circCDK13-loaded small extracellular vesicles accelerate healing in preclinical diabetic wound models.

Chronic wounds are a major complication in patients with diabetes. Here, we identify a therapeutic circRNA and load it into small extracellular vesicles (sEVs) to treat diabetic wounds in preclinical models. We show that circCDK13 can stimulate the proliferation and migration of human dermal fibroblasts and human epidermal keratinocytes by interacting with insulin-like growth factor 2 mRNA binding protein 3 in an N6-Methyladenosine-dependent manner to enhance CD44 and c-MYC expression. We engineered sEVs that overexpress circCDK13 and show that local subcutaneous injection into male db/db diabetic mouse wounds and wounds of streptozotocin-induced type I male diabetic rats could accelerate wound healing and skin appendage regeneration. Our study demonstrates that the delivery of circCDK13 in sEVs may present an option for diabetic wound treatment.

MiR-17-5p-engineered sEVs Encapsulated in GelMA Hydrogel Facilitated Diabetic Wound Healing by Targeting PTEN and p21.

Delayed wound healing is a major complication of diabetes, and is associated with impaired cellular functions. Current treatments are unsatisfactory. Based on the previous reports on microRNA expression in small extracellular vesicles (sEVs), miR-17-5p-engineered sEVs (sEVs17-OE) and encapsulated them in gelatin methacryloyl (GelMA) hydrogel for diabetic wounds treatment are fabricated. SEVs17-OE are successfully fabricated with a 16-fold increase in miR-17-5p expression. SEVs17-OE inhibited senescence and promoted the proliferation, migration, and tube formation of high glucose-induced human umbilical vein endothelial cells (HG-HUVECs). Additionally, sEVs17-OE also performs a promotive effect on high glucose-induced human dermal fibroblasts (HG-HDFs). Mechanism analysis showed the expressions of p21 and phosphatase and tensin homolog (PTEN), as the target genes of miR-17-5p, are downregulated significantly by sEVs17-OE. Accordingly, the downstream genes and pathways of p21 and PTEN, are activated. Next, sEVs17-OE are loaded in GelMA hydrogel to fabricate a novel bioactive wound dressing and to evaluate their effects on diabetic wound healing. Gel-sEVs17-OE effectively accelerated wound healing by promoting angiogenesis and collagen deposition. The cellular mechanism may be associated with local cell proliferation. Therefore, a novel bioactive wound dressing by loading sEVs17-OE in GelMA hydrogel, offering an option for chronic wound management is successfully fabricated.

MiR-141-3p-Functionalized Exosomes Loaded in Dissolvable Microneedle Arrays for Hypertrophic Scar Treatment.

Hypertrophic scar (HS) is a common fibroproliferative disease caused by abnormal wound healing after deep skin injury. However, the existing approaches have unsatisfactory therapeutic effects, which promote the exploration of newer and more effective strategies. MiRNA-modified functional exosomes delivered by dissolvable microneedle arrays (DMNAs) are expected to provide new hope for HS treatment. In this study, a miRNA, miR-141-3p, which is downregulated in skin scar tissues and in hypertrophic scar fibroblasts (HSFs), is identified. MiR-141-3p mimics inhibit the proliferation, migration, and myofibroblast transdifferentiation of HSFs in vitro by targeting TGF-ß2 to suppress the TGF-ß2/Smad pathway. Subsequently, the engineered exosomes encapsulating miR-141-3p (miR-141-3pOE -Exos) are isolated from adipose-derived mesenchymal stem cells transfected with Lv-miR-141-3p. MiR-141-3pOE -Exos show the same inhibitive effects as miR-141-3p mimics on the pathological behaviors of HSFs in vitro. The DMNAs for sustained release of miR-141-3pOE -Exos are further fabricated in vivo. MiR-141OE -Exos@DMNAs effectively decrease the thickness of HS and improve fibroblast distribution and collagen fiber arrangement, and downregulate the expression of α-SMA, COL-1, FN, TGF-ß2, and p-Smad2/3 in the HS tissue. Overall, a promising, effective, and convenient exosome@DMNA-based miRNA delivery strategy for HS treatment is provided.

Circular RNA circHERC4 as a novel oncogenic driver to promote tumor metastasis via the miR-556-5p/CTBP2/E-cadherin axis in colorectal cancer.

BACKGROUND: The main cause of death in colorectal cancer patients is metastasis. Accumulating evidences suggest that circRNA plays pivotal roles in cancer initiation and development. However, the underlying molecular mechanisms of circRNAs that orchestrate cancer metastasis remain vague and need further clarification. METHODS: Two paired CRC and adjacent normal tissues were used to screen the upregulated circRNAs by circRNA-seq; then, cell invasion assay was applied to confirm the functional invasion-related circRNAs. According to the above methods, circHERC4 (hsa_circ_0007113) was selected for further research. Next, we investigated the clinical significance of circHERC4 in a large cohort of patients with CRC. The oncogenic activity of circHERC4 was investigated in both CRC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying circHERC4 as a malignant driver. RESULTS: We demonstrated that circHERC4 was aberrantly elevated in CRC tissues (P < 0.001), and was positively associated with lymph node metastasis and advanced tumor grade (P < 0.01). Notably, the expression of circHERC4 was associated with worse survival in patients with CRC. Silencing of circHERC4 significantly inhibited the proliferation and migration of two highly aggressive CRC cell lines and reduced liver and lung metastasis in vivo. Mechanistically, we revealed that circHERC4 inactivated the tumor suppressor, miR-556-5p, leading to the activation of CTBP2/E-cadherin pathway which promotes tumor metastasis in CRC. CONCLUSIONS: CircHERC4 exerts critical roles in promoting tumor aggressiveness through miR-556-5p/CTBP2/E-cadherin pathway and is a prognostic biomarker of the disease, suggesting that circHERC4 may serve as an exploitable therapeutic target for patients with CRC.

