Dynamic Hybrid Module-Driven NK Cell Stimulation and Release for Tumor Immunotherapy.

Natural killer (NK) cells have become a powerful candidate for adoptive tumor immunotherapy, while their therapeutic efficacy in solid tumors remains unsatisfactory. Here, we developed a hybrid module with an injectable hydrogel and hydroxyapatite (HAp) nanobelts for the controlled delivery of NK cells to enhance the therapy of solid tumors. Surface-functionalized HAp nanobelts modified with agonistic antibodies against NKG2D and 4-1BB and cytokines IL-2 and IL-21 support survival and dynamic activation. Thus, the HAp-modified chitosan (CS) thermos-sensitive hydrogel not only improved the retention of NK cells for more than 20 days in vivo but also increased NK cell function by more than one-fold. The unique architecture of this biomaterial complex protects NK cells from the hostile tumor environment and improves antitumor efficacy. The generation of a transient inflammatory niche for NK cells through a biocompatible hydrogel reservoir may be a conversion pathway to prevent cancer recurrence of resectable tumors.

Increased Siglec-9/Siglec-9L interactions on NK cells predict poor HCC prognosis and present a targetable checkpoint for immunotherapy.

BACKGROUND & AIMS: Natural killer (NK) cell-based anti-hepatocellular carcinoma (HCC) therapy is an increasingly attractive approach that warrants further study. Siglec-9 interacts with its ligand (Siglec-9L) and restrains NK cell functions, suggesting it is a potential therapeutic target. However, in situ Siglec-9/Siglec-9L interactions in HCC have not been reported, and a relevant interventional strategy is lacking. Herein, we aim to illustrate Siglec-9/Siglec-9L-mediated cell sociology and identify small-molecule inhibitors targeting Siglec-9 that could improve the efficacy of NK cell-based immunotherapy for HCC. METHODS: Multiplexed immunofluorescence staining was performed to analyze the expression pattern of Siglec-7, -9 and their ligands in HCC tissues. Then we conducted docking-based virtual screening combined with bio-layer interferometry assays to identify a potent small-molecule Siglec-9 inhibitor. The therapeutic potential was further evaluated in vitro and in hepatoma-bearing NCG mice. RESULTS: Siglec-9 expression, rather than Siglec-7, was markedly upregulated on tumor-infiltrating NK cells, which correlated significantly with reduced survival of patients with HCC. Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival, further suggesting that Siglec-9/Siglec-9L interactions are a potential therapeutic target in HCC. In addition, we identified a small-molecule Siglec-9 inhibitor MTX-3937 which inhibited phosphorylation of Siglec-9 and downstream SHP1 and SHP2. Accordingly, MTX-3937 led to considerable improvement in NK cell function. Notably, MTX-3937 enhanced cytotoxicity of both human peripheral and tumor-infiltrating NK cells. Furthermore, transfer of MTX-3937-treated NK92 cells greatly suppressed the growth of hepatoma xenografts in NCG mice. CONCLUSIONS: Our study provides the rationale for HCC treatment by targeting Siglec-9 on NK cells and identifies a promising small-molecule inhibitor against Siglec-9 that enhances NK cell-mediated HCC surveillance. IMPACT AND IMPLICATIONS: Herein, we found that Siglec-9 expression is markedly upregulated on tumor-infiltrating natural killer (TINK) cells and correlates with reduced survival in patients with hepatocellular carcinoma (HCC). Moreover, the number of Siglec-9L+ cells neighboring Siglec-9+ NK cells was increased in HCC tissues and was also associated with tumor recurrence and reduced survival. More importantly, we identified a small-molecule inhibitor targeting Siglec-9 that augments NK cell functions, revealing a novel immunotherapy strategy for liver cancer that warrants further clinical investigation.

Tim-4 alleviates acute hepatic injury by modulating homeostasis and function of NKT cells.

