Objective To construct human secreted apoptosis-related protein 1 (SARP1) gene yeast two-hybrid bait vector so as to study the biological functions of the SARP1 gene in the scar tissue. Methods The target gene from SARP1 gene full-length DNA segment was amplified by PCR, the upstream and downstream primers of the SARP1 gene with restriction enzymes Nde I and Sal I were designed. pGBKT7-SARP1 recombination plasmid was constructed by ligating the vector and the PCR production and identified by PCR and sequencing. Further more, pGBKT7-SARP1 was transformed into competent AH109 which contained kanamycin for selecting positive clones and screened the positive clony on the plate of SD/-Trp. The toxicity and transcriptional activation were tested by the phenotype assay. Results SARP1 was amplified and cloned into pGBKT7 successfully, SARP1 gene sequence in recombinant plasmid pGBKT7-SARP1 was verified by gel electrophoresis and DNA sequencing analysis. The sequence of inserted SARP1 gene was the same as the corresponding sequence found in GenBank database. The recombinant pGBKT7-SARP1 plasmids and empty pGBKT7 vector could form white colonies on SD/-Trp plates and none could survive on SD/-Leu plates. Conclusion The recombinant pGBKT7-SARP1 gene yeast two-hybrid bait vector is successfully constructed.
Objective To investigate the effect of chondroitinase ABC (ChABC) on the expression of growth associated protein 43 (GAP-43) and gl ial fibrillary acidic protein (GFAP) after spinal cord injury (SCI) in rats. Methods A total of 150 adult female SD rats, weighing 250-300 g, were randomly divided into ChABC treatment group (group A), sal ine treatment group (group B), and sham operation group (group C) with 50 rats in each group. In groups A and B, the rats were made the SCI models and were treated by subarachnoid injection of ChABC and sal ine; in group C, the rats were not treated as a control. At 1, 3, 7, 14, and 21 days after operation, the Basso, Beattie, and Bresnahan (BBB) score system was used toevaluate the motion function, and immunofluorescent histochemical staining was used to observe the expressions of GAP-43 and GFAP. Results At different time points, the BBB scores of groups A and B were significantly lower than those of group C (P lt; 0.05); there was no significant difference in BBB score between groups A and B after 1, 3, and 7 days of operation (P gt; 0.05), but the BBB score of group A was significantly higher than that of group B after 14 and 21 days of operation (P lt; 0.01). At different time points, the GAP-43 and GFAP positive neurons of groups A and B were significantly higher than those of group C (P lt; 0.05). After 14 and 21 days of operation, the GAP-43 positive neurons of group A were more than those of group B (P lt; 0.01). After 7, 14, and 21 days of operation, the GFAP positive neurons of group A were significantly less than those of group B (P lt; 0.01). Conclusion ChABC can degrade gl ial scar, improve the microenvironment of the injured region and enhance the expression of GAP-43, which promotes axonal growth and extension.
Objective To dynamically study the formation of multidrug resistance(MDR) of human hepatocellular carcinoma cell SMMC-7721 induced by Adriamycin (ADM) and the role of multidrug resistance-associated protein(MRP) in its mechanisms.Methods Hepatocellular carcinoma cell SMMC-7721 was cultured in RPMI-1640 medium containing ADM with progressively increased concentration or directly cultured in medium containing different concentrations of ADM. Resistant index of drug-resistant variants of SMMC-7721 cell was determined by drawing cell dosage-reaction curves.Levels of MRP mRNA expression were detected by reverse transcription-polymerase chain reaction(RTPCR). Intracellular rubidomycin(DNR) concentration was examined by flow cytometry(FCM).Results With progressive increasing of ADM concentration in medium resistant index and levels of MRP mRNA expression were correspondingly increased but intracellular DNR concentration was markly reduced. When parental cells were directly cultured in medium containing different concentrations of ADM, the higher the ADM concentration, the higher the level of MRP mRNA expression, but intracellular DNR concentration was kept at the similar high level and most cells died. Conclusion ADM may progressively induce SMMC-7721 cell resistant to multiple chemotherapeutic drugs with reduced intracellular DNR accumulation associated with the overexpression of MRP gene.
Objective To study the effect of secreted frizzled-related protein 1 (SFRP1) on airway remodeling in chronic obstructive pulmonary disease (COPD). Methods Forty-five patients with stable COPD and 21 healthy controls were collected in Jiangsu Province Hospital from June 2014 to June 2016. The level of SFRP1 in serum was detected by ELISA. The expression of SFRP1, E-cadherin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (16HBEs) were detected by Western blot. Results The level of SFRP1 in serum of the COPD patients was significantly higher than that in the healthy controls [(80.65 ± 15.01) pg/ml vs. (23.89 ± 2.59) pg/ml, P<0.05]. There were negative correlations between serum SFRP1 levels and lung function parameters (FEV 1, FEV 1%pred, and FVC) in the COPD patients (correlation coefficient r value were –0.368 9, –0.382 6 and –0.417 3, respectively, all P<0.05). The level of serum SFRP1 in the COPD smokers was significantly higher than that in the COPD non-smokers [(97.74 ± 21.95) pg/mlvs. (52.51 ± 14.87) pg/ml, P<0.05]. The expression of SFRP1 could be up-regulated by cigarette smoke extract in 16HBEs. The expression of E-cadherin of 16HBEs was up-regulated by recombinant SFRP1, but the expression of α-SMA of 16HBEs was down-regulated by recombinant SFRP1. Conclusion SFRP1 may be involved in the pathogenesis of airway remodeling of COPD by regulating the expression of E-cadherin and α-SMA of bronchial epithelial cells.
Objective To identify the effects of single immunoglobin IL-1 receptor related protein （SIGIRR） on inflammation induced by high mobility group box 1 （HMGB1） in A549 derived from human alveolar epithelial cells. Methods Eukaryotic expression vectors pCDNA3.1（+） constructed with SIGIRR cDNA were transiently transfected into A549 cells，in which SIGIRR was forced to be over-expressed. Western blot and RT-PCR were applied to detect the expression level of SIGIRR after transfection. After the stimulation by HMGB1，the transcriptional activity of NF-κB in A549 cells was detected by dual-luciferase reporter assay system，and the protein levels of inflammatory cytokine TNF-α and IL-1β were measured by ELISA. Results The expression level of SIGIRR increased significantly in A549 cells transfected with SIGIRR vectors. The transcriptional activity of NF-κB was enhanced obviously after HMGB1 treatment in A549 cells by dual-luciferase reporter assay system，while the transfection of SIGIRR vectors decreased the activity. The protein levels of TNF-α and IL-1β were down-regulated in A549 cells over-expressing SIGIRR after HMGB1 stimulation compared with the non-transfected cells. Conclusions Up-regulated SIGIRR expression can inhibit HMGB1-induced proinlammatory cytