Genetic Biomarkers For Hepatocellular Carcinoma In The Era Of Precision Medicine.

Being one of the most lethal cancers that exhibit high levels of heterogeneity, hepatocellular carcinoma (HCC) is associated with diverse oncogenic pathways underpinned by varied driver genes. HCC can be induced by different etiological factors including virus infection, toxin exposure or metabolic disorders. Consequently, patients may display varied genetic profiles, and may respond differently to the treatments involving inhibition of target pathways. These DNA/RNA mutations, copy number variations, chromatin structural changes, aberrant expression of non-coding RNAs and epigenetic modifications were considered as biomarkers in the application of precision medication. To explore how genetic testing could contribute to early diagnosis, prognosis, treatment and postoperative monitoring of HCC, we conducted a systematic review of genetic markers associated with different pathologies. Moreover, we summarized on-going clinical trials for HCC treatment, including the trials for multiple kinase inhibitors and immune checkpoint blockade (ICB). The efficacy of ICB treatment in HCC is not as good as what was observed in lung cancer and melanoma, which might be due to the heterogeneity of the microenvironment of the liver.

Synergism of open chromatin regions involved in regulating genes in Bombyx mori.

The dynamic variability of transcription factors (TFs) and their binding sites makes it challenging to conduct genome-wide transcription regulation research. The silkworm Bombyx mori, which produces silk, is one of the most valuable model insects in the order Lepidoptera. The "opening" and "closing" of chromatin in different silk yield strains is associated with changes in silk production, making this insect a good model for studying the transcriptional regulation of genes. However, few studies have examined the open chromatin regions (OCRs) of silkworms, and studying OCR synergism and their function in silk production remains challenging. Here, we performed formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate OCRs from the silk glands of fifth-instar larvae of the DaZao and D872 strains. In total, 128,908 high confidence OCRs were identified and approximately 80% of OCRs were located in non-coding regions. OCRs upregulated adjacent genes and showed signal-dependent vulnerability to single-nucleotide polymorphisms. Mid- and low-signal OCRs were more likely to have single-nucleotide polymorphisms (SNP). Further, OCRs interacted with each other within a distance of 5 kb. We named the OCR interaction complex as the "cluster of related regions" (COREs). The functions of the CORE and its harbored OCRs showed some differences. Additionally, COREs enriched many silk protein synthesis-associated genes, some of which were upregulated. This study identified numerous high confidence regulation sites and synergistic regulatory modes of OCRs that affect adjacent genes. These results provide insight into silkworm transcriptional regulation and improve our understanding of cis-element cooperation.

Gene Expression Pattern and Regulatory Network of α-Toxin Treatment in Bombyx mori.

Bacillus bombyseptieus is a pathogen of Bombyx mori; it can cause bacterial septicemia in silkworm. One of the components of the parasporal crystal toxin of B. bombyseptieus, α-toxin, plays an important role in the process of infection in silkworm. In this study, we investigated the immune response of silkworm induced by α-toxin by using RNA-seq. We compared the changes in gene expression in the midgut, fatbody, and hemocytes of silkworm and in the B. mori embryonic cell line (BmE) after treatment with α-toxin and identified 952 differentially expressed genes and 353 differentially expressed long noncoding RNAs (lncRNAs). These regulated genes in different tissues were found to be enriched in different pathways. The upregulated genes in the midgut were mainly involved in peptidoglycan catabolic process and tyrosine kinase signaling pathway, whereas the downregulated genes were mainly involved in chitin metabolic pathways. The upregulated genes in fatbody were also involved in peptidoglycan catabolic process, but they were for a different peptidoglycan subtype. Further, genes encoding cecropins were enriched in the fatbody. The downregulated genes were mainly involved in the metabolic pathways of fundamental substances such as cellular protein metabolic process and nucleobase-containing compound metabolic process. These results suggest that α-toxin can induce various immune responses in silkworm, and further studies are warranted to understand the mechanism of α-toxin action in silkworm. Further, lncRNAs and differentially expressed genes were correlated using coexpression network analysis. Our findings revealed potential candidate genes and lncRNAs that might play important physiological functions in the immune response to α-toxins in silkworm.

[Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin].

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Transcriptome analysis of the response of silkworm to drastic changes in ambient temperature.