T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim-4) is an immune checkpoint molecule, which involves in numerous inflammatory diseases. Tim-4 is mainly expressed on antigen presenting cells. However, increasing evidences have shown that Tim-4 is also expressed on natural killer T (NKT) cells. The role of Tim-4 in maintaining NKT cell homeostasis and function remains unknown. In this study, we explored the effect of Tim-4 on NKT cells in acute liver injury. This study found that Tim-4 expression on hepatic NKT cells was elevated during acute liver injury. Tim-4 deficiency enhanced IFN-γ, TNF-α expression while impaired IL-4 production in NKT cells. Loss of Tim-4 drove NKT cell effector lineages to be skewed to NKT1 subset. Furthermore, Tim-4 KO mice were more susceptible to α-GalCer challenge. In conclusion, our findings indicate that Tim-4 plays an important role in regulating homeostasis and function of NKT cells in acute liver injury. Therefore, Tim-4 might become a new regulator of NKT cells and a potential target for the therapy of acute hepatitis.

VEGFR2 blockade inhibits glioblastoma cell proliferation by enhancing mitochondrial biogenesis.

BACKGROUND: Glioblastoma is an aggressive brain tumor linked to significant angiogenesis and poor prognosis. Anti-angiogenic therapies with vascular endothelial growth factor receptor 2 (VEGFR2) inhibition have been investigated as an alternative glioblastoma treatment. However, little is known about the effect of VEGFR2 blockade on glioblastoma cells per se. METHODS: VEGFR2 expression data in glioma patients were retrieved from the public database TCGA. VEGFR2 intervention was implemented by using its selective inhibitor Ki8751 or shRNA. Mitochondrial biogenesis of glioblastoma cells was assessed by immunofluorescence imaging, mass spectrometry, and western blot analysis. RESULTS: VEGFR2 expression was higher in glioma patients with higher malignancy (grade III and IV). VEGFR2 inhibition hampered glioblastoma cell proliferation and induced cell apoptosis. Mass spectrometry and immunofluorescence imaging showed that the anti-glioblastoma effects of VEGFR2 blockade involved mitochondrial biogenesis, as evidenced by the increases of mitochondrial protein expression, mitochondria mass, mitochondrial oxidative phosphorylation (OXPHOS), and reactive oxygen species (ROS) production, all of which play important roles in tumor cell apoptosis, growth inhibition, cell cycle arrest and cell senescence. Furthermore, VEGFR2 inhibition exaggerated mitochondrial biogenesis by decreased phosphorylation of AKT and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which mobilized PGC1α into the nucleus, increased mitochondrial transcription factor A (TFAM) expression, and subsequently enhanced mitochondrial biogenesis. CONCLUSIONS: VEGFR2 blockade inhibits glioblastoma progression via AKT-PGC1α-TFAM-mitochondria biogenesis signaling cascade, suggesting that VEGFR2 intervention might bring additive therapeutic values to anti-glioblastoma therapy.

NAD + salvage governs mitochondrial metabolism, invigorating natural killer cell antitumor immunity.

BACKGROUND AND AIMS: Natural killer (NK) cells are key players in tumor immunosurveillance, and metabolic adaptation manipulates their fate and functional state. The nicotinamide adenine dinucleotide (NAD + ) has emerged as a vital factor to link cellular metabolism and signaling transduction. Here, we identified NAD + metabolism as a central hub to determine the homeostasis and function of NK cells. APPROACH AND RESULTS: NAD + level was elevated in activated NK cells. NAD + supplementation not only enhanced cytokine production and cytotoxicity but also improved the proliferation and viability of NK cells. Intriguingly, the salvage pathway was involved in maintaining NAD + homeostasis in activated NK cells. Genetic ablation or pharmacological blockade of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD + salvage pathway, markedly destroyed the viability and function of NK cells. Mechanistically, NAD + salvage dictated the mitochondrial homeostasis and oxidative phosphorylation activity to support the optimal function of NK cells. However, in human HCC tissues, NAMPT expression and NAD + level were significantly down-regulated in tumor-infiltrating NK cells, which negatively correlated with patient survival. And lactate accumulation in the tumor microenvironment was at least partially responsible for the transcriptional repression of NAMPT in NK cells. Further, deficiency of Nampt in NK cells accelerated the growth of HCC and melanoma. Supplementation of the NAD + precursor nicotinamide mononucleotide (NMN) significantly improved NK antitumor response in both mouse and human cell-derived xenografts. CONCLUSIONS: These findings reveal NAD + salvage as an essential factor for NK-cell homeostasis and function, suggesting a potential strategy for invigorating NK cell-based immunotherapy.

