siRNAs Targeting Mouse-Specific lncRNA AA388235 Induce Human Tumor Cell Pyroptosis/Apoptosis.

Species-specific lncRNAs significantly determine species-specific functions through various ways, such as epigenetic regulation. However, there has been no study focusing on the role of species-specific lncRNAs in other species yet. Here, we found that siRNAs targeting mouse-specific lncRNA AA388235 could significantly induce death of human tumor cells, although it has no effect on mouse tumor cells and normal human cells. The mechanism studies showed that these siRNAs could activate the response of human tumor cells to exogenous nucleic acids, induce pyroptosis and apoptosis in the presence of GSDME, but induce apoptosis in the absence of GSDME. They also significantly inhibited the growth of human tumor cells in vivo. 17 siRNAs were designed for seven more mouse-specific lncRNAs selected randomly, among which 12 siRNAs targeting five lncRNAs induced death in human tumor cell. Our study not only demonstrates that the siRNAs designed for knocking down mouse-specific lncRNA AA388235 can be potential tumor therapeutic drugs, but also suggests that non-human species-specific lncRNAs are a huge potential library that can be used to design siRNAs for tumor treatment. Large-scale screening based on this is promising.

Contraction-dependent TGF-ß1 activation is required for thrombin-induced remodeling in human airway smooth muscle cells.

AIMS: Thrombin is a serine proteinase that is not only involved in coagulation cascade, but also mediates a number of biological responses relevant to tissues repair, and induces bronchoconstriction. TGF-ß plays a pivotal role in airway remodeling due to its effects on airway smooth muscle proliferation and extracellular matrix (ECM) deposition. Recently, bronchoconstriction itself is found to constitute a form of strain and is highly relevant to asthmatic airway remodeling. However, the underlying mechanisms remain unknown. Here, we investigated the role of contraction- dependent TGF-ß activation in thrombin-induced remodeling in human airway smooth muscle (HASM) cells. MATERIALS AND METHODS: Primary HASM cells were treated with or without thrombin in the absence or presence of anti-TGF-ß antibody, cytochalasin D and formoterol. CFSE labeling index or CCK-8 assay were performed to test cell proliferation. RT-PCR and Western blotting were used to examined ECM mRNA level and collagen Iα1, α-actin protein expression, respectively. Immunofluorescence was also used to confirm contraction induced by thrombin in HASM cells. KEY FINDING: Thrombin stimulation enhanced HASM cells proliferation and activated TGF-ß signaling. Thrombin induced ECM mRNA and collagen Iα1 protein expression, and these effects are mediated by TGF-ß. Abrogation of TGF-ß activation by contraction inhibitors cytochalasin D and formoterol prevents the thrombin-induced effects. SIGNIFICANCE: These findings suggest that contraction-dependent TGF-ß activation could be a mechanism by which thrombin leads to the development of asthmatic airway remodeling. Blocking physical forces with bronchodilator would be an intriguing way in reducing airway remodeling in asthma.

Adenoviral delivery of recombinant soluble human tumor necrosis factor receptor 1 partially normalized mouse model of asthma.

