Potent immunogenicity in BRCA1‐mutated patients with high‐grade serous ovarian carcinoma

High‐grade serous ovarian carcinomas (HGSOCs) were among the tumours with an unsatisfactory outcome of immune checkpoint inhibitors (ICIs). It is imperative to develop feasible biomarker for identifying responsive candidates and guiding precise immunotherapy for HGSOC patients. Here, we analysed genomic data of patients with HGSOCs to depict their immunological phenotype of tumour microenvironment (TME) and figure out the major determinants of immunogenicity. In comparison with other solid tumours, we observed the lowest levels of PD‐L1, total mutation burden (TMB) and cytolytic molecules in HGSOCs. Surprisingly, TMB is not certainly positively related to tumour immune response as it failed to predict the response to ICIs in a considerable portion of patients in previous clinical trials. By a machine learning approach in search of biomarkers for immunotherapy implications for HGSOCs, we identified the ten most dominant factors determining the immunogenicity of HGSOCs. Interestingly, we found that BRCA1 mutated tumours presented a potent immunogenic phenotype, independent of TMB, meeting the criteria of both our dominant factors and the determinants of immunogenicity established before. Our findings provide evidence that BRCA1‐mutation may be served as a predictive biomarker in guiding ICI therapies for the patients with HGSOCs.     

Tumour cell‐intrinsic CTLA4 regulates PD‐L1 expression in non‐small cell lung cancer

Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD‐1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non‐small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD‐1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti‐CTLA4 antibody can induce PD‐L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti‐CTLA4 antibody was not able to induce PD‐L1 expression in NSCLC cells. Moreover, anti‐CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell‐intrinsic CTLA4 can regulate PD‐L1 expression and cell proliferation, and that anti‐CTLA4 antibody, by binding to the tumour cell‐intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

HULC: an oncogenic long non‐coding RNA in human cancer

Highly up‐regulated in liver cancer (HULC) was originally identified as the most overexpressed long non‐coding RNA in hepatocellular carcinoma. Since its discovery, the aberrant up‐regulation of HULC has been demonstrated in other cancer types, including gastric cancer, pancreatic cancer, osteosarcoma and hepatic metastasis of colorectal cancer. Recent discoveries have also shed new light on the upstream molecular mechanisms underlying HULC deregulation. As an oncogene, HULC promotes tumorigenesis by regulating multiple pathways, such as down‐regulation of EEF1E1, promotion of abnormal lipid metabolism, and up‐regulation of sphingosine kinase 1. Pertinent to clinical practice, a genetic variant in the HULC gene has been found to alter the risk for hepatocellular carcinoma and oesophageal cancer, whereas cancer patients with high or low expression of HULC exhibit different clinical outcome. These findings highlighted the pathogenic role and clinical utility of HULC in human cancers. Further efforts are warranted to promote the development of HULC‐directed therapeutics.     

Long non‐coding RNAs in non‐small cell lung cancer as biomarkers and therapeutic targets

Lung cancer‐associated mortality is the most common cause of cancer death worldwide. Non‐coding RNAs (ncRNAs), with no protein‐coding ability, have multiple biological roles. Long non‐coding RNAs (lncRNAs) are a recently characterized class of ncRNAs that are over 200 nucleotides in length. Many lncRNAs have the ability of facilitating or inhibiting the development and progression of tumours, including non‐small cell lung cancer (NSCLC). Because of their fundamental roles in regulating gene expression, along with their involvement in the biological mechanisms underlying tumourigenesis, they are a promising class of tissue‐ and/or blood‐based cancer biomarkers. In this review, we highlight the emerging roles of lncRNAs in NSCLC, and discuss their potential clinical applications as diagnostic and prognostic markers and as therapeutic targets.     

Aberrantly expressed long non‐coding RNAs in air pollution‐induced congenital defects

Air pollution has been a serious public health issue over the past few decades particularly in developing countries. Air pollution exposure during pregnancy poses potential threat to offspring as the deleterious substances might pass through placenta to alter foetal development. A growing number of studies have demonstrated that long non‐coding RNAs (lncRNAs) participate in the development of many diseases, including congenital defects. Here, we used RNA sequencing to identify differentially expressed lncRNAs in air pollution‐exposed rat embryos compared with control group. Our data suggested that 554 lncRNAs (216 up‐regulated and 338 down‐regulated) were significantly differentially expressed in the air pollution‐exposed embryos. Moreover, potential cellular functions of these deregulated lncRNAs were predicted via KEGG signal pathway/GO enrichment analyses, which suggested the possible involvements of neurological process, sensory perception of smell and the G‐protein signalling pathway. Furthermore, potential functional network of deregulated lncRNAs and their correlated mRNAs in the development of congenital spinal abnormality was established. Our data suggested that lncRNAs may play a vital role in the pathophysiology of air pollution‐exposed congenital spinal malformation.     