Microbiological characteristics of the lower airway in adults with bronchiectasis: a prospective cohort study.

BACKGROUND: Microbial infection and colonization are frequently associated with disease progression and poor clinical outcomes in bronchiectasis. Identification of pathogen spectrum is crucial for precision treatment at exacerbation of bronchiectasis. METHODS: We conducted a prospective cohort study in patients with bronchiectasis exacerbation onset and stable state. Bronchoalveolar lavage fluid (BALF) was collected for conventional microbiological tests (CMTs) and metagenomic Next-Generation Sequencing (mNGS). Bronchiectasis patients were monitored for documenting the time to the next exacerbation during longitudinal follow-up. RESULTS: We recruited 168 eligible participants in the exacerbation cohorts, and 38 bronchiectasis patients at stable state at longitudinal follow-up. 141 bronchiectasis patients at exacerbation onset had definite or probable pathogens via combining CMTs with mNGS reports. We identified that Pseudomonas aeruginosa, non-tuberculous mycobacteria, Haemophilus influenzae, Nocardia spp, and Staphylococcus aureus were the top 5 pathogens with a higher detection rate in our cohorts via combination of CMTs and mNGS analysis. We also observed strong correlations of Pseudomonas aeruginosa, Haemophilus influenzae, non-tuberculous mycobacteria with disease severity, including the disease duration, Bronchiectasis Severity Index, and lung function. Moreover, the adjusted pathogenic index of potential pathogenic microorganism negatively correlated (r = -0.7280, p < 0.001) with the time to the next exacerbation in bronchiectasis. CONCLUSION: We have revealed the pathogenic microbial spectrum in lower airways and the negative correlation of PPM colonization with the time to the next exacerbation in bronchiectasis. These results suggested that pathogens contribute to the progression of bronchiectasis.

T-cell immunoglobulin- and mucin-domain-containing molecule-4 maintains adipose tissue homeostasis by orchestrating M2 macrophage polarization via nuclear factor kappa B pathway.

Obesity is generally associated with low-grade inflammation. Adipose tissue macrophages (ATMs) orchestrate metabolic inflammation. The classical (M1-like) or alternative (M2-like) activation of ATMs is functionally coupled with the metabolic status of fat tissues. It has been found that T-cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) inhibits inflammation by regulating macrophages. However, the exact role of Tim-4 in macrophage polarization and obesity remains unknown. Here, we identified Tim-4 as a critical switch governing macrophage M1/M2 polarization and energy homeostasis. Tim-4 deletion led to spontaneous obesity in elder mice and promoted obesity severity of db/db mice. Obesity microenvironment enhanced the expression of Tim-4 in white adipose tissue and ATMs. In vitro, we detected an increase in M1-like cells and decrease in M2-like cells in both peritoneal macrophages and bone marrow-derived macrophages from Tim-4 knockout mice. Mechanistically, we demonstrated that Tim-4 promoted M2-like macrophages polarization via suppressing nuclear factor kappa B (NF-κB) signaling pathway. In addition, we found that Tim-4 promoted TLR4 internalization, which might contribute to regulation of NF-κB signaling. Collectively, these results indicated that Tim-4 maintained adipose tissue homeostasis by regulating macrophage polarization via NF-κB pathway, which would provide a new target for obesity intervention.

SREBF2-STARD4 axis confers sorafenib resistance in hepatocellular carcinoma by regulating mitochondrial cholesterol homeostasis.

