G-MDSC-derived exosomes mediate the differentiation of M-MDSC into M2 macrophages promoting colitis-to-cancer transition.

BACKGROUNDS: In inflammatory bowel disease microenvironment, transdifferentiation of myeloid-derived suppressor cells (MDSCs) and M2 macrophage accumulation are crucial for the transition of colitis-to-cancer. New insights into the cross-talk and the underling mechanism between MDSCs and M2 macrophage during colitis-to-cancer transition are opening new avenues for colitis-associated cancer (CAC) prevention and treatment. METHODS: The role and underlying mechanism that granulocytic MDSCs (G-MDSCs) or exosomes (Exo) regulates the differentiation of monocytic MDSCs (M-MDSCs) into M2 macrophages were investigated using immunofluorescence, FACS, IB analysis, etc, and employing siRNA and antibodies. In vivo efficacy and mechanistic studies were conducted with dextran sulfate sodium-induced CAC mice, employed IL-6 Abs and STAT3 inhibitor. RESULTS: G-MDSCs promote the differentiation of M-MDSC into M2 macrophages through exosomal miR-93-5 p which downregulating STAT3 activity in M-MDSC. IL-6 is responsible for miR-93-5 p enrichment in G-MDSC exosomes (GM-Exo). Mechanistically, chronic inflammation-driven IL-6 promote the synthesis of miR-93-5 p in G-MDSC via IL-6R/JAK/STAT3 pathway. Early use of IL-6 Abs enhances the effect of STAT3 inhibitor against CAC. CONCLUSIONS: IL-6-driven secretion of G-MDSC exosomal miR-93-5 p promotes the differentiation of M-MDSC into M2 macrophages and involves a STAT3 signaling mechanism that promote colitis-to-cancer transition. Combining STAT3 inhibitors with strategies that inhibit IL-6-mediated G-MDSC exosomal miR-93-5 p production is beneficial for the prevention and treatment of CAC.

Research Advances on Anti-Cancer Natural Products.

Malignant tumors seriously threaten people's health and life worldwide. Natural products, with definite pharmacological effects and known chemical structures, present dual advantages of Chinese herbs and chemotherapeutic drug. Some of them exhibit favorable anti-cancer activity. Natural products were categorized into eight classes according to their chemical structures, including alkaloids, terpenoids and volatile oils, inorganic salts, phenylpropanoids, flavonoids and isoflavones, quinone, saponins and polysaccharides. The review focused on the latest advances in anti-cancer activity of representative natural products for every class. Additionally, anti-cancer molecular mechanism and derivatization of natural products were summarized in detail, which would provide new core structures and new insights for anti-cancer new drug development.

Research Advances on Matrine.

Matrine is an alkaloid extracted from traditional Chinese herbs including Sophora flavescentis, Sophora alopecuroides, Sophora root, etc. It has the dual advantages of traditional Chinese herbs and chemotherapy drugs. It exhibits distinct benefits in preventing and improving chronic diseases such as cardiovascular disease and tumors. The review introduced recent research progresses on extraction, synthesis and derivatization of Matrine. The summary focused on the latest research advances of Matrine on anti-atherosclerosis, anti-hypertension, anti-ischemia reperfusion injury, anti-arrhythmia, anti-diabetic cardiovascular complications, anti-tumor, anti-inflammatory, anti-bacterium, anti-virus, which would provide new core structures and new insights for new drug development in related fields.

Targeted Bisulfite Sequencing Reveals DNA Methylation Changes in Zinc Finger Family Genes Associated With KRAS Mutated Colorectal Cancer.

Background: Colorectal cancer (CRC) is a leading cause of cancer death, and early diagnosis of CRC could significantly reduce its mortality rate. Previous studies suggest that the DNA methylation status of zinc finger genes (ZFGs) could be of potential in CRC early diagnosis. However, the comprehensive evaluation of ZFGs in CRC is still lacking. Methods: We first collected 1,426 public samples on genome-wide DNA methylation, including 1,104 cases of CRC tumors, 54 adenomas, and 268 para-tumors. Next, the most differentially methylated ZFGs were identified and validated in two replication cohorts comprising 218 CRC patients. Finally, we compared the prediction capabilities between the ZFGs and the SEPT9 in all CRC patients and the KRAS + and KRAS- subgroup. Results: Five candidate ZFGs were selected: ESR1, ZNF132, ZNF229, ZNF542, and ZNF677. In particular, ESR1 [area under the curve (AUC) = 0.91] and ZNF132 (AUC = 0.93) showed equivalent or better diagnostic capability for CRC than SEPT9 (AUC = 0.91) in the validation dataset, suggesting that these two ZFGs might be of potential for CRC diagnosis in the future. Furthermore, we performed subgroup analysis and found a significantly higher diagnostic capability in KRAS + (AUC ranged from 0.97 to 1) than that in KRAS- patients (AUC ranged from 0.74 to 0.86) for all these five ZFGs, suggesting that these ZFGs could be ideal diagnostic markers for KRAS mutated CRC patients. Conclusion: The methylation profiles of the candidate ZFGs could be potential biomarkers for the early diagnosis of CRC, especially for patients carrying KRAS mutations.

