Development and performance evaluations of an HER-2 kit.

Objective To develop a kit for detecting human epidermal growth factor receptor 2 (HER-2) in the human body. Methods The HER-2 kit was evaluated based on an automated magnetic particle chemiluminescence platform. The kit was developed using the double antibody sandwich-complexation method. Results The kit showed a linear range of 0.01-800 ng/mL, with a linear R2 of >0.999. The limit of the blank was 0.0039 ng/mL, and the precision at 1.00 ng/mL was 9.4%. The recovery rate at 10.00 ng/mL was 97.81-101.81%. The negative serum reference range was 0-8.23 ng/mL. Conclusions The kit had a wide linear range, high accuracy, good precision, and high sensitivity, indicating that it has good application prospects.

Identification of circRNA-miRNA-mRNA Regulatory Network in Bladder Cancer by Integrated Analysis.

INTRODUCTION: Bladder cancer (BC) is a common malignant tumor in the urinary system with high mortality and recurrence rates. This study sought to identify crucial circular RNAs (circRNAs) associated with BC. METHODS: The mRNA, miRNA, and circRNA expression profiles of BC were downloaded from GEO database. The differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), and circRNAs (DEcircRNAs) were identified using bioinformatics method. Combining circRNA-miRNA pairs with miRNA-mRNA pairs, the competing endogenous RNA (ceRNA; DEcircRNA-DEmi-RNA-DEmRNA) regulatory network was constructed. Functional annotation of host gene of DEcircRNAs and DEmRNAs in ceRNA regulatory network were performed. qRT-PCR validation was performed. RESULTS: A total of 4,003 DEmRNAs, 25 DEmiRNAs, and 119 DEcircRNAs were obtained. The ceRNA network contained 18 circRNA-miRNA pairs and 699 mi-RNA-mRNA pairs, including 17 circRNAs, 4 miRNAs, and 624 mRNAs. Functional annotation of DEmRNAs in ceRNA regulatory network revealed that these DEmRNAs were significantly enriched in glycerolipid metabolism, p53 signaling pathway, and oocyte meiosis. Except for hsa_circ_0028173, expression of the others in the qRT-PCR results was consistent with that in our integrated analysis, generally. CONCLUSION: We speculate that hsa_circ_0008035/hsa-miR-107/MSRB3 and hsa_circ_0028173/hsa-miR-338-3p/TPX2/GATA3 interaction pairs may play a vital role in BC.

A novel hexamerin with an unexpected contribution to the prophenoloxidase activation system of the Chinese oak silkworm, Antheraea pernyi.

Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap-hexamerin), which shows high homology with reported insect methionine-rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap-hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap-hexamerin to immune responses. In further immune functional studies, Ap-hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen-associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap-hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.

Knockdown of ZNF280A inhibits cell proliferation and promotes cell apoptosis of bladder cancer.

OBJECTIVE: ZNF280A is a member of the zinc finger protein family, whose role in human cancers is little known and rarely reported. This study aimed to investigate the role of ZNF280A in bladder cancer. METHODS: Immunohistochemical analysis was performed to detect the expression of ZNF280A in clinical samples. ZNF280A knockdown cell models were constructed by transfection of shRNA-expressing lentivirus. MTT assay and flow cytometry were performed for detecting cell proliferation, apoptosis and cycle. Wound healing and Transwell assays were operated to detect cell migration. Western blotting and Human Apoptosis Antibody Microarray were used to measure expression of related proteins. A mouse xenograft model was constructed for in vivo study. RESULTS: Our study demonstrated that ZNF280A was up-regulated in bladder cancer tissues compared with normal tissues, whose high expression was significantly correlated with advanced malignant grade. Knockdown of ZNF280A inhibited cell proliferation and cell migration, promoted cell apoptosis and G1/G2 phase arrest. The tumor growth in vivo was also proved to be inhibited by ZNF280A. Moreover, ZNF280A may promote bladder cancer through regulation of MAPK9, Cyclin D1 and the Akt pathway. CONCLUSIONS: In this study, ZNF280A was shown as a potential tumor promoter and prognosis indicator for bladder cancer. Targeting ZNF280A may be a promising strategy for the development of novel bladder cancer treatment.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Oridonin inhibits bladder cancer survival and immune escape by covalently targeting HK1.