Sorafenib resistance limits its survival benefit for treatment of hepatocellular carcinoma (HCC). Cholesterol metabolism is dysregulated in HCC, and its role in sorafenib resistance of HCC has not been fully elucidated. Aiming to elucidate this, in vitro and in vivo sorafenib resistant models were established. Sterol regulatory element binding transcription factor 2 (SREBF2), the key regulator of cholesterol metabolism, was activated in sorafenib resistant HepG2 and Huh7 cells. Knockdown of SREBF2 resensitized sorafenib resistant cells and xenografts tumors to sorafenib. Further study showed that SREBF2 positively correlated with StAR related lipid transfer domain containing 4 (STARD4) in our sorafenib resistant models and publicly available datasets. STARD4, mediating cholesterol trafficking, not only promoted proliferation and migration of HepG2 and Huh7 cells, but also increased sorafenib resistance in liver cancer. Mechanically, SREBF2 promoted expression of STARD4 by directly binding to its promoter region, leading to increased mitochondrial cholesterol levels and inhibition of mitochondrial cytochrome c release. Importantly, knockdown of SREBF2 or STARD4 decreased mitochondrial cholesterol levels and increased mitochondrial cytochrome c release, respectively. Moreover, overexpression of STARD4 reversed the effect of SREBF2 knockdown on mitochondrial cytochrome c release and sorafenib resistance. In conclusion, SREBF2 promotes STARD4 transcription, which in turn contributes to mitochondrial cholesterol transport and sorafenib resistance in HCC. Therefore, targeting the SREBF2-STARD4 axis would be beneficial to a subset of HCC patients with sorafenib resistance.

Spatial distribution and functional analysis define the action pathway of Tim-3/Tim-3 ligands in tumor development.

The spatial organization of immune cells within the tumor microenvironment (TME) largely determines the anti-tumor immunity and also highly predicts tumor progression and therapeutic response. Tim-3 is a well-accepted immune checkpoint and plays multifaceted immunoregulatory roles via interaction with distinct Tim-3 ligands (Tim-3L), showing great potential as an immunotherapy target. However, the cell sociology mediated by Tim-3/Tim-3L and their contribution to tumor development remains elusive. Here, we analyzed the spatial distribution of Tim-3/Tim-3L in TME using multiplex fluorescence staining and revealed that despite the increased Tim-3 expression in various tumor-infiltrated lymphocytes, Tim-3+CD4+ cells were more accumulated in parenchymal/tumor region compared with stromal region and exhibited more close association with patient survival. Strikingly, CD4 T cells surrounding Tim-3L+ cells expressed higher Tim-3 than other cells in cancerous tissues. In vivo studies confirmed that depletion of CD4 T cells completely abrogated the inhibition of tumor growth and metastasis, as well as the functional improvement of CD8 T and NK, mediated by Tim-3 blockade, which was further validated in peripheral lymphocytes from patients with hepatocellular carcinoma. In conclusion, our findings unravel the importance of CD4 T cells in Tim-3/Tim-3L-mediated immunosuppression and provide new thoughts for Tim-3 targeted cancer immunotherapy.

ZHX2 inhibits thyroid cancer metastasis through transcriptional inhibition of S100A14.

BACKGROUND: Thyroid cancer is the most common malignant endocrine tumour, and metastasis has become the main reason for treatment failure. However, the underlying molecular mechanism of thyroid cancer metastasis remains poorly understood. We investigated the role of the tumour suppressor zinc fingers and homeoboxes 2 (ZHX2) in the metastasis of thyroid cancer. METHODS: To study the role of ZHX2 in thyroid cancer metastasis, we evaluated the EMT process using cell migration, wound healing and lung metastatic tumour formation in vitro and in vivo models. RESULTS: ZHX2 expression was significantly decreased in thyroid cancer tissues, which correlated with poor prognosis of thyroid cancer patients. ZHX2 knockdown significantly promoted the migration of thyroid cancer cells. Mechanistically, ZHX2 associated with the S100 calcium binding protein A14 (S100A14) promoter to decrease the transcription of S100A14. Moreover, S100A14 was highly expressed in human thyroid cancer samples, and its expression negatively correlated with ZHX2 expression. CONCLUSIONS: Inhibition of S100A14 attenuated the ZHX2 knockdown-induced enhanced metastasis of thyroid cancer cells both in vitro and in vivo. The evidence presented here suggests that ZHX2 inhibits the progression of thyroid cancer by blocking S100A14-mediated metastasis.

Zhx2 Accelerates Sepsis by Promoting Macrophage Glycolysis via Pfkfb3.

