Metformin regulates autophagy via LGMN to inhibit choriocarcinoma.

Choriocarcinoma has the problem of chemotherapy insensitivity and recurrence. Metformin may be a promising candidate to restrict choriocarcinoma progress because of its indirect and direct beneficial role on inhabitations of cancer cells without severe adverse side effects. In this study, metformin pressed the proliferation and invasion of choriocarcinoma JAR cells in vitro and the growth of the JAR subcutaneous xenografts in vivo. The high throughput sequencing and bioinformatics technology identified the low expression of legumain (LGMN) in lysosomal pathway caused by metformin, which was upregulated in human choriocarcinoma tissues compared with the early pregnancy tissues. As elevating metformin concentration and treatment time, the mRNA and protein expression of LGMN both depressed in two choriocarcinoma cell lines (JAR and JEG-3). LGMN was involved in metformin-mediated inhibition of cell proliferation and invasion. Furthermore, metformin induced autophagy via inhibiting LGMN through AKT/mTOR/LC3II signaling pathway of choriocarcinoma. Autophagy inhibitor could depress metformin-induced autophagy and improve cell proliferation and invasion ability dropped by metformin, while autophagy inducer could partially reverse the change of cell proliferation and invasion modulated by combination of metformin and LGMN overexpression. These results indicated that metformin inhibited cell proliferation and invasion ability by inducing autophagy in a LGMN-dependent manner so as to play a role in the treatment of choriocarcinoma.

Downregulation of miR-21 is Involved in the Pathogenesis of Infection-Induced Preterm Birth by Targeting NF-κB.

Infection-induced preterm birth (PTB) is contributing to the main factors of increased maternal and fetal morbidity and mortality. Infections and inflammation are often accompanied by histologic chorioamnionitis. Recently, several studies have uncovered that miR-21 and NF-κB are associated with pathological processes of pregnant women. However, the role of miR-21 in infection-induced PTB remains unclear. This study aimed to determine whether miR-21 is involved in the pathogenesis of infection-induced PTB by regulating NF-κB. In this study, we found that the expression of miR-21 was significantly decreased in placental tissues of lipopolysaccharides (LPS)-induced infectious PTB mice model, accompanied by the increase of NF-κB, IL-6, and TNF-α (P < 0.05). Luciferase reporter gene assays showed that NF-κB was a validated target of miR-21. Furthermore, cell transfection experiments showed that miR-21 overexpression significantly decreased NF-κB mRNA expression compared with the miR-control group and blank group. Conversely, miR-21 inhibitor can enhance NF-κB mRNA expression. After the treatment of miR-21 mimics, miR-21 expression was obviously increased compared with the LPS group, accompanied by the decrease of NF-κB, TNF-α, and IL-6 mRNA expression (P < 0.05). What's more, miR-21 expression was negatively correlated with NF-κB (r=-0.87, P < 0.01). Overall, the study findings indicate that miR-21 may contribute to the pathogenesis of infection-induced PTB by upregulating the target NF-κB and that miR-21 may be a new potential therapeutic target for infection-induced PTB.

In situ inflammatory-regulated drug-loaded hydrogels for promoting pelvic floor repair.

Biomedical hydrogel has been widely used as regenerative biomaterials, however, an immune inflammatory response of hydrogel constantly crops up in body due to crosslinking agent, external stimulus or small molecule residues. Here we present a strategy to treat pelvic organ prolapse (POP) by combining both anti-inflammatory and promote tissue regeneration, using drug-loaded hydrogel to reconstruct the pelvic floor and minimize multiple inflammations. Photo-crosslinked gelatin hydrogel (GelMA) loaded with Puerarin (Pue) regulate inflammation by inhibiting the aggregation of neutrophils and eosinophils, simultaneously intervene the matrix regenerating/remodeling via TGF-ß/MMPs pathway to repair the fascia of pelvic floor in rabbit models (POP model). The assessment of inflammatory cytokines expression (IL-3, IL-6, TNF-α, TGF-ß1) in human uterus fibroblasts (HUVs), and extracellular matrix (ECM) related factors (COL-1, COL-3, MMP2, MMP9) was performed in rabbit. Immune microenvironment was analyzed by immunohistochemistry in rabbit samples. Pue-loaded GelMA (Pue@GelMA) down regulate inflammatory cytokines (IL-3 and IL-6) and matrix metalloproteinase 2/9 (MMP 2/9), and up regulate 1/3 type collagen (COL-1/3) in vitro. In this study, Pue@GelMA was able to regulate immune microenvironment through restricting the aggregation of neutrophils and eosinophils and remodel the distribution of ECM collagen in vivo. In the POP model, Pue@GelMA can effectively inhibits the inflammatory response caused by material implanted and promote fascia regenerate. This Hydrogel drug loading system was considered as an safe and effective method to treat POP without persistent complications, and it can also be applied to other prolapse diseases (e.g., intestinal hernia) or complex diseases treatment.

Klotho Protein Reduced the Expression of Matrix Metalloproteinase-1 (MMP-1) and Matrix Metalloproteinase-3 (MMP-3) in Fibroblasts from Patients with Pelvic Organ Prolapse (POP) by Down-Regulating the Phosphorylation of ERK1/2.

BACKGROUND Pelvic organ prolapse (POP) is due to age-related atrophy and the weakening of the tissues of the pelvic floor, with degradation of collagen and extracellular matrix (ECM) by metalloproteinases (MMPs). This study aimed to investigates the role of the age-related enzyme klotho, encoded by the KL gene, in cultured fibroblasts obtained from patients with POP and the levels of reactive oxygen species (ROS), interleukin-6 (IL-6), and MMPs. MATERIAL AND METHODS Pelvic floor fibroblasts were obtained from connective tissue from three patients with POP and three normal subjects. Cell proliferation and ROS production were measured using a cell counting kit-8 (CCK-8) assay and flow cytometry. Levels of interleukin-6 (IL-6), klotho, metalloproteinase-1 (MMP-1), MMP-3, extracellular signal-regulated kinases 1/2 (ERK1/2), and p-ERK1/2 were measured by enzyme-linked immunoassay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. RESULTS In cultured pelvic floor fibroblasts from patients with POP, the expression of klotho protein and klotho mRNA were significantly down-regulated in fibroblasts from patients with POP compared with normal fibroblasts. Klotho supplementation in cultured fibroblasts for patients with POP included increased cell growth, reduced expression of ROS reduction, and reduced the secretion of IL-6. Using qRT-PCR and Western blot, klotho supplementation of fibroblasts from patients with POP increased cell growth and reduced the levels of IL-6 and ROS in a dose-dependent way. CONCLUSIONS Klotho protein reduced the expression of MMP-1 and MMP-3 in fibroblasts from patients with POP by down-regulating the phosphorylation of ERK1/2.