BACKGROUND: Hexokinase I (HK1) is highly expressed in a variety of malignancies, regulates glycolytic pathway in cancer cells, and thus considered to be one of the promising molecular targets for cancer therapy. Nonetheless, the development of a specific inhibitor against HK1 remains elusive. PURPOSE: This study aims to elucidate the mechanism by which oridonin inhibits the proliferation and immune evasion of bladder cancer cells, specifically through the suppression of HK1. METHODS: To examine the mechanisms by which oridonin directly binds to cysteines of HK1 and inhibits bladder cancer growth, this study utilized a variety of methods. These included the Human Proteome Microarray, Streptavidin-agarose affinity assay, Biolayer Interferometry (BLI) ainding analysis, Mass Spectrometry, Cellular Thermal Shift Assay, Extracellular Acidification Rate measurement, and Xenotransplant mouse models. RESULTS: As indicated by our current findings, oridonin forms a covalent bond with Cys-813, located adjacently to glucose-binding domain of HK1. This suppresses the enzymatic activity of HK1, leading to an effective reduction of glycolysis, which triggers cell death via apoptosis in cells derived from human bladder cancer. Significantly, oridonin also inhibits lactate-induced PD-L1 expression in bladder cancer. Furthermore, pairing oridonin with a PD-L1 inhibitor amplifies the cytotoxicity of CD8+ T cells against bladder cancer. CONCLUSION: This research strongly suggests that oridonin serves as a covalent inhibitor of HK1. Moreover, it indicates that functional cysteine residue of HK1 could operate as viable targets for selective inhibition. Consequently, oridonin exhibits substantial potential for the evolution of anti-cancer agents targeting the potential therapeutic target HK1 via metabolism immunomodulation.

Immune functions of pattern recognition receptors in Lepidoptera.

Pattern recognition receptors (PRRs), as the "sensors" in the immune response, play a prominent role in recognizing pathogen-associated molecular patterns (PAMPs) and initiating an effective defense response to pathogens in Lepidoptera. It is becoming increasingly clear that damage-associated molecular patterns (DAMPs) normally play a physiological role within cells; however, when exposed to extracellular, they may become "part-time" critical signals of the immune response. Based on research in recent years, we review herein typical PRRs of Lepidoptera, including peptidoglycan recognition protein (PGRP), gram-negative binding protein (GNBP), ß-1,3-glucan recognition protein (ßGRP), C-type lectin (CTL), and scavenger receptor (SR). We also outline the ways in which DAMPs participate in the immune response and the correlation between PRRs and immune escape. Taken together, these findings suggest that the role of PRRs in insect innate immunity may be much greater than expected and that it is possible to recognize a broader range of signaling molecules.

A Versatile Hemolin With Pattern Recognitional Contributions to the Humoral Immune Responses of the Chinese Oak Silkworm Antheraea pernyi.

Hemolin is a distinctive immunoglobulin superfamily member involved in invertebrate immune events. Although it is believed that hemolin regulates hemocyte phagocytosis and microbial agglutination in insects, little is known about its contribution to the humoral immune system. In the present study, we focused on hemolin in Antheraea pernyi (Ap-hemolin) by studying its pattern recognition property and humoral immune functions. Tissue distribution analysis demonstrated the mRNA level of Ap-hemolin was extremely immune-inducible in different tissues. The results of western blotting and biolayer interferometry showed recombinant Ap-hemolin bound to various microbes and pathogen-associated molecular patterns. In further immune functional studies, it was detected that knockdown of hemolin regulated the expression level of antimicrobial peptide genes and decreased prophenoloxidase activation in the A. pernyi hemolymph stimulated by microbial invaders. Together, these data suggest that hemolin is a multifunctional pattern recognition receptor that plays critical roles in the humoral immune responses of A. pernyi.

Hepatitis C Virus Core Protein Promotes the Metastasis of Human Hepatocytes by Activating the MAPK/ERK/PEA3-SRF/c-Fos/MMPs Axis.

BACKGROUND AND AIM: Previous studies have shown that the hepatitis C virus (HCV) core protein plays an important role in the metastasis of hepatocellular carcinoma (HCC) cells. This study aimed to identify the potential mechanism of HCV core protein in HCC. METHODS: A transcription factor microarray analysis was performed to identify the factors regulated by the HCV core protein. A comprehensive bioinformatics analysis approach was utilized to predict the functions, regulatory signaling pathways and downstream target genes of the differentially regulated transcription factors. Dual-luciferase assays, qPCR, Western blotting, ERK pathway inhibition experiments and siRNA knockdown experiments were performed to verify the effects of the HCV core protein on PEA3, SRF and c-Fos, as well asthe underlying mechanism. The migration/invasion assay and scratch assay served to confirm the metastasis-promoting mechanism of the HCV core protein. RESULTS: The results demonstrated that altered expression of PEA3, SRF and c-Fos mediated by the HCV core protein were associated with the MAPK/ERK pathway. c-Fos was a downstream target protein of PEA3 and SRF. Knockdown of PEA3-SRF/c-Fos expression and ERK pathway components suppressed the migration and invasion activity of hepatocytes by affecting MMP2 and MMP9 expression. CONCLUSION: We provided preliminary evidence that the role of the HCV core protein in promoting metastasis is at least partially dependent on the activation of the MAPK/ERK/PEA3-SRF/c-Fos/MMP2/MMP9 axis. These findings reveal a novel mechanism by which the HCV core protein promotes HCC metastasis and may provide new therapeutic targets for patients with metastatic HCC.