Sepsis is a life-threatening condition with limited therapeutic options, characterized as excessive systemic inflammation and multiple organ failure. Macrophages play critical roles in sepsis pathogenesis. Metabolism orchestrates homeostasis of macrophages. However, the precise mechanism of macrophage metabolism during sepsis remains poorly elucidated. In this study, we identified the key role of zinc fingers and homeoboxes (Zhx2), a ubiquitous transcription factor, in macrophage glycolysis and sepsis by enhancing 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3) expression. Mice with myeloid Zhx2-specific deletion (abbreviated as MKO) showed more resistance to cecal ligation and puncture and LPS-induced sepsis, exhibiting as prolonged survival, attenuated pulmonary injury, and reduced level of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1ß. Interestingly, Zhx2 deletion conferred macrophage tolerance to LPS-induced glycolysis, accompanied by reduced proinflammatory cytokines and lactate. Consistently, treatment of glycolytic inhibitor 2-deoxyglucose almost completely abrogated the protection of mice from LPS-induced sepsis initiated by Zhx2 deletion in macrophages. RNA sequencing and chromatin immunoprecipitation assays confirmed that Zhx2 enhanced transcription of Pfkfb3, the glycolysis rate-limiting enzyme, via binding with Pfkfb3 promoter. Furthermore, Pfkfb3 overexpression not only rescued the reduction of macrophage glycolysis caused by Zhx2 deficiency, displaying as extracellular acidification rates and lactate production but also destroyed the resistance of mice to LPS-induced sepsis initiated by transfer of bone marrow-derived macrophages from MKO mice. These findings highlight the novel role of transcription factor Zhx2 in sepsis via regulating Pfkfb3 expression and reprogramming macrophage metabolism, which would shed new insights into the potential strategy to intervene sepsis.

ZHX2 inhibits SREBP1c-mediated de novo lipogenesis in hepatocellular carcinoma via miR-24-3p.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Lipogenesis has been considered as a critical player in HCC initiation and progression. However, the underlying mechanism is still not fully understood. Here, we identified zinc fingers and homeoboxes 2 (ZHX2), an HCC-associated tumor suppressor, as an important repressor of de novo lipogenesis. Ectopic expression of ZHX2 significantly inhibited de novo lipogenesis in HCC cells and decreased expression of FASN, ACL, ACC1, and SCD1. In accordance with this, ZHX2 was negatively associated with SREBP1c, the master regulator of de novo lipogenesis, in HCC cell lines and human specimens. Results from silencing and overexpression demonstrated that ZHX2 inhibited de novo lipogenesis and consequent HCC progression via repression of SREBP1c. Furthermore, treatment with the SREBP1c inhibitor fatostatin dampened the spontaneous formation of tumors in liver-specific Zhx2 knockout mice. Mechanistically, ZHX2 increased expression of miR-24-3p transcriptionally, which targeted SREBP1c and led to its degradation. In conclusion, our data suggest a novel mechanism through which ZHX2 suppresses HCC progression, which may provide a new strategy for the treatment of HCC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tim-4 Inhibits NLRP3 Inflammasome via the LKB1/AMPKα Pathway in Macrophages.

Nonalcoholic fatty liver disease (NAFLD), characterized by excessive inflammation and lipid deposition, is one of the most common metabolic liver diseases. The expression of NLRP3 inflammasome in macrophages is significantly increased in NAFLD, and its activation aggravates NAFLD greatly. Tim-4, as the phosphatidylserine (PS) receptor, is expressed highly in macrophages, and macrophage Tim-4 inhibits inflammation under various conditions of immune activation. However, the precise role of Tim-4 in NLRP3 inflammasome regulation and NAFLD pathogenesis remains completely unknown. Using NAFLD mice models, we confirmed that the expression of Tim-4 was increased in liver tissues by Western blot, real-time PCR, immunohistochemistry, and immunofluorescence, especially higher expression in liver macrophages, and Tim-4 knockout mice displayed more severe liver inflammation and hepatic steatosis than controls in NAFLD mice model. In vitro, we found that Tim-4 could inhibit NLRP3 inflammasome activation, and the inhibition was dependent on PS binding domain in the IgV domain. Mechanistically, Tim-4 induced the degradation of NLRP3 inflammasome components through activating AMPKα-mediated autophagy. Specifically, Tim-4 promoted AMPKα phosphorylation by interacting with LKB1 and AMPKα. In addition, PS binding motif was responsible for Tim-4-mediated AMPKα and LKB1 interaction. In conclusion, NAFLD microenvironments upregulate Tim-4 expression in macrophages, and elevated Tim-4, in turn, suppresses NLRP3 inflammasome activation by activating LKB1/AMPKα-mediated autophagy, thereby ameliorating the release of IL-1ß and IL-18. Collectively, this study unveils the novel function of Tim-4 in suppressing NLRP3 inflammasome, which would shed new lights on intervention of NAFLD or inflammatory liver diseases by targeting Tim-4.

