BACKGROUND: Lung cancer is a major threat to human health. The molecular mechanisms related to the occurrence and development of lung cancer are complex and poorly known. Exploring molecular markers related to the development of lung cancer is helpful to improve the effect of early diagnosis and treatment. Long non-coding RNA (lncRNA) THAP7-AS1 is known to be highly expressed in gastric cancer, but has been less studied in other cancers. The aim of the study is to explore the role and mechanism of methyltransferase-like 3 (METTL3) mediated up-regulation of N6-methyladenosine (m6A) modified lncRNA THAP7-AS1 expression in promoting the development of lung cancer. METHODS: Samples of 120 lung cancer and corresponding paracancerous tissues were collected. LncRNA microarrays were used to analyze differentially expressed lncRNAs. THAP7-AS1 levels were detected in lung cancer, adjacent normal tissues and lung cancer cell lines by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The diagnostic value of THAP7-AS1 in lung cancer and the relationship between THAP7-AS1 expression and survival rate and clinicopathological parameters were analyzed. Bioinformatics analysis, methylated RNA immunoprecipitation (meRIP), RNA pull-down and RNA-immunoprecipitation (RIP) assay were used to investigate the molecular regulation mechanism of THAP7-AS1. Cell proliferation, migration, invasion and tumorigenesis of SPC-A-1 and NCI-H1299 cells were determined by MTS, colony-formation, scratch, Transwell and xenotransplantation in vivo, respectively. Expression levels of phosphoinositide 3-kinase/protein kenase B (PI3K/AKT) signal pathway related protein were detected by Western blot. RESULTS: Expression levels of THAP7-AS1 were higher in lung cancer tissues and cell lines (P<0.05). THAP7-AS1 has certain diagnostic value in lung cancer [area under the curve (AUC)=0.737], and its expression associated with overall survival rate, tumor size, tumor-node-metastasis (TNM) stage and lymph node metastasis (P<0.05). METTL3-mediated m6A modification enhanced THAP7-AS1 expression. The cell proliferation, migration, invasion and the volume and mass of transplanted tumor were all higher in the THAP7-AS1 group compared with the NC group and sh-NC group of SPC-A-1 and NCI-H1299 cells, while the cell proliferation, migration and invasion were lower in the sh-THAP7-AS1 group (P<0.05). THAP7-AS1 binds specifically to Cullin 4B (CUL4B). The cell proliferation, migration, invasion, and expression levels of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), phosphoinositide-3 kinase, catalytic subunit delta (PIK3CD), phospho-phosphatidylinositol 3-kinase (p-PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) were higher in the THAP7-AS1 group compared with the Vector group of SPC-A-1 and NCI-H1299 cells (P<0.05). CONCLUSIONS LncRNA THAP7-AS1 is stably expressed through m6A modification mediated by METTL3, and combines with CUL4B to activate PI3K/AKT signal pathway, which promotes the occurrence and development of lung cancer.
Humans ; Lung Neoplasms/pathology* ; RNA, Long Noncoding/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Up-Regulation ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Methyltransferases/metabolism* ; Cullin Proteins/genetics*

OBJECTIVE: To investigate the effect and possible mechanism of hydroxysafflor yellow A (HSYA) on human immortalized keratinocyte cell proliferation and migration. METHODS: HaCaT cells were treated with HSYA. Cell proliferation was detected by the cell counting kit-8 assay, and cell migration was measured using wound healing assay and Transwell migration assay. The mRNA and protein expression levels of heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), mammalian target of rapamycin (mTOR), and hypoxia-inducible factor-1α (HIF-1α) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA. The expression of circ_0084443 was detected by qRT-PCR. RESULTS: HSYA (800 µmol/L) significantly promoted HaCaT cell proliferation and migration (P<0.05 or P<0.01). It also increased the mRNA and protein expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and increased the phosphorylation levels of PI3K and AKT (P<0.05 or P<0.01). Furthermore, HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/mTOR signaling pathways (P<0.01). Circ_0084443 attenuated the mRNA expression levels of HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α (P<0.05). HSYA inhibited the circ_0084443 expression, further antagonized the inhibition of circ_0084443 on HBEGF, EGFR, PI3K, AKT, mTOR and HIF-1α, and promoted the proliferation of circ_0084443-overexpressing HaCaT cells (P<0.05 or P<0.01). However, HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration (P>0.05). CONCLUSION HSYA played an accelerative role in HaCaT cell proliferation and migration, which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways, and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositol 3-Kinases/metabolism* ; ErbB Receptors/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; RNA, Messenger/genetics* ; Cell Movement ; Cell Line, Tumor ; Chalcone/analogs & derivatives* ; Quinones

