (ACC1, encoded by the ACACA gene) and pyruvate carboxylase (PC). Differentially expressed lncRNAs between non-small cell lung cancer (NSCLC) and paired adjacent nontumor tissues were identified by a microarray and validated using quantitative real-time polymerase chain reaction. CTD-2245E15.3 was significantly upregulated in NSCLC and was mainly located in the cytoplasm. Knockdown of CTD-2245E15.3 by specific antisense oligonucleotides suppressed cell growth in vitro and in vivo, largely due to cell-cycle arrest and induction of apoptosis. Overexpression of CTD-2245E15.3 in an orthotopic model of lung cancer led to a significant increase in total tumor burden. CTD-2245E15.3 exerted its oncogenic function by binding ACC1 and PC, which are key anabolic factors for biomolecule synthesis in rapidly proliferating tumor cells. Knockdown of CTD-2245E15.3 increased phosphorylation of ACC1 at an inhibitory site for enzymatic activity and promoted PC degradation via ubiquitination. Supplements of palmitate or oxaloacetate, products of ACC1 and PC, alleviated the suppression of cell growth caused by loss of CTD-2245E15.3. These findings reveal the important role of CTD-2245E15.3 as an oncogenic lncRNA in the anabolic process for tumor growth. SIGNIFICANCE: These findings demonstrate a novel lncRNA CTD-2245E15.3 that binds and positively regulates anabolic enzymes ACC1 and PC to promote tumor growth. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3509/F1.large.jpg.

PD-L1 lncRNA splice isoform promotes lung adenocarcinoma progression via enhancing c-Myc activity.

BACKGROUND: Although using a blockade of programmed death-ligand 1 (PD-L1) to enhance T cell immune responses shows great promise in tumor immunotherapy, the immune-checkpoint inhibition strategy is limited for patients with solid tumors. The mechanism and efficacy of such immune-checkpoint inhibition strategies in solid tumors remains unclear. RESULTS: Employing qRT-PCR, Sanger sequencing, and RNA BaseScope analysis, we show that human lung adenocarcinoma (LUAD) all produce a long non-coding RNA isoform of PD-L1 (PD-L1-lnc) by alternative splicing, regardless if the tumor is positive or negative for the protein PD-L1. Similar to PD-L1 mRNA, PD-L1-lnc in various lung adenocarcinoma cells is significantly upregulated by IFNγ. Both in vitro and in vivo studies demonstrate that PD-L1-lnc increases proliferation and invasion but decreases apoptosis of lung adenocarcinoma cells. Mechanistically, PD-L1-lnc promotes lung adenocarcinoma progression through directly binding to c-Myc and enhancing c-Myc transcriptional activity. CONCLUSIONS: In summary, the PD-L1 gene can generate a long non-coding RNA through alternative splicing to promote lung adenocarcinoma progression by enhancing c-Myc activity. Our results argue in favor of investigating PD-L1-lnc depletion in combination with PD-L1 blockade in lung cancer therapy.

Five-lncRNA signature in plasma exosomes serves as diagnostic biomarker for esophageal squamous cell carcinoma.

Changes in expression of long non-coding RNAs (lncRNAs) in plasma exosomes can be useful for diagnosis of cancer patients. Here, we conducted a four-stage study to identify plasma exosome lncRNAs with diagnostic potential in esophageal squamous cell carcinoma (ESCC). First, plasma exosome lncRNA expression profiles were examined in ESCC patients, esophagitis patients, and healthy controls using RNA sequencing. Differentially expressed plasma exosome lncRNAs from the lncRNA expression profile were then evaluated by qRT-PCR in a large cohort of ESCC patients, esophagitis patients, and healthy controls. Expression levels of the lncRNAs NR_039819, NR_036133, NR_003353, ENST00000442416.1, and ENST00000416100.1 were significantly higher in exosomes from ESCC patients than non-cancer controls. We also confirmed that levels of these five plasma exosome lncRNAs decreased markedly in ESCC patients after surgery. Our results suggest that these five exosome lncRNAs may serve as highly effective, noninvasive biomarkers for ESCC diagnosis.

3'-Terminal 2'-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute 2.

Methylation of miRNAs at the 2'-hydroxyl group on the ribose at 3'-end (2'-O-methylation, 2'Ome) is critical for miRNA function in plants and Drosophila. Whether this methylation phenomenon exists for mammalian miRNA remains unknown. Through LC-MS/MS analysis, we discover that majority of miR-21-5p isolated from human non-small cell lung cancer (NSCLC) tissue possesses 3'-terminal 2'Ome. Predominant 3'-terminal 2'Ome of miR-21-5p in cancer tissue is confirmed by qRT-PCR and northern blot after oxidation/ß-elimination procedure. Cancerous and the paired non-cancerous lung tissue miRNAs display different pattern of 3'-terminal 2'Ome. We further identify HENMT1 as the methyltransferase responsible for 3'-terminal 2'Ome of mammalian miRNAs. Compared to non-methylated miR-21-5p, methylated miR-21-5p is more resistant to digestion by 3'→5' exoribonuclease polyribonucleotide nucleotidyltransferase 1 (PNPT1) and has higher affinity to Argonaute-2, which may contribute to its higher stability and stronger inhibition on programmed cell death protein 4 (PDCD4) translation, respectively. Our findings reveal HENMT1-mediated 3'-terminal 2'Ome of mammalian miRNAs and highlight its role in enhancing miRNA's stability and function.