Multi‐targeting of viral RNAs with synthetic trans‐acting small interfering RNAs enhances plant antiviral resistance

RNA interference (RNAi)‐based tools are used in multiple organisms to induce antiviral resistance through the sequence‐specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi‐based tools include artificial microRNAs (amiRNAs) and synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). syn‐tasiRNAs have emerged as a promising antiviral tool allowing for the multi‐targeting of viral RNAs through the simultaneous expression of several syn‐tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn‐tasiRNA construct expressing four different syn‐tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn‐tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn‐tasiRNA lines was not exclusive of lines with high syn‐tasiRNA accumulation. Collectively, these results suggest that syn‐tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn‐tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn‐tasiRNA.     

Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.     

MiR‐520b promotes the progression of non‐small cell lung cancer through activating Hedgehog pathway

Although the non‐small cell lung cancer (NSCLC) is one of the most malignant tumours worldwide, the mechanisms controlling NSCLC tumourigenesis remain unclear. Here, we find that the expression of miR‐520b is up‐regulated in NSCLC samples. Further studies have revealed that miR‐520b promotes the proliferation and metastasis of NSCLC cells. In addition, miR‐520b activates Hedgehog (Hh) pathway. Inhibitor of Hh pathway could relieve the oncogenic effect of miR‐520b upon NSCLC cells. Mechanistically, we demonstrate that miR‐520b directly targets SPOP 3′‐UTR and decreases SPOP expression, culminating in GLI2/3 stabilization and Hh pathway hyperactivation. Collectively, our findings unveil that miR‐520b promotes NSCLC tumourigenesis through SPOP‐GLI2/3 axis and provide miR‐520b as a potential diagnostic biomarker and therapeutic target for NSCLC.     

Liquid chromatography high‐resolution mass spectrometry for fatty acid profiling

Quantification of fatty acids has been crucial to elucidate lipid biosynthesis pathways in plants. To date, fatty acid identification and quantification has relied mainly on gas chromatography (GC) coupled to flame ionization detection (FID) or mass spectrometry (MS), which requires the derivatization of samples and the use of chemical standards for annotation. Here we present an alternative method based on a simple procedure for the hydrolysis of lipids, so that fatty acids can be quantified by liquid chromatography mass spectrometry (LC‐MS) analysis. Proper peak annotation of the fatty acids in the LC‐MS‐based methods has been achieved by LC‐MS measurements of authentic standard compounds and elemental formula annotation supported by ¹³C isotope‐labeled Arabidopsis. As a proof of concept, we have compared the analysis by LC‐MS and GC‐FID of two previously characterized Arabidopsis thaliana knock‐out mutants for FAD6 and FAD7 desaturase genes. These results are discussed in light of lipidomic profiles obtained from the same samples. In addition, we performed untargeted LC‐MS analysis to determine the fatty acid content of two diatom species. Our results indicate that both LC‐MS and GC‐FID analyses are comparable, but that because of higher sensitivity and selectivity the LC‐MS‐based method allows for a broader coverage and determination of novel fatty acids.     

Identification of a recently active Prunus‐specific non‐autonomous Mutator element with considerable genome shaping force

Miniature inverted‐repeat transposable elements (MITEs) are known to contribute to the evolution of plants, but only limited information is available for MITEs in the Prunus genome. We identified a MITE that has been named Falling Stones, FaSt. All structural features (349‐bp size, 82‐bp terminal inverted repeats and 9‐bp target site duplications) are consistent with this MITE being a putative member of the Mutator transposase superfamily. FaSt showed a preferential accumulation in the short AT‐rich segments of the euchromatin region of the peach genome. DNA sequencing and pollination experiments have been performed to confirm that the nested insertion of FaSt into the S‐haplotype‐specific F‐box gene of apricot resulted in the breakdown of self‐incompatibility (SI). A bioinformatics‐based survey of the known Rosaceae and other genomes and a newly designed polymerase chain reaction (PCR) assay verified the Prunoideae‐specific occurrence of FaSt elements. Phylogenetic analysis suggested a recent activity of FaSt in the Prunus genome. The occurrence of a nested insertion in the apricot genome further supports the recent activity of FaSt in response to abiotic stress conditions. This study reports on a presumably active non‐autonomous Mutator element in Prunus that exhibits a major indirect genome shaping force through inducing loss‐of‐function mutation in the SI locus.