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 β in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 β (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.
Mice ; Male ; Animals ; Proto-Oncogene Proteins c-akt/metabolism* ; Lipopolysaccharides ; Phosphatidylinositol 3-Kinases/metabolism* ; Interleukin-1beta/metabolism* ; Vascular Endothelial Growth Factor A/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Claudin-5/metabolism* ; Acute Lung Injury/chemically induced* ; Lung/pathology* ; Interleukin-6/metabolism* ; Drugs, Chinese Herbal

OBJECTIVE: To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. METHODS: Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR. RESULTS: The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01). CONCLUSIONS EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.
Rats ; Male ; Animals ; Rats, Sprague-Dawley ; Electroacupuncture ; Phosphatidylinositol 3-Kinase/metabolism* ; Facial Nerve Injuries/therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Beclin-1 ; Glial Cell Line-Derived Neurotrophic Factor ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Autophagy ; Mammals/metabolism*

This paper aimed to investigate the role and potential mechanism of p53 on primordial follicle activation. Firstly, the p53 mRNA expression in the ovary of neonatal mice at 3, 5, 7 and 9 days post-partum (dpp) and the subcellular localization of p53 were detected to confirm the expression pattern of p53. Secondly, 2 dpp and 3 dpp ovaries were cultured with p53 inhibitor Pifithrin-μ (PFT-μ, 5 μmol/L) or equal volume of dimethyl sulfoxide for 3 days. The function of p53 in primordial follicle activation was determined by hematoxylin staining and whole ovary follicle counting. The proliferation of cell was detected by immunohistochemistry. The relative mRNA levels and protein levels of the key molecules involved in the classical pathways associated with the growing follicles were examined by immunofluorescence staining, Western blot and real-time PCR, respectively. Finally, rapamycin (RAP) was used to intervene the mTOR signaling pathway, and ovaries were divided into four groups: Control, RAP (1 μmol/L), PFT-μ (5 μmol/L), PFT-μ (5 μmol/L) + RAP (1 μmol/L) groups. The number of follicles in each group was determined by hematoxylin staining and whole ovary follicle counting. The results showed that the expression of p53 mRNA was decreased with the activation of primordial follicles in physiological condition. p53 was expressed in granulosa cells and oocyte cytoplasm of the primordial follicles and growing follicles, and the expression of p53 in the primordial follicles was higher than that in the growing follicles. Inhibition of p53 promoted follicle activation and reduced the primordial follicle reserve. Inhibition of p53 promoted the proliferation of the granulosa cells and oocytes. The mRNA and protein expression levels of key molecules in the PI3K/AKT signaling pathway including AKT, PTEN, and FOXO3a were not significantly changed after PFT-μ treatment, while the expression of RPS6/p-RPS6, the downstream effectors of the mTOR signaling pathway, was upregulated. Inhibition of both p53 and mTOR blocked p53 inhibition-induced primordial follicle activation. Collectively, these findings suggest that p53 may inhibit primordial follicle activation through the mTOR signaling pathway to maintain the primordial follicle reserve.
Female ; Animals ; Mice ; Tumor Suppressor Protein p53/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Hematoxylin ; Signal Transduction/physiology* ; TOR Serine-Threonine Kinases ; Sirolimus ; RNA, Messenger

Pentaxin 3 (PTX3), as a multifunctional glycoprotein, plays an important role in regulating inflammatory response, promoting tissue repair, inducing ectopic calcification and maintaining bone homeostasis. The effect of PTX3 on bone mineral density (BMD) may be affected by many factors. In PTX3 knockout mice and osteoporosis (OP) patients, the deletion of PTX3 will lead to decrease of BMD. In Korean community "Dong-gu study", it was found that plasma PTX3 was negatively correlated with BMD of femoral neck in male elderly patients. In terms of bone related cells, PTX3 plays an important role in maintaining the phenotype and function of osteoblasts (OB) in OP state;for osteoclast (OC), PTX3 in inflammatory state could stimulate nuclear factor κ receptor activator of nuclear factor-κB ligand (RANKL) production and its combination with TNF-stimulated gene 6(TSG-6) could improve activity of osteoclasts and promote bone resorption;for mesenchymal stem cells (MSCs), PTX3 could promote osteogenic differentiation of MSCs through PI3K/Akt signaling pathway. In recent years, the role of PTX3 as a new bone metabolism regulator in OP and fracture healing has been gradually concerned by scholars. In OP patients, PTX3 regulates bone mass mainly by promoting bone regeneration. In the process of fracture healing, PTX3 promotes fracture healing by coordinating bone regeneration and bone resorption to maintain bone homeostasis. In view of the above biological characteristics, PTX3 is expected to become a new target for the diagnosis and treatment of OP and other age-related bone diseases and fracture healing.
Animals ; Male ; Mice ; Bone Resorption/metabolism* ; Cell Differentiation ; Fracture Healing/genetics* ; Osteoblasts ; Osteoclasts ; Osteogenesis ; Osteoporosis/genetics* ; Phosphatidylinositol 3-Kinases/pharmacology*