Effects of early cold stress on gene expression in Chlamydomonas reinhardtii.

Cold stress imposes a great impact on the growth of nearly all photosynthetic organisms, including Chlamydomonas reinhardtii (C. reinhardtii). Despite prior studies on the mechanism of stress acclimation in plants, little has been done on the early events of cold sensing in C. reinhardtii. Here, we used C. reinhardtii as a model to study early events of cold signal transduction. By analyzing transcriptomic changes of C. reinhardtii exposed to cold, we found that 3471 genes were differentially expressed after 1 h of cold exposure. These genes were associated with a wide range of biological events and processes such as protein synthesis, cell cycle and protein kinase-based phosphorylation. Besides, the promoter of one gene (named as crAP2) which belongs to AP2/EREBP family and was significantly induced by cold was cloned, and functional analysis was conducted using GUS activity analysis through Agrobacterium-mediated transient assay in tobacco leaves.

Interior circular RNA.

Formed by back splicing or back fusion of linear RNAs, circular RNAs (circRNAs) constitute a new class of non-coding RNAs of eukaryotes. Recent studies reveal a spliceosome-dependent biogenesis of circRNAs where circRNAs arise at the intron-exon junctions of mRNAs. In this study, using a novel de novo identification method, we show that circRNAs can originate from the interior regions of exons, introns, and intergenic transcripts in human, mouse and rice, which were referred to as interior circRNAs (i-circRNAs). Many i-circRNAs have some remarkable characteristics: multiple i-circRNAs may arise from the same genomic locus; their back fusion points may not be associated with the AG/GT splicing sites, but rather a new pair of motif AC/CT, their back fusion points are adjacent to complementary sequences; and they may circulate on short homologous sequences. We validated several i-circRNAs in HeLa cells by Polymerase Chain Reaction followed by Sanger sequencing. Our results combined showed that i-circRNAs are bona fide circRNAs, indicated novel biogenesis pathways independent of the splicing apparatus, and expanded our understanding of the origin, diversity, and complexity of circRNAs.

Phylogenetic and expression analysis of histone acetyltransferases in Brachypodium distachyon.

Histone acetylation is an important post-translational modification in eukaryotes and is regulated by two antagonistic enzymes, namely histone acetyltransferase (HAT) and histone deacetylase (HDAC). However, little has been done on the HAT superfamily in Brachypodium distachyon (B. distachyon), a new model plant of Poaceae. In this study, eight HATs were identified from B. distachyon and classified into four major families. Subcellular localization analysis showed that a majority of BdHATs were predominantly localized in the nucleus. Syntenic and phylogenetic analysis indicated there may be two common ancestral CREB-binding protein (p300/CBP, HAC) genes prior to the separation of monocots and dicots. Expression analysis revealed that the potential roles of BdHATs in B. distachyon development and responses to four abiotic stresses. Protein-protein network analysis identified some potential interactive genes with BdHATs. Thus, our results will provide solid basis for further study the function of HAT genes in B. distachyon and other monocot plants.

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis.

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3'UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3'-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment.

Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening.

BACKGROUND: Host genetic backgrounds affect gene functions. The genetic backgrounds of genetically engineered organisms must be identified to confirm their genetic backgrounds identity with those of recipients. Marker-assisted backcrossing (MAB), transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) editing are three commonly used genetic engineering techniques. However, methods for genetic background screening between genetically engineered organisms and corresponding recipients suffer from low efficiency, low accuracy or high cost. RESULTS: Here, we improved our previously reported AmpSeq-SSR method, an amplicon sequencing-based simple sequence repeat (SSR) genotyping method, by selecting SSR loci with high polymorphism among varieties. Ultimately, a set of 396 SSRs was generated and applied to evaluate the genetic backgrounds identity between rice lines developed through MAB, transgenesis, and CRISPR/Cas9 editing and the respective recipient rice. We discovered that the percentage of different SSRs between the MAB-developed rice line and its recipient was as high as 23.5%. In contrast, only 0.8% of SSRs were different between the CRISPR/Cas9-system-mediated rice line and its recipient, while no SSRs showed different genotypes between the transgenic rice line and its recipient. Furthermore, most differential SSRs induced by MAB technology were located in non-coding regions (62.9%), followed by untranslated regions (21.0%) and coding regions (16.1%). Trinucleotide repeats were the most prevalent type of altered SSR. Most importantly, all altered SSRs located in coding regions were trinucleotide repeats. CONCLUSIONS: This method is not only useful for the background evaluation of genetic resources but also expands our understanding of the unintended effects of different genetic engineering techniques. While the work we present focused on rice, this method can be readily extended to other organisms.