DNA hypermethylation contributes to colorectal cancer metastasis by regulating the binding of CEBPB and TFCP2 to the CPEB1 promoter.

BACKGROUND: Aberrant DNA methylation has been firmly established as a factor contributing to the pathogenesis of colorectal cancer (CRC) via its capacity to silence tumour suppressor genes. However, the methylation status of multiple tumour suppressor genes and their roles in promoting CRC metastasis are not well characterised. METHODS: We explored the methylation and expression profiles of CPEB1 (the gene encoding cytoplasmic polyadenylation element-binding protein 1), a candidate CRC tumour suppressor gene, using The Cancer Genome Atlas (TCGA) database and validated these results in both CRC cell lines and cells from Han Chinese CRC patients (n = 104). The functional role of CPEB1 in CRC was examined in experiments performed in vitro and in vivo. A candidate transcription factor capable of regulating CPEB1 expression was predicted in silico and validated by luciferase reporter, DNA pull-down, and electrophoretic mobility shift assays. RESULTS: Hypermethylation and decreased expression of CPEB1 in CRC tumour tissues were revealed by TCGA database. We also identified a significant inverse correlation (Pearson's R = - 0.43, P < 0.001) between promoter methylation and CPEB1 expression. We validated these results in CRC samples and two CRC cell lines. We also demonstrated that up-regulation of CPEB1 resulted in significantly decreased tumour growth, migration, invasion, and tumorigenicity and promoted tumour cell apoptosis both in vitro and in vivo. We identified the transcription factors CCAAT enhancer-binding protein beta (CEBPB) and transcription factor CP2 (TFCP2) as critical regulators of CPEB1 expression. Hypermethylation of the CPEB1 promoter resulted in a simultaneous increase in the capacity for TFCP2 binding and a decreased likelihood of CEBPB binding, both of which led to diminished expression of CPEB1. CONCLUSIONS: Our results identified a novel tumour-suppressive role of CPEB1 in CRC and found that hypermethylation of the CPEB1 promoter may lead to diminished expression due to decreased chromatin accessibility and transcription factor binding. Collectively, these results suggest a potential role for CPEB1 in the diagnosis and treatment of CRC.

Association between serum NPTX2 and cognitive function in patients with vascular dementia.

OBJECTIVE: Neuronal Pentraxin 2 (NPTX2) has recently been widely reported as a novel biomarker for Alzheimer's disease (AD), but its correlation with vascular dementia (VaD) has not been elucidated. This study aimed to explore the correlation between NPTX2 and the cognitive function of VaD patients. METHODS: 112 VaD patients and 76 healthy controls were included in the study. Upon admission, clinical baseline data for all subjects were collected. Serum NPTX2 levels were determined using enzyme-linked immunosorbent assay (ELISA). At the same time, the Montreal cognitive assessment (MoCA) scale was used to measure cognitive function. Multivariate regression analysis was used to determine the relationship between serum NPTX2 level and the cognitive function of VaD patients. RESULTS: Compared with healthy controls, VaD patients had lower serum NPTX2 levels (p < .001). The results of Spearman's correlation analysis showed that serum NPTX2 levels in VaD patients were positively correlated with MoCA scores (r = .347, p = .042). The results of multivariate regression analysis showed that after adjusting for common risk factors, serum NPTX2 levels in VaD patients were still significantly associated with MoCA scores (ß = 0.346, p = .039). CONCLUSIONS: Serum NPTX2 level was independently associated with cognitive function in patients with VaD. Serum NPTX2 level may be a novel predictor for cognitive function in VaD.

Rational discovery of novel type-III FTF antagonists to competitively suppress TIF-2 coactivation in liver cancer.

The α-fetoprotein transcription factor (FTF) is a member of the nuclear receptor NR5A subfamily, which is involved in the pathogenesis of liver cancer and some other gastrointestinal cancers. The protein's transcriptional activity is regulated by binding TIF-2 coactivator at its coactivator-interacting site (CIS); suppression of the transcriptional activity has been recognized as a potential therapeutic strategy against cancer. Previously, small-molecule antagonists have been developed to target the ligand-binding site (LBS) of FTF ligand-binding domain, which simply occupy the site to exclusively block natural ligand entry (type-I antagonists) or destabilize the agonist conformation of activation helix 12 of the domain (type-II antagonists). Here, we describe the use of small-molecule competitors (type-III antagonists) to directly disrupt FTF-TIF-2 interaction by competitively targeting FTF CIS site. High-throughput virtual screening is performed against a structurally diverse, commercially available compound library to identify FTF CIS binders as competitor candidates, from which 12 hits are manually selected and their competitive potency with TIF-2 core binding sequence for FTF CIS site is tested with CC50 values up to 2.5 µM. Structural modeling analysis revealed that the competitive ligands can form a complicated network of noncovalent interactions to specifically or nonspecifically pack against FTF CIS site, thus preventing TIF-2 from binding to the site.