Objective To investigate the effect of Echinococcus granulosus cyst fluid(EgCF) on the cytoskeletal rearrangement and phagocytosis and the migration of macrophages induced by lipopolysaccharide(LPS). Methods Peritoneal macrophages of C57BL/6 mice were isolated and cultured in vitro, and divided into control group and LPS group and LPS combined with EgCF group. After 48 hours of treatment, filamentous actin (F-actin) changes were observed with rhodamine-labelled phalloidin staining and fluorescence microscopy; Transwell^TM chamber was used to test cell migration ability and flow cytometry to test cell phagocytosis. After 1 hour of treatment, PI3K and AKT, phosphorylated AKT (p-AKT), Rac1, guanosine triphospho-Rac1 (GTP-Rac1), WASP and Arp2 protein expressions were detected with Western blot analysis. Results Compared with the control group, after LPS stimulation, macrophages were deformed significantly; pseudopodia increased; actin cytoskeleton increased and was more distributed in pseudopodia; the ability of migration and phagocytosis were significantly improved, and the expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 proteins significantly increased. EgCF treatment caused cell shrinkage and disappearance of pseudopodia protrusions of LPS-activated cells, and led to the reduced phagocytic and migratory of cells; the protein expression of PI3K, p-AKT, GTP-Rac1, WASP and Arp2 decreased significantly compared with the LPS group. Conclusion LPS induces the migration and enhances phagocytosis of macrophages while EgCF inhibits these effects, which is related to actin cytoskeleton rearrangement.
Mice ; Animals ; Lipopolysaccharides/pharmacology* ; Echinococcus granulosus/metabolism* ; Proto-Oncogene Proteins c-akt ; Cyst Fluid/metabolism* ; Mice, Inbred C57BL ; Macrophages/metabolism* ; Phagocytosis ; Actins/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Guanosine Triphosphate/pharmacology*

Objective To investigate the ameliorative effect of salidroside on diabetes retinopathy (DR) rats and its mechanism. Methods Male SD rats were randomly divided into blank group, model group, low-dose and high-dose salidroside treatment groups. Except for the blank group, other groups were modeled by intraperitoneal injection of streptozotocin. After successful modeling, treatment groups were injected intraperitoneally with [50 mg/(kg.d)] and [100 mg/(kg.d)] salidroside respectively, for 4 weeks; the blank group and model group were injected with corresponding doses of saline. ELISA was used to measure the expression levels of antioxidant-related enzyme activity and inflammatory factors in blood glucose and serum of rats in each group. Retinal tissue lesions were detected by HE staining, and the expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) in retinal tissues were detected by immunohistochemical staining. Western blot analysis was used to detect the expression of phosphatidylinositol 3 kinase (PI3K) , nuclear factor κB p65 (NF-κB p65), phosphorylated p38 MAPK (p-p38 MAPK), and phosphorylated protein kinase B (p-AKT) proteins. Results Compared with model group, salidroside could significantly reduce blood glucose level and increase body mass in DR rats. The serum levels of superoxide dismutase (SOD) and catalase (CAT) were significantly increased, while the levels of malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-1β were reduced. The protein expression of VEGF, ICAM-1, NF-κB p65 and p-p38 MAPK was significantly decreased, while the protein expression of PI3K and p-AKT was increased. Conclusion Salidroside can reduce DR in rats by inhibiting oxidative stress and immune inflammatory response, which may be related to the reduction of abnormal expression of VEGF and ICAM-1 and the activation of PI3K/AKT signaling pathway.
Animals ; Male ; Rats ; Blood Glucose ; Diabetes Mellitus ; Inflammation/metabolism* ; Intercellular Adhesion Molecule-1/metabolism* ; NF-kappa B/metabolism* ; Oxidative Stress ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats, Sprague-Dawley ; Retinal Diseases ; Tumor Necrosis Factor-alpha/metabolism* ; Vascular Endothelial Growth Factor A/metabolism*