Monocarboxylate transporter 1 promotes classical microglial activation and pro-inflammatory effect via 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3.

BACKGROUND: Microglia, the resident macrophages of central nervous system, have been initially categorized into two opposite phenotypes: classical activation related to pro-inflammatory responses and alternative activation corresponding with anti-inflammatory reactions and tissue remodeling. The correlation between metabolic pattern and microglial activation has been identified. However, little is known about the mechanism of metabolism-mediated microglia polarization and pro-inflammatory effect. METHODS: Metabolic alteration was analyzed in different phenotypes of microglia in vitro. LPS-induced neuroinflammation and sickness behavior mouse model was used to investigate the effect of lactate on classical microglial activation in vivo. RESULTS: Glycolysis-related regulators, monocarboxylate transporter 1 (MCT1), MCT4, and pro-glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), were specifically increased in LPS-stimulated primary microglia and microglia cell line BV2. Knockdown of MCT1 suppressed glycolysis rate and decreased LPS-induced expression of iNOS, interleukin-1ß (IL-1ß), IL-6, and phosphorylation of STAT1 in BV2 cells. Importantly, MCT1 promoted PFKFB3 expression via hypoxia-inducible factor-1α (Hif-1α), and overexpression of PFKFB3 restored the classical activation of BV2 cells suppressed by MCT1 silence. All above strongly suggested that MCT1/PFKFB3 might accelerate LPS-induced classical polarization of microglia probably by promoting glycolysis. Interestingly, additional administration of moderate lactate, which may block the transport function of MCT1, decreased LPS-induced classical activation and expression of PFKFB3 in BV2 cells. Intracerebroventricular injection of lactate ameliorated LPS-induced sickness behavior and classical polarization of microglia in mice. CONCLUSIONS: Our results demonstrate the key role of MCT1 in microglial classical activation and neuroinflammation in pathological conditions. In addition, lactate administration may be a potential therapy to suppress neuroinflammation by altering microglial polarization.

An accurate and efficient method for large-scale SSR genotyping and applications.

Accurate and efficient genotyping of simple sequence repeats (SSRs) constitutes the basis of SSRs as an effective genetic marker with various applications. However, the existing methods for SSR genotyping suffer from low sensitivity, low accuracy, low efficiency and high cost. In order to fully exploit the potential of SSRs as genetic marker, we developed a novel method for SSR genotyping, named as AmpSeq-SSR, which combines multiplexing polymerase chain reaction (PCR), targeted deep sequencing and comprehensive analysis. AmpSeq-SSR is able to genotype potentially more than a million SSRs at once using the current sequencing techniques. In the current study, we simultaneously genotyped 3105 SSRs in eight rice varieties, which were further validated experimentally. The results showed that the accuracies of AmpSeq-SSR were nearly 100 and 94% with a single base resolution for homozygous and heterozygous samples, respectively. To demonstrate the power of AmpSeq-SSR, we adopted it in two applications. The first was to construct discriminative fingerprints of the rice varieties using 3105 SSRs, which offer much greater discriminative power than the 48 SSRs commonly used for rice. The second was to map Xa21, a gene that confers persistent resistance to rice bacterial blight. We demonstrated that genome-scale fingerprints of an organism can be efficiently constructed and candidate genes, such as Xa21 in rice, can be accurately and efficiently mapped using an innovative strategy consisting of multiplexing PCR, targeted sequencing and computational analysis. While the work we present focused on rice, AmpSeq-SSR can be readily extended to animals and micro-organisms.