No abstract

The present study aimed to investigate the roles of the microRNA-29a/DNA methyltransferase 3B/suppressor of cytokine signalling 1 (miR-29a/DNMT3B/SOCS1) axis in the invasion and the migration of osteosarcoma (OS). The expression levels of miR-29a, DNMT3B and SOCS1 were determined in tissue samples and OS cell lines by reverse transcription-quantitative polymerase chain reaction (PCR). Apoptosis was measured using flow cytometry analysis. Transwell and wound healing assays were conducted to measure the invasion and migration abilities of OS cells, respectively. A dual-luciferase reporter assay was also conducted to determine the interaction between DNMT3B and miR-29a, while methylation-specific PCR was used to detect the methylation of SOCS1. Western blotting was performed to determine the protein levels of DNMT3B and SOCS1, as well as the levels of proteins associated with epithelial-mesenchymal transition (EMT), apoptosis and the nuclear factor (NF)-κB signalling pathway. The results demonstrated that miR-29a and SOCS1 were downregulated, and DNMT3B was upregulated in both OS tissues and cell lines. The expression of miR-29a and SOCS1 was found to be associated with advanced clinical stage and distant metastasis. In addition, the dual-luciferase reporter assay revealed that DNMT3B was a direct target of miR-29a. Overexpression using miR-29a mimics decreased DNMT3B expression and the methylation level of SOCS1, which resulted in the upregulation of SOCS1 in U2OS and MG-63 cells, while miR-29a inhibition led to the opposite results. Transfection with miR-29a mimics also promoted the apoptosis, and inhibited the invasion, migration and EMT process of OS cells, while it markedly reduced the nuclear translocation of p65 and IκB-α degradation. Treatment with 5-aza-2′-deoxycytidine worked together with miR-29a mimics to synergistically enhance the aforementioned effects. By contrast, the effects induced by miR-29a were partly reversed upon co-transfection with SOCS1 siRNA. In conclusion, miR-29a promoted the apoptosis, and inhibited the invasion, migration and EMT process of OS cells via inhibition of the SOCS1/NF-κB signalling pathway by directly targeting DNMT3B.

ProblemChlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact.Method of studyWe used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot.ResultsqPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect.ConclusionOur data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.