Oridonin attenuates lung inflammation and fibrosis in silicosis via covalent targeting iNOS.

Silicosis, the most common type of pneumoconiosis, exhibits a high incidence in workers who are chronically exposed to crystalline silica (CS). No specific remedy for cure as yet. The terpenoid oridonin exerts multiple modulatory functions in neoplasms and inflammations as a natural compound. In this study, we explored the effect of oridonin on silicosis and revealed the underlying molecular mechanism. An experimental silicosis mouse model was established to evaluate the effects of oridonin on pneumonia and pulmonary fibrosis. In addition, the impact of oridonin on alveolar macrophages (AMs) was examined in the MH-S cell line. Its molecular target, inducible nitric oxide synthase (iNOS), was identified by chemobiological means, and virus-mediated gene overexpression systems confirmed that oridonin directly restrained iNOS protein levels. Oridonin alleviated pneumonia and pulmonary fibrosis in silicosis mice with no obvious systemic toxicity. These effects were partially related to oridonin inhibition of CS-induced AMs injury and inflammation. Furthermore, oridonin suppressed iNOS enzymatic expression and activity by covalently binding to the Thr109 residue of the iNOS target. Thus, our results indicate oridonin as a potential iNOS enzymatic suppressor in experimental silicosis that attenuates pneumonia and pulmonary fibrosis progression, which provides a therapeutic avenue for silicosis prevention and treatment.

Rab6c is a new target of miR­218 that can promote the progression of bladder cancer.

Bladder cancer has high morbidity and mortality rates among the male genitourinary system tumor types. MicroRNA­218 (miR­218) is associated with the development of a variety of cancer types, including bladder cancer. Rab6c is a member of the Rab family and is involved in drug resistance in MCF7 cells. The aim of the present study was to clarify the relationship between Rab6c and miR­218 in bladder cancer cell lines. In this study, the expression levels of miR­218 and Rab6c were evaluated via reverse transcription­quantitative PCR and western blotting, respectively. The association between Rab6c and miR­218 was recognized via TargetScan analysis and dual luciferase reporter gene detection. Cell proliferation was analyzed using Cell Counting Kit­8 and colony formation assays, and the invasive ability was measured via Transwell assays. Rab6c was highly expressed in bladder cancer, while miR­218 had abnormally low expression in bladder cancer. In addition, there was a mutual regulation between Rab6c and miR­218 in bladder cancer. It was found that overexpression of Rab6c significantly enhanced the proliferation, colony formation and invasion of T24 and EJ cells. Furthermore, miR­218 overexpression blocked the promoting effects of Rab6c on the malignant behavior of bladder cancer cells. Thus, Rab6c promotes the proliferation and invasion of bladder cancer cells, while miR­218 has the opposite effect, which may provide a novel insight for the treatment of bladder cancer.

The diversity of pattern recognition receptors (PRRs) involved with insect defense against pathogens.

Through evolution, selection pressures cause both insects and the pathogens attacking them to adapt so that they will both survive and this has been called the co-evolutionary 'arms race'. Insects expand their repertoire of pattern recognition receptors (PRRs), a fundamental and core component of their immune systems, to adapt to the constantly changing and unpredictable diversity of pathogens. In this review, we discuss the diversity of PRRs based on studies conducted in recent years. The strategies associated with PRR diversity summarized here are genetic evolution, isoform diversity based on alternative splicing, 'part-time' PRRs, PRRs with opsonin function, and regulation of complex signaling pathways. Taken together, these data indicate that the function of PRRs in insect immunity is far more complex and possesses more features than originally thought.

Innate immune responses in the Chinese oak silkworm, Antheraea pernyi.

Innate immunity, the evolutionarily conserved defense system, has been extensively analyzed in insect models over recent decades. The significant progress in this area has formed our dominant conceptual framework of the innate immune system, but critical advances in other insects have had a profound impact on our insights into the mystery of innate immunity. In recent years, we focused on the immune responses in Antheraea pernyi, an important commercial silkworm species reared in China. Here, we review the immune responses of A. pernyi based on immune-related gene-encoded proteins that are divided into five categories, namely pattern recognition receptors, hemolymph proteinases and their inhibitors, prophenoloxidase, Toll pathway factors and antimicrobial peptides, and others. Although the summarized information is limited since the research on A. pernyi immunity is in its infancy, we hope to provide evidence for further exploration of innate immune mechanisms.

A novel peptidoglycan recognition protein involved in the prophenoloxidase activation system and antimicrobial peptide production in Antheraea pernyi.