Bombyx mori is a poikilothermic insect and is economically important for silk production. Drastic changes in the ambient temperature have a negative impact on sericulture. However, the reason as to why high temperature is associated with the occurrence of diseases in silkworm and the response of silkworm to low temperature remain unclear and were the focus of the present study. Dazao silkworm exposed to 13 °C (DZ-13), 25 °C (DZ-25), and 37 °C (DZ-37) were used for RNA-seq analysis. There were 478 and 194 upregulated differentially expressed genes (DEGs) in DZ-13 and DZ-37 while 49 and 273 downregulated DEGs in DZ-13 and DZ-37, respectively. Eight DEGs were co-upregulated, in which seven genes were for heat shock proteins (Hsps), implying that Hsps play important roles in the tolerance of silkworm to high and low temperature. Gene ontology analysis revealed that the developmental process was downregulated in DZ-13. All the DEGs in the oxidative phosphorylation and insulin signaling pathways were upregulated in DZ-13. Several cuticular proteins and ATP synthesis-related genes were upregulated in DZ-13, suggesting that thickening of the cuticle and increase in the ATPase expression would help silkworms to protect themselves from low temperature-induced stress. Several immune-related genes, such as BmRel and BmSerpin-2, were downregulated in DZ-37, revealing that the resistance of silkworm is decreased under high temperature shock resulting in susceptibility to pathogens. Thus, the increase in the thermo-tolerance of silkworm should be related to the enhancement in the pathogen resistance.

The genome-wide transcriptional regulatory landscape of ecdysone in the silkworm.

BACKGROUND: The silkworm, Bombyx mori, a typical representative of metamorphic insects, is of great agricultural and economic importance. The steroid hormone ecdysone (20-hydroxyecdysone, 20E) is the central regulator of insect developmental transitions, and its nuclear receptors are crucial for numerous biological processes, including reproduction, metabolism, and immunity. However, genome-wide DNA regulatory elements and the ecdysone receptor (EcR) that control these programs of gene expression are not well defined. RESULTS: In this study, we investigated the alterations in three types of histone modification in silkworm embryonic cells treated with 20E by chromatin immunoprecipitation sequencing (ChIP-seq). We identified enhancers using histone modifications and derived genome-wide ecdysone-dependent enhancer activity maps in the silkworm. We found enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) that showed dynamic changes in acetylation of histone H3 Lys27 (H3K27ac) after 20E treatment and functioned to regulate the transcription of specific genes. EcR regulated transcription by binding not only to proximal promoters but also to the distal enhancers of target genes. Moreover, only 52.65% EcR peaks contained ecdysone response element (EcRE) motif, suggesting that EcR regulates the expression of target genes not only by binding directly to EcRE, but also by binding with other transcription factor. CONCLUSIONS: Our findings provide novel insights into the complex regulatory landscape of hormone-responsive cell activity and a basis for understanding the complex transcriptional regulatory processes of ecdysone.

Circular transcriptome sequencing of the middle silk gland and posterior silk gland in the Bombyx mori.

Circular transcriptome sequencing of the middle silk gland (MSG) and posterior silk gland (PSG) in the Bombyx mori are presented. The middle silk gland and posterior silk gland were collected from the third day of fifth-instar B.mori larvae. The circular RNAs enriched by using RNase R to degrade the linear RNA molecules, and circular RNA sequencing (circRNA-seq) was performed using an Illumina Hiseq. 2500 sequencing platform. Samples are described in the SRA portal (SRP100385) and FASTQ files have been deposited in Sequence Read Archive (accession numbers: SRX2577343 and SRX2577342). The interpretation of these data is presented in the following research article: "Identification of circular RNA in the Bombyx mori silk gland" [1] (Gan et al., 2017).

[Interaction of aminopeptidase (BmAPN5) and parasporal crystal (PC) toxin isolated from Bacillus bombysepticus].

Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.

Identification of circular RNA in the Bombyx mori silk gland.

Bombyx mori is an economically important holometabolous lepidopteran insect. In B. mori endogenous noncoding RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs play crucial biological functions in metamorphosis and sex determination. In addition, circular RNAs (circRNAs) have been recently identified as noncoding RNAs in most common model organisms and show potential as gene regulators. However, to date, there have been few studies on the circRNAs present in the B. mori genome conducted to date. Here, we identified 3916 circRNAs by deep circular transcriptome sequencing using the silk gland of B. mori. 3155 circRNAs were found to be derived from 1727 parental genes. The circRNAs displayed tissue-specific expression between the middle silk gland (MSG) and posterior silk gland (PSG), with 2532 and 880 being upregulated circRNAs in the MSG and PSG, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that the parental genes from the MSG and PSG were generally annotated to similar categories and pathways. The interaction network of circRNAs and miRNAs showed that circRNAs might act as miRNA sponges or interact with miRNAs in some other way. Overall, the results revealed the complicated patterns of circRNAs in the B. mori silk gland providing a new angle from which to explore the mechanisms of complex gene regulation and efficient silk protein synthesis.