BACKGROUND: Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that has been implicated in the airway pathology of asthma and result in resistance to hormone therapy. Tumor necrosis factor α inhibitors have become a major research focus in the treatment of asthma. METHODS: Recombinant adenovirus (Ad-sTNFR1-IgGFc) expressing a fusion protein (sTNFR1-IgGFc), which was consisted of the soluble extracellular region of TNF receptor 1 and Fc fragment of IgG (sTNFR1-IgGFc), was used to transduce primary human airway smooth muscle cells (HASMCs). Enzyme-linked immunosorbent assay, flow cytometry, and immunocytochemistry confirmed the expression of sTNFR1-IgGFc. MTT was used to test the effect of sTNFR1-IgGFc to antagonism TNF-α-induced proliferates of HASMCs. To investigate the in vivo effectiveness of sTNFR1-IgGFc, mouse model of asthma was established. Ad-sTNFR1-IgGFc was delivered to the lung via nasal spray. Expression of sTNFR1-IgGFc in the tissue was confirmed by in situ hybridization and immunohistochemistry. The 2 major cell types that are involved in the inflamed asthmatic airway, neutrophils and eosinophils, in bronchoalveolar lavage fluid were observed. RESULTS: The sTNFR1-IgGFc isolated from transduced HASMC culture supernatant was able to antagonize HASMC proliferation stimulated by TNF-α. Asthma-induced pathologies and alterations in the cell composition in bronchoalveolar lavage fluid were reduced in mice subjected to Ad-sTNFR1-IgGFc therapy. CONCLUSIONS: The soluble extracellular region of TNF receptor 1 and Fc fragment of IgG was able to functionally antagonize TNF-α in vitro and showed promise as a therapeutic agent for the localized treatment of severe refractory asthma.

[Activation of Rho-kinase pathway is involved in angiotensin II-induced contraction of human airway smooth muscle cells].

OBJECTIVE: To investigate of the regulatory effect of Rho-kinase pathway activation on angiotensin II (Ang II)-induced contraction of human airway smooth muscle cells (HASMCs) in vitro. METHODS: Cultured primary HASMCs were divided into control group, AngII group, AngII + irbesartan group and AngII + Y-27632 group with corresponding treatment. AngII-induced contraction of HASMCs was evaluated using collagen gel lattices and observed morphologically using immunofluorescence assay. Western Blotting was significantly performed to examine the protein expression of Rho-kinase signal pathway. RESULTS: AngII-induced HASMC contraction was inhibited by treatments with irbesartan and Y-27632 as shown by gel contraction assay (P<0.001). Y-27632 treatment produced a stronger inhibitory effect than irbesartan on the expression of phosphorylated moesin, a substrate of Rho kinase (P<0.05). CONCLUSION: AngII induces the contraction of HASMCs partially as a result of activation of Rho-kinase pathway.

[The comparison between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice on lung tissue pathological change and vasopermeability in acute lung injury].

OBJECTIVE: To compare the change of lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice in acute lung injury. METHODS: The mice with vascular endothelial cell-specific expression of cre recombinase were crossed with cdc42(flox/flox) mice. The cdc42(flox/+)Cre(+/-) F1 offspring mice were crossed back with cdc42(flox/flox) mice, resulting in the F2 generation mice with three genotypes, namely cdc42(flox/+)Cre(+/-), cdc42(flox/flox)Cre(-/-) and cdc42(flox/+)Cre(+/-). The heterozygous mice with cdc42(flox/+)Cre(+/-) genotype were selected as the model mice, with the other two genotype groups as the control. After intratracheal instillation of 2 mg/kg LPS to induce acute lung injury, the mice were sacrificed to examine the lung pathologies, lung wet/dry ratio and lung microvascular permeability. RESULTS: The heterozygous mice with cdc42 gene knockout (cdc42(flox/+)Cre(+/-)) showed no significant differences from the two control groups in the lung pathological score, lung wet/dry ratio or the lung microvascular permeability coefficient. CONCLUSION: There were no significant difference on lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice.

[Application of flexirigid thoracoscopy in the diagnosis of pleural disease with unknown etiology].

OBJECTIVE: To investigate the diagnostic accuracy of flexirigid thoracoscopy for pleural diseases and the patients' compliance. METHODS: Forty-seven patients with pleural effusion and thickening of unknown etiology underwent examinations with flexirigid thoracoscopy with subsequent pathological examination, and the diagnostic accuracy and the patients' compliance were observed. RESULTS: Thoracoscopy identified lesions in the pleural and/or diaphragm in 42 patients and no lesions in 5 patients. Malignancy was confirmed in 21 (44.7%), tuberculosis in 17 (36.2%), idiopathic hypereosinophilic syndrome in 1 (2.1%), nocardiasis in 1 (2.1%), constrictive pericarditis in 1 (2.1%), chronic empyema in 2 (4.3%), splenic artery embolization in 1 (2.1%), and negative result in 3 (6.4%) of the cases. The diagnostic accuracy rate of flexirigid thoracoscopy reached 93.6%, and no serious complications in relation to the examination was found. CONCLUSION: Flexirigid thoracoscopy is efficient and relatively safe for diagnosis of pleural diseases with or without hydrothorax.