Neutrophil extracellular traps in gastrointestinal cancer.

Gastrointestinal (GI) cancer is a high-risk malignancy and is characterized by high mortality and morbidity worldwide. Neutrophil extracellular traps (NETs), a weblike structure consisting of chromatin DNA with interspersed cytoplasmic and granule proteins, are extruded by activated neutrophils to entrap and kill bacteria and fungi. However, accumulating evidence shows that NETs are related to the progression and metastasis of cancer. In clinical studies, NETs infiltrate primary GI cancer tissues and are even more abundant in metastatic lesions. The quantity of NETs in peripheral blood is revealed to be associated with ascending clinical tumour stages, indicating the role of NETs as a prognostic markers in GI cancer. Moreover, several inhibitors of NETs or NET-related proteins have been discovered and used to exert anti-tumour effects in vitro or in vivo, suggesting that NETs can be regarded as targets in the treatment of GI cancer. In this review, we will focus on the role of NETs in gastric cancer and colorectal cancer, generalizing their effects on tumour-related thrombosis, invasion and metastasis. Recent reports are also listed to show the latest evidences of how NETs affect GI cancer. Additionally, notwithstanding the scarcity of systematic studies elucidating the underlying mechanisms of the interaction between NETs and cancer cells, we highlight the potential importance of NETs as biomarkers and anti-tumour therapeutic targets.

The mechanisms of colorectal cancer cell mesenchymal-epithelial transition induced by hepatocyte exosome-derived miR-203a-3p.

BACKGROUND: Liver metastasis is the most common cause of death in patients with colorectal cancer (CRC). Phosphatase of regenerating liver-3 induces CRC metastasis by epithelial-to-mesenchymal transition, which promotes CRC cell liver metastasis. Mesenchymal-to-epithelial transition (MET), the opposite of epithelial-to-mesenchymal transition, has been proposed as a mechanism for the establishment of metastatic neoplasms. However, the molecular mechanism of MET remains unclear. METHODS: Using Immunohistochemistry, western blotting, invasion assays, real-time quantitative PCR, chromatin immunoprecipitation, luciferase reporter assays, human miRNA arrays, and xenograft mouse model, we determined the role of hepatocyte exosome-derived miR-203a-3p in CRC MET. RESULTS: In our study, we found that miR-203a-3p derived from hepatocyte exosomes increased colorectal cancer cells E-cadherin expression, inhibited Src expression, and reduced activity. In this way miR-203a-3p induced the decreased invasion rate of CRC cells. COCLUSION: MiR-203a-3p derived from hepatocyte exosomes plays an important role of CRC cells to colonize in liver.

Mesenchymal and Phosphatase of Regenerating Liver-3 Status in Circulating Tumor Cells May Serve as a Crucial Prognostic Marker for Assessing Relapse or Metastasis in Postoperative Patients With Colorectal Cancer.

INTRODUCTION: Circulating tumor cells (CTCs) and phosphatase of regenerating liver-3 (PRL-3) have been considered to be significant prognostic indicators in metastatic colorectal cancer (CRC). This study discusses the prognostic significance of mesenchymal CTCs with PRL-3 (M+ PRL-3+ CTCs) in postoperative patients with CRC. METHODS: We detected CTC subtypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) and PRL-3 in CTCs from the peripheral blood samples of 156 patients. Receiver operating characteristic curve analysis, Kaplan-Meier analysis, and Cox proportional hazards regression analysis were performed to identify the prognostic value of mesenchymal CTCs with PRL-3+. Immunohistochemistry was used to detect the expression of PRL-3 in tumor tissues from some of the patients to explore the connection between CTCs and tissues. RESULTS: All CTCs were positive in all samples, both mesenchymal CTCs and PRL-3-positive cells. The count of mesenchymal and PRL-3+ CTCs was significantly associated with recurrence, and the optimal cutoff value was 2 (area under the curve = 0.690, P < 0.001). In addition, these patients had a significantly shorter median disease-free survival than those who did not fulfill the criteria (8.5 vs 24 months, P < 0.001) according to multivariable and multinomial logistic regression. Immunohistochemistry was applied to explore the associations between PRL-3 expression and significant prognostic risk factors, including recurrence (R = 0.566; P < 0.001), and M+ PRL-3+ status in CTCs (R = 0.452; P = 0.001). DISCUSSION: The status of M+ PRL-3+ in CTCs may serve as a crucial prognostic marker for assessing clinical outcomes in CRC.

Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.