Highly sensitive detection of microRNA-21 by nitrogen-doped carbon dots-based ratio fluorescent probe via nuclease-assisted rolling circle amplification strategy.

Highly sensitive and selective detection of microRNA-21 (miRNA-21) in biological samples is critical for the disease diagnosis and cancer treatment. In this study, a nitrogen-doped carbon dots (N-CDs)-based ratio fluorescence sensing strategy was constructed for miRNA-21 detection with high sensitivity and excellent specificity. Bright-blue N-CDs (λex/λem = 378 nm/460 nm) were synthesized by facile one-step microwave-assisted pyrolysis method by using uric acid as the single precursor, and the absolute fluorescence quantum yield and fluorescence lifetime of N-CDs were 35.8% and 5.54 ns separately. The padlock probe hybridized with miRNA-21 firstly and then was cyclized by T4 RNA ligase 2 to form a circular template. At the present of dNTPs and phi29 DNA polymerase, the oligonucleotide sequence in miRNA-21 was prolonged to hybridize with the surplus oligonucleotide sequences in circular template, generating long and reduplicated oligonucleotide sequences containing abundant guanine nucleotides. Separate G-quadruplex sequences were generated after the addition of Nt.BbvCI nicking endonuclease, and then hemin bound with G-quadruplex sequence to construct the G-quadruplex DNAzyme. Such G-quadruplex DNAzyme catalyzed the redox reaction of o-phenylenediamine (OPD) with H2O2, finally producing the yellowish-brown 2,3-diaminophenazine (DAP) (λem = 562 nm). Due to the inner filter effect between N-CDs and DAP, the ratio fluorescence signal of DAP with N-CDs was utilized for sensitive detection of miRNA-21 with detection limit of 0.87 pM. Such approach has practical feasibility and excellent specificity for miRNA-21 analysis during highly homological miRNA family in HeLa cell lysates and human serum samples.

Circ_0000228 Promotes Cervical Cancer Progression via Regulating miR-337-3p /TGFBR1 Axis.

Objective: This study aims to investigate the biological function of circular RNA (circRNA) circ_0000228 in the cervical cancer (CC). Materials and Methods: In this experimental study, the GSE113696 dataset was downloaded from the Gene Expression Omnibus (GEO). GEO2R was employed to obtain differentially expressed circRNA between CC tissues and matched paracancerous tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were employed to detect circ_0000228, microRNA-337-3p (miR-337-3p ) and transforming growth factor, beta receptor I (TGFBR1) expression levels in the CC tissues and cells. Following gain-of-function and loss-of-function models establishment, CCK-8 and BrdU tests were conducted to examine cell proliferation. Transwell experiment was executed to examine CC cells migration and invasion. A lung metastasis model was utilized to determine the ability of circ_0000228 on the lung metastasis. Bioinformatics analysis, dual-luciferase reporter experiment and RNA immunoprecipitation (RIP) assay were applied to verify the targeting relationship among miR-337-3p , circ_0000228, and TGFBR1. Results: Circ_0000228 expression in the CC tissues and cells was up-modulated. Circ_0000228 overexpression markedly enhanced cell proliferation, migration, and invasion, while knocking down circ_0000228 remarkably repressed cell proliferation, migration, and invasion. MiR-337-3p could be adsorbed by circ_0000228. TGFBR1 was identified as a target gene of miR-337-3p that indirectly and positively modulated bycirc_0000228 in the CC cells. Conclusion: Circ_0000228 up-modulates TGFBR1 by targeting miR-337-3p to enhance CC cell proliferation, migration and invasion. Also, Circ_0000228 is a promising therapeutic target for the CC.

[Mechanism of carbon monoxide affecting the expression of cellular adhesion molecule under stimulation of inflammatory cytokines to human gingival fibroblasts].