The generation of the endothelial specific cdc42-deficient mice and the effect of cdc42 deletion on the angiogenesis and embryonic development.

BACKGROUND: High microvascular permeability plays an essential role in pathological process of multiple diseases such as septic shock, acute lung injury and acute respiratory distress syndrome, and burns. Inhibiting hyperpermeability is significant for controlling these conditions. Cdc42, as a main member of the small Rho GTPase family, plays a critical role in controlling and regulating the endothelial junctional permeability. We aimed to generate and identify endothelial specific cdc42-deficient mice by the Cre/loxp recombination approach, for examination in an animal model of the contribution of the cdc42 gene in the microvascular barrier function. METHODS: We crossed cdc42(Flox/Flox) mice with mice expressing endothelial cell-specific Cre recombinase, and the offspring with the genotype cdc42(Flox/+)Tie2Cre(+/-) were back-crossed with the cdc42(Flox/Flox) mice. The cdc42(Flox/Flox)Tie2Cre(+/-) mice in the F2 generation were the target mice. If the cdc42 deficient mice did not survive, we would observe the cdc42 deficient mice embryos, and compare them with wild-type mice embryos. RESULTS: Cdc42(flox/+)Cre(+/-) mice were mated with the cdc42(Flox/Flox) mice and among the living offspring there were no cdc42(Flox/Flox)Cre(+/-) target mice. We found the endothelial special cdc42 deficient embryos at the E7.5-E16.5 stage. We observed that cdc42 deficient embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. CONCLUSIONS: Endothelial specific knockout of cdc42 caused embryonic lethality and the mice did not survive to birth. The target embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. These results demonstrated that the cdc42 plays an important role in development of embryos and in development of microvessels as well as microvascular permeability.

[Perindopril and losartan attenuate bleomycin A5-induced pulmonary fibrosis in rats].

OBJECTIVE: To evaluate the effects of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor (AT-1 receptor) blocker on the progression of rat pulmonary fibrosis induced by bleomycin A5. METHODS: Twenty-four male Wistar rats were randomized into pulmonary fibrosis model, perindopril treatment, losartan treatment and control groups. In the former 3 groups, pulmonary fibrosis was induced via intratracheal injection of bleomycin A5 (5 mg/kg), after which the rats in the perindopril and losartan groups received intragastric administration of the corresponding agents at the daily dose of 2 mg/kg and 10 m/kg, respectively. The rats in the control group had intratracheal injection of normal saline only. In the 4th week, the histological changes of the lung tissues were examined microscopically with Masson staining. Hydroxyproline content in the lungs was measured, and the protein expressions of AT-1 receptor, TGF-beta1 and IkappaBalpha were examined using Western blotting. DNA binding activity of NF-kappaB was analyzed with electrophoretic gel mobility shift assay (EMSA), and zymography was used to assess the activity of matrix metalloproteinase-2 and 9 (MMP-2, 9). RESULTS: Both perindopril and losartan treatment significantly reduced the pulmonary fibrosis score, content of hydroxyproline, protein expression of TGF-beta1, DNA binding activity of NF-kappaB and MMP-2, 9 activity, and increased cytoplasmic protein expression of IkappaBalpha. Perindopril treatment lowered the protein level of AT-1 receptor. CONCLUSION: Perindopril and losartan may inhibit bleomycin A5-induced pulmonary fibrosis in rats by reducing the protein expression of TGF-beta1 and suppressing the DNA binding activity of NF-kappaB and MMP-2, 9 activity.