Preparation, Structural Analysis, and Intestinal Probiotic Properties of a Novel Oligosaccharide from Enzymatic Degradation of Huangshui Polysaccharide.

Huangshui polysaccharide (HSP) has attracted more and more interest due to its potential health benefits. Despite being an excellent source for the preparation of oligosaccharides, there are currently no relevant research reports on HSP. In the present study, a novel oligosaccharide (HSO) with a molecular weight of 1791 Da and a degree of polymerization of 11 was prepared through enzymatic degradation of crude HSP (cHSP). Methylation and NMR analyses revealed that the main chain of HSO was (1 → 4)-α-d-glucose with two O-6-linked branched chains. Morphological observations indicated that HSO exhibited smooth surface with lamellar and filamentary structure, and the glycan size ranged from 0.03 to 0.20 µm. Notably, HSO significantly promoted the proliferation of Bifidobacterium, Bacteroides, and Phascolarctobacterium, thereby making positive alterations in intestinal microbiota composition. Moreover, HSO markedly increased the content of short-chain fatty acids during in vitro fermentation. Metabolomics analysis illustrated the important metabolic pathways primarily involving glucose metabolism, amino acid metabolism, and fatty acid metabolism.

Exosome-derived miR-372-5p promotes stemness and metastatic ability of CRC cells by inducing macrophage polarization.

Colorectal cancer (CRC) is the most common malignancy in the digestive system, and tumor metastasis is the main cause of death in clinical patients with CRC. It has been shown that exosomes promote phenotypic changes in macrophages and tumor metastasis in the CRC tumor microenvironment. In this study, we used miRNA-seq technology to screen out the highly expressed miR-372-5p among the miRNAs differentially expressed in plasma exosomes of clinical CRC patients. It was found that miR-372-5p highly expressed in HCT116 exosomes could be phagocytosed by macrophages and promote their polarization into M2 macrophages by regulating the PTEN/AKT pathway. Meanwhile, co-culture of CRC cells with conditioned medium (CM) of macrophages enhanced the EMT, stemness and metastasis of CRC cells. Mechanistically, CRC cells exosome-derived miR-372-5p induced polarized M2 macrophages to secrete chemokine C-X-C-Motif Ligand 12 (CXCL12), which activated the WNT/ß-catenin pathway to promote the EMT, stemness and metastatic ability of CRC cells. In summary, this study elucidated the molecular mechanism of exosomal miR-372-5p promoting metastasis and stemness in CRC, which may provide new therapeutic targets for CRC metastasis and prognosis assessment.

Colorectal cancer tumor cell-derived exosomal miR-203a-3p promotes CRC metastasis by targeting PTEN-induced macrophage polarization.

(1) Background: Tumor-associated macrophages (TAMs) are important immunocytes associated with liver metastasis of colorectal cancer (CRLM). However, the molecular processes underpinning the interaction between the TME and the tumour-derived exosomal miRNAs in CRLM are not being fully understood; (2) Methods: Transmission electron microscopy was utilized to confirm the existence of exosomes after differential ultracentrifugation. To determine the roles of exosomal miR-203a-3p, an in vivo and in vitro investigation was conducted. The mechanism by which exosomal miR-203a-3p governs the interaction between CRC cells and M2 macrophages was investigated using a dual-luciferase reporter assay, western blot, and other techniques; (3) Results: Overexpression of miR-203a-3p was associated with poor prognosis and liver metastasis in CRC patients. Exosomal miR-203a-3p was upregulated in the plasma of CRC patients and highly metastatic CRC cells HCT116, and it could be transported to macrophages via exosomes. Exosomal miR-203a-3p induced M2 polarization of macrophages by controlling PTEN and activating the PI3K/Akt signaling pathway. M2-polarized macrophages secreted the CXCL12, which increased cancer metastasis and resulted in pre-metastatic niches in CRLM by CXCL12/CXCR4/NF-κB signaling pathway. Co-culture of macrophages with miR-203a-3p-transfected or exosome-treated cells increased the ability of HCT116 cells to metastasize both in vitro and in vivo; (4) Conclusions: Exosomes produced by highly metastatic CRC cells and rich in miR-203a-3p may target PTEN and alter the TME, promoting liver metastasis in CRC patients. These findings offer fresh understanding of the liver metastatic process in CRC.