Objective To investigate the effect of viral myocarditis serum exosomal miR-320 on apoptosis of cardiomyocytes and its mechanism. Methods The model of viral myocarditis mice was established by intraperitoneal injection of Coxsackie virus B3. Serum exosomes were extracted by serum exosome extraction kit and co-cultured with cardiomyocytes. The uptake of exosomes by cardiomyocytes was detected by laser confocal microscopy. Cardiomyocytes were transfected with miR-320 inhibitor or mimic, and the expression level of miR-320 was detected by real-time quantitative PCR. Flow cytometry was used to detect cardiomyocyte apoptosis rate, and the expression levels of B cell lymphoma 2 (Bcl2) and Bcl2-related X protein (BAX) were tested by Western blot analysis. The prediction of miR-320 target genes and GO and KEGG enrichment analysis were tested by online database. The relationship between miR-320 and its target gene phosphoinositide-3-kinase regulatory subunit 1(Pik3r1) was examined by luciferase reporter gene. The effect of miR-320 on AKT/mTOR pathway protein was detected by Western blot analysis. Results Viral myocarditis serum exosomes promoted cardiomyocyte apoptosis, and increased the level of BAX while the level of Bcl2 was decreased. miR-320 was significantly up-regulated in myocardial tissue of viral myocarditis mice, and both pri-miR-320 and mature of miR-320 were up-regulated greatly in cardiomyocytes. The level of miR-320 in cardiomyocytes treated with viral myocarditis serum exosomes was significantly up-regulated, while transfection of miR-320 inhibitor counteracted miR-320 overexpression and reduced apoptosis rate caused by exosomes. Pik3r1 is the target gene of miR-320, and its overexpression reversed cardiomyocyte apoptosis induced by miR-320 up-regulation. The overexpression of miR-320 inhibited AKT/mTOR pathway activation. Conclusion Viral myocarditis serum exosome-derived miR-320 promotes apoptosis of mouse cardiomyocytes by inhibiting AKT/mTOR pathway by targeting Pik3r1.
Mice ; Animals ; Myocytes, Cardiac ; Phosphatidylinositol 3-Kinase/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Myocarditis/pathology* ; Exosomes/metabolism* ; bcl-2-Associated X Protein/metabolism* ; MicroRNAs/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis/genetics*

Male diabetic individuals present a marked impairment in fertility; however, knowledge regarding the pathogenic mechanisms and therapeutic strategies is unsatisfactory. The new hypoglycemic drug dapagliflozin has shown certain benefits, such as decreasing the risk of cardiovascular and renal events in patients with diabetes. Even so, until now, the effects and underlying mechanisms of dapagliflozin on diabetic male infertility have awaited clarification. Here, we found that dapagliflozin lowered blood glucose levels, alleviated seminiferous tubule destruction, and increased sperm concentrations and motility in leptin receptor-deficient diabetic db/db mice. Moreover, the glucagon-like peptide-1 receptor (GLP-1R) antagonist exendin (9-39) had no effect on glucose levels but reversed the protective effects of dapagliflozin on testicular structure and sperm quality in db/db mice. We also found that dapagliflozin inhibited the testicular apoptotic process by upregulating the expression of the antiapoptotic protein B-cell lymphoma 2 (BCL2) and X-linked inhibitor of apoptosis protein (XIAP) and inhibiting oxidative stress by enhancing the antioxidant status, including total antioxidant capacity, total superoxide dismutase (SOD) activity, and glutathione peroxidase (GPx) activity, as well as decreasing the level of 4-hydroxynonenal (4-HNE). Exendin (9-39) administration partially reversed these effects. Furthermore, dapagliflozin upregulated the glucagon-like peptide-1 (GLP-1) level in plasma and GLP-1R expression by promoting AKT8 virus oncogene cellular homolog (Akt) phosphorylation in testicular tissue. Exendin (9-39) partially inhibited Akt phosphorylation. These results suggest that dapagliflozin protects against diabetes-induced spermatogenic dysfunction via activation of the GLP-1R/phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Our results indicate the potential effects of dapagliflozin against diabetes-induced spermatogenic dysfunction.
Mice ; Animals ; Male ; Proto-Oncogene Proteins c-akt/metabolism* ; Antioxidants ; Phosphatidylinositol 3-Kinases/metabolism* ; Semen/metabolism* ; Diabetes Mellitus