OBJECTIVE: To investigate the mechanism by which carbon monoxide inhibits the expression of adhesion molecules on human gingival fibroblasts (HGF) stimulated with inflammatory cytokines. METHODS: HGF were cultured in vitro, and stimulated with 50 ng x mL tumor necrosis factor-alpha (TNF-alpha) and 10 ng x mL(-1) interleukin-1beta (IL-1beta) concurrently in the presence or absence of carbon monoxide releasing molecule-3 (CORM-3) at 500 micromol x L-1. Expression of phosphorylated extracellular regulated protein kinase (ERK), phosphorylated c-Jun N-terminal kinase (NK) and phosphorylated p38 in mitogen-activated protein kinase(MAPK) pathway was studied by Western blot at 10 min and 20 min, respectively. Nuclear expression of nuclear factor-kappaB (NF-kappaB) was checked by Western blot after 4 h stimulation. In some experiments, cells were prestimulated by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) for 8 h before cytokine stimulation and the expression of intercellular adhesion molecule-1 (ICAM-1) was checked by Western blot after 24 h. RESULTS: CORM-3 significantly inhibited the phosphorylation of MAPK p38 after 10 min stimulation with cytokines, but had no signifi-cant effect on the phosphorylation of ERK and JNK. CORM-3 significantly inhibited the nuclear expression of NF-KB-p65 on HGF after 4 h stimulation by inflammatory cytokines. The inhibitory effect of CORM-3 on the expression of ICAM-1 was not influenced by guanylate cyclase inhibitor ODQ. CONCLUSION: The inhibitory effect of carbon monoxide on the expression of adhesion molecules might be exerted by its inhibitory effect on the NF-kappaB activity and MAPK p38 phosphorylation.

[Expression of high mobility group box 1 in gingival tissues of chronic periodontitis].

OBJECTIVE: To investigate the expression of high mobility group box 1 (HMGB1) in gingival tissues of chronic periodontitis. METHODS: Human peripheral blood mononuclear cells(PBMC) were stimulated with 1 microg x mL(-1) lipopolysaccharide (LPS) for 24 h or 48 h. Expression and release of HMGB1 were checked by immunofluorescence and enzyme-linked immunosorbent assay (ELISA), respectively. PBMC were stimulated with 100 ng x mL(-1) HMGB1 or 50 ng x mL(-1) tumor necrosis factor-alpha (TNF-alpha), the expressions of TNF-alpha and HMGB1 in the supernatant were studied by ELISA. Gingival tissues and gingival crevicular fluids (GCF) were collected from patients and healthy people. Expression of HMGB1 in gingival tissues and GCF was studied using immunofluorescence and ELISA, respectively. RESULTS: HMGB1 was translocated from nucleus to cytosol in PBMC after LPS stimulation for 24 h. The content of HMGB1 in the supernatant from stimulated cells was significantly higher than that from unstimulated cells after 48 h (P < 0.01). HMGB1 was released by PBMC in response to TNF-alpha stimulation, it also stimulated PBMC to release TNF-alpha (P < 0.01). Translocation of HMGB1 from nucleus to cytosol was also found in infiltrated cells in gingival tissues from patients, and HMGB1 in GCF from patients was significantly higher than that from healthy people P < 0.01). CONCLUSION: The results suggest that HMGB1 may play an important role in the pathological progress of chronic periodontitis.

[Influence of carbon monoxide on the expression of adhesion molecules stimulated with tumor necrosis factor-alpha and interleukin-1beta on human gingival fibroblasts].

OBJECTIVE: To investigate the influence of carbon monoxide on the expression of adhesion molecules stimulated by inflammatory cytokines on human gingival fibroblasts. METHODS: Human gingival fibroblasts were stimulated with 50 ng x mL(-1) tumor necrosis factor (TNF)-alpha and 10 ng x mL(-1) interleukin (IL)-1beta concurrently in the presence or absence of 500 micromol x L(-1) carbon monoxide releasing molecule (CORM). Expression of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 at protein and mRNA level was examined by Western blot and reverse transcription polymerase chain reaction (RT-PCR), respectively. Activity of transcription factor NF-kappaB was evaluated by reporter gene assay. RESULTS: Expression of ICAM-1 and VCAM-1 on human gingival fibroblasts increased dramatically after concurrent stimulation of TNF-alpha and IL-1beta, while CORM inhibited the upregulation of ICAM-1 and VCAM-1. CORM decreased the activity of NF-KB stimulated by TNF-alpha and IL-1beta. CONCLUSION: Carbon monoxide could be a promising way in treating of periodontitis.