Construction and optimization of an efficient amplification method of a random ssDNA library by asymmetric emulsion PCR.

Construction of a random ssDNA sublibrary is an important step of the aptamer screening process. The available construction methods include asymmetric PCR, biotin-streptavidin separation, and lambda exonuclease digestions, in which PCR amplification is a key step. The main drawback of PCR amplification is overamplification increasing nonspecific hybridization among different products and by-products, which may cause the loss of potential high-quality aptamers, inefficient screening, and even screening failure. Cycle number optimization in PCR amplification is the main way to avoid overamplification but does not fundamentally eliminate the nonspecific hybridization, and the decreased cycle number may lead to insufficient product amounts. Here, we developed a new method, "asymmetric emulsion PCR," which could overcome the shortcomings of conventional PCR. In asymmetric emulsion PCR, different templates were separated by emulsion particles, allowing single-molecule PCR, in which each template was separately amplified, and the nonspecific hybridization was avoided. Overamplification or formation of by-products was not observed. The method is so simple that direct amplification of 40 or more cycles can provide a high-quality ssDNA library. Therefore, the asymmetric emulsion PCR would improve the screening efficiency of systematic evolution of ligands by exponential enrichment.

Amelioration of colorectal cancer using negative lipidoid nanoparticles to encapsulate siRNA against APRIL by enema delivery mode.

A proliferation-inducing ligand (APRIL) is a key cell proliferation-regulatory molecule and have been investigated well enough in immunity regulation and a few of immune diseases. APRIL can stimulate tumor cell growth and is up-expressed in cancer tissues, especially in CRC (colorectal cancer). However, whether inhibition of APRIL can regulate tumor-relative genes expression in vivo and subsequently ameliorate the pathological progress of CRC remains obscure. To address this question, we developed a novel negative lipidoid nanoparticles (NLNs) encapsulating small interference RNA (siRNA) for selectively silencing APRIL in the parenchyma of CRC focus in vivo, which uptake proceeded through a lipid raft endocytotic pathway. Local enema delivery of APRIL-NLNs silenced APRIL in CRC cells and animal models, and then ameliorated experimentally the progress of CRC by suppressing CRC cell proliferation, metastasis, and apoptosis-related cytokine expression and did not affect the function of liver and kidneys and not trigger the immune response of CRC models. This study reveals APRIL to be a potential anti-CRC target by in vivo experiments, and suggests that the application of similar modes of siRNA delivery may be feasible in other therapeutic settings.

Simultaneous knockdown of APRIL via multiple shRNAs reduces the malignancy of SW480 cells.

A proliferation-inducing ligand (APRIL) is a key factor involved in the tumor development and progression in some tumor tissues and cells. Its overexpression and as gene target in SW480 colon carcinoma cells was confirmed in our previous study. To seek a more potent way to treat colon carcinoma using a gene therapy method, herein, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against the APRIL gene in SW480 cells. APRIL expression levels and cell biological behavior were detected after transfection with different kinds of vectors. As expected, we found that our multiple shRNA vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA. Furthermore, our findings indicate that silencing APRIL expression in SW480 cells decreased their malignancy by reducing proliferation, invasion and adhesion, as well as inducing apoptosis. Based on our findings, vectors containing multiple shRNAs to silence the expression of APRIL may be exploited as a novel therapeutic strategy for tumors.

Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection.

Aptamers are short RNA or DNA oligonucleotides which can bind with different targets. Typically, they are selected from a large number of random DNA sequence libraries. The main strategy to obtain aptamers is systematic evolution of ligands by exponential enrichment (SELEX). Low efficiency is one of the limitations for conventional PCR amplification of random DNA sequence library in aptamer selection because of relative low products and high by-products formation efficiency. Here, we developed emulsion PCR for aptamer selection. With this method, the by-products formation decreased tremendously to an undetectable level, while the products formation increased significantly. Our results indicated that by-products in conventional PCR amplification were from primer-product and product-product hybridization. In emulsion PCR, we can completely avoid the product-product hybridization and avoid the most of primer-product hybridization if the conditions were optimized. In addition, it also showed that the molecule ratio of template to compartment was crucial to by-product formation efficiency in emulsion PCR amplification. Furthermore, the concentration of the Taq DNA polymerase in the emulsion PCR mixture had a significant impact on product formation efficiency. So, the results of our study indicated that emulsion PCR could improve the efficiency of SELEX.