Layered Double Hydroxide-Loaded miR-30a for the Treatment of Breast Cancer In Vitro and In Vivo.

MicroRNAs (miRNAs) play an essential role in cancer therapy, but the disadvantages of its poor inherent stability, rapid clearance, and low delivery efficiency affect the therapeutic efficiency. Loading miRNAs by nanoformulations can improve their bioavailability and enhance therapeutic efficiency, which is an effective miRNA delivery strategy. In this study, we synthesized layered double hydroxides (LDH), which are widely used as carriers of drugs or genes due to the characteristics of good biocompatibility, high loading capacity, and pH sensitivity. We loaded the suppressor oncogene miR-30a on LDH nanomaterials (LDH@miR-30a) and determined the mass ratio of miRNA binding to LDH by agarose gel electrophoresis. LDH@miR-30a was able to escape the lysosomal pathway and was successfully phagocytosed by breast cancer SKBR3 cells and remained detectable in the cells after 24 h of co-incubation. In vitro experiments showed that LDH@miR-30a-treated SKBR3 cells showed decreased proliferation and cell cycle arrest in the G0/G1 phase and LDH@miR-30a was able to regulate the epithelial-mesenchymal transition (EMT) process and inhibit cell migration and invasion by targeting SNAI1. Meanwhile, in vivo experiments showed that nude mice treated with LDH@miR-30a showed a significant reduction in their solid tumors and no significant impairment of vital organs was observed. In conclusion, LDH@miR-30a is an effective drug delivery system for the treatment of breast cancer.

Huangshui Polysaccharide Exerts Intestinal Barrier Protective Effects through the TLR4/MyD88/NF-κB and MAPK Signaling Pathways in Caco-2 Cells.

Several reports have demonstrated that natural polysaccharides exert protective effects on intestinal barrier function. In our previous study, we isolated a polysaccharide named HSP-W from Huangshui (HS). In the present study, the protective role of HSP-W in LPS-induced intestinal barrier dysfunction was determined by several molecular biological techniques. The results showed that HSP-W treatment alleviated the deduced TEER and increased the permeability of intestinal epithelial cells induced by LPS through inhibiting the release of inflammatory cytokines and enhancing the expression of tight junction (TJ) proteins. The underlying molecular mechanisms were elucidated by RNA-seq technique, which indicated that the differentially expressed genes (DEGs) between the LPS-treated and LPS+HSP-W-treated groups were enriched in the "MAPK" signaling pathway. Notably, the overlapping DEGs reversed by HSP-W intervention highlighted the pathways of the "Toll-like receptor" and "NF-κB" signaling pathways. The suppression of p38 and NF-κB were mediated by the inhibition of MyD88. Furthermore, HSP-W treatment prevented the translocation of NF-κB to nucleus, thus inhibiting the release of TNF-α, IL-6, and IL-1ß. Overall, HSP-W has beneficial effects on LPS-induced inflammation; it protects the intestinal barrier from injury in Caco-2 cells through inhibiting the TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.

Exosomal lncRNA NEAT1 induces paclitaxel resistance in breast cancer cells and promotes cell migration by targeting miR-133b.

The lncRNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) has been associated with the development, metastasis and drug resistance of breast cancer (BC). However, the mechanisms underlying NEAT1-induced paclitaxel resistance in the microenvironment of BC remain unclear. In this study, NEAT1 expression was found to be high in paclitaxel-resistant BC cells (SKBR3/PR cells) and exosomes derived from these cells. NEAT1 promoted the migration of BC cells and their resistance to paclitaxel, whereas its downregulation reduced the drug resistance. In addition, downregulation of NEAT1 decreased the migration and proliferation of BC cells by inhibiting the expression of CXCL12 by reducing the adsorption of miR-133b. Furthermore, inhibition of miR-133b reversed the interference of NEAT1 and CXCL12 in paclitaxel resistance, migration and proliferation of BC cells. Knockdown of NEAT1 in a xenograft-bearing mouse model remarkably inhibited cancer progression and improved the response to paclitaxel. Altogether, this study revealed that SKBR3/PR cell-derived exosomal lncRNA NEAT1 can induce paclitaxel resistance and cell migration and growth in the tumour microenvironment of BC and may serve as a new target for the clinical treatment of BC.