Pattern recognition receptors (PRRs) are employed in insects to defend against infectious pathogens by triggering various immune responses. Peptidoglycan recognition proteins (PGRPs), a vital family of PRRs, are widely distributed and highly conserved from vertebrates to invertebrates. To date, five PGRP genes have been identified in Antheraea pernyi, but their biochemical roles still remain unknown. In this study, we focused on the immune functions of PGRP-SA in A. pernyi (ApPGRP-SA), which was confirmed to be immune-related according to its significantly up-regulated expression level post microbial injection. In addition, the binding properties of ApPGRP-SA were investigated using a recombinant protein produced in a prokaryotic expression system, revealing that rApPGRP-SA displayed a multi-binding ability to various microbes, including the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and fungus Candida albicans, together with their surface pathogen associated molecular patterns (PAMPs). Further studies showed that after recognition, the mixture of rApPGRP-SA/PAMP remarkably stimulated prophenoloxidase (PPO) activation in the hemolymph of A. pernyi in vitro, while the ds-PGRP-SA-treated hemolymph exhibited a lower sensitivity to PAMPs in comparison to the native sample. Moreover, the transcriptional level of the three antimicrobial peptides was also decreased in PGRP-SA knock-down larvae in response to immune-challenge. In summary, we conclude that ApPGRP-SA is a novel identified PGRP in A. pernyi that might act as a broad-spectrum pattern recognition receptor and is involved in the PPO activation system as well as antimicrobial peptide production.

Cloning and characterization of an insect apolipoprotein (apolipophorin-II/I) involved in the host immune response of Antheraea pernyi.

Apolipoproteins are protein components of lipoprotein particles, and are increasingly recognized to be functioning in the innate immune systems of both insects and mammals. Mammalian apolipoprotein B (apoB) is associated with a diverse range of innate immune defenses including suppression of bacterial pathogenesis, virus toxicity neutralization, and inhibition of cytokine releases from immune cells. However, little is known about apoB homologous insect apolipophorin-II/I (apoLp-II/I) in controlling of specific pathogen-host encounters such as microbial infections. In the present study, we describe cDNA cloning and characterization of an apoLp-II/I from Chinese oak silk worm, Antheraea pernyi. The apoLp-II/I cDNA is 10237bp in length, which possesses an open reading frame encoding 3305 amino acids. A consensus cleavage site RGRR presenting from Arg710 to Arg713 implies posttranslational cleavage of this protein. Ap-apoLp-II/I shares high sequence identities with apoLp-II/I in lepidoptera and other insects. In addition, considerable similarities also exist between Ap-apoLp-II/I and human apoB, which basically positioned in first 1000 residues of the amino termini. Tissue distribution and time-course expression results demonstrate that Ap-apoLp-II/I transcripts were detected predominantly in the fat body, less in epidermis and rarely in midgut, while the synthetic apoLp-II/I protein was abundant in hemocytes and plasma instead of the fatbody. Expression of Ap-apoLp-II/I was stimulated in response to bacterial challenge. In addition, our preliminary studies established a novel role for Ap-apoLp-II/I in regulating prophenoloxidase activation system. Taken together, apoLp-II/I may play an essential role in innate responses of Antheraea pernyi.

The functions of serpin-3, a negative-regulator involved in prophenoloxidase activation and antimicrobial peptides expression of Chinese oak silkworm, Antheraea pernyi.

Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae.

Involvement of a versatile pattern recognition receptor, apolipophorin-III in prophenoloxidase activation and antibacterial defense of the Chinese oak silkworm, Antheraea pernyi.

Apolipophorin-III (apoLp-III) is an exchangeable apolipoprotein found in many insect species and functions as a lipid transport vehicle. Recent studies have shown that apoLp-III is a multifunctional molecule involved in not only lipid transportation but also innate immune responses. In the present study, the pattern recognition properties of Antheraea pernyi apoLp-III were investigated. Recombinant Ap-apoLp-III was bound to different species of microbes and further study showed the rAp-apoLp-III is capable of interacting with pathogen associated molecular patterns (PAMPs) on the microbial cell surface. In addition, an Ap-apoLp-III/PAMP mixture stimulated the prophenoloxidase (PPO) activation of A. pernyi hemolymph in vitro, to a greater extent than PAMP alone while Ap-apoLp-III itself failed to activate the PPO system, indicating that Ap-apoLp-III up-regulates PPO activation by combining with PAMP. After pathogen invasion following an injection of Staphylococcus aureus, RNAi-mediated silencing of apoLp-III decreased the transcriptional abundance of three antimicrobial peptide genes. These data suggest that apoLp-III is a versatile pattern recognition receptor and may play important roles in the innate immune responses of Antheraea pernyi.