Bioinformatics analysis to determine the prognostic value and prospective pathway signaling of miR-126 in non-small cell lung cancer.

BACKGROUND: MicroRNAs (miRNAs) have been demonstrated to play crucial roles in the initiation and development of non-small cell lung cancer (NSCLC). However, further investigation of the specific role of miR-126 in NSCLC is still required. METHODS: An analysis of miR-126 expression in NSCLC was carried out using the Gene Expression Omnibus (GEO) database, and a literature review was also performed. The differentially expressed genes (DEGs) in three mRNA datasets, GSE18842, GSE19804, and GSE101929, from GEO were identified. Following the prediction of hsa-miR-126-5p target genes by TargetScan, the overlap of miR-126 target genes with DEGs in NSCLC was examined. After that, Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed. Finally, an analysis to identify the impact of hub genes on the prognosis of NSCLC was carried out on the basis of a protein-protein interaction (PPI) network constructed using STRING and Cytoscape. RESULTS: The data in the literature review revealed a trend that miR126 was downregulated in NSCLC. The number of both NSCLC-related and miR-126-related DEGs was 187. Dozens of DEGs were significantly enriched in biological regulation, cell membrane binding, and signal receptor binding. In the PPI network analysis, 3 of 10 identified hub genes, namely NCAPG, MELK, and KIAA0101, were obviously related to poor prognosis in NSCLC; the survival rate was low among patients with high expression levels of these genes. Furthermore, through network analysis, TPX2, HMMR, and ANLN were identified as recessive miR-126-related genes that may be involved in NSCLC. CONCLUSIONS: MiR-126 plays an essential role in the biological processes of NSCLC through binding to target genes and influences the prognosis of patients with the disease.

Long noncoding antisense RNA FAM83A-AS1 promotes lung cancer cell progression by increasing FAM83A.

The abnormal expression of long noncoding RNAs (lncRNAs) is closely associated with human cancers. As one special group of lncRNAs, natural antisense transcripts (NATs) can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Their expression levels are altered in many cancers, but their roles are poorly understood. We strove to find NATs involved in human non-small-cell lung cancer (NSCLC) and to reveal their mechanism of action in cancer. We analysed the NATs in NSCLC from the TCGA database by circlncRNAnet. One NAT, family with sequence similarity 83 member A antisense RNA 1 (FAM83A-AS1), was found to be markedly upregulated and positively correlated with its cognate sense counterpart, FAM83A, in NSCLC. Moreover, overexpression of FAM83A-AS1 increased FAM38A protein levels and induced ERK1/2 phosphorylation downstream of FAM83A in cells. Finally, overexpression of FAM83A-AS1 promoted LUAD cell proliferation and invasion. In summary, lncRNA FAM83A-AS1 promotes LUAD by increasing FAM83A expression.

Study and application progress of function of tubular stomach after esophagectomy / 中国胸心血管外科临床杂志

@#Esophageal cancer is one of the most common malignant tumors in China. The comprehensive treatment of esophageal cancer based on operation is important. In recent ten years, with the development of surgical techniques and medical instruments, tubular stomach has been widely used. Although the advantages of tubular stomach are becoming more and more obvious, there are still many details and problems for the function and application of tubular stomach worthy of further discussion and study. In this paper, the technical progress, advantages, functions and applications of tubular stomach are reviewed and discussed, and the future prospect is predicted.

The necessity of gastrointestinal decompression after Ivor Lewis esophagectomy: A randomized controlled trial / 中国胸心血管外科临床杂志

@#Objective To explore the feasibility and safety of non-gastrointestinal decompression after esophagectomy and the necessity of gastric tube or the time to remove gastric tube. Methods Thirty patients with esophageal cancer who underwent surgical treatment in the Department of Thoracic and Cardiovascular Surgery, Nanjing Drum Tower Hospital, were included in the trial from June to October 2017. The patients were randomly and equally assigned to a trial group (non-gastrointestinal decompression) or a control group (gastrointestinal decompression). There was no significant difference in age (P=1.000), sex (P=1.000), tumor location (P=0.732), pathological type (P=1.000), pathological stage (P=0.507), and operation time (P=0.674) between the two groups. The clinical effect between the two groups were compared. Results There was no statistical difference in incidences of anastomotic leakage (P=1.000), anastomotic bleeding (P=1.000), gastroesophageal reflux (P=1.000) between the two groups. And there was no statistical difference in time of the first flatus (P=0.629) and the first bowel movement (P=0.599) after operation between the two groups. Conclusion Without gastrointestinal decompression after Ivor Lewis esophagectomy does not increase the incidences of anastomotic leakage, anastomotic bleeding and gastroesophageal reflux, and has no significant effect on the recovery of gastrointestinal function. Without gastrointestinal decompression after Ivor Lewis esophagectomy is safe and feasible. Removing gastric tube on the second day after operation is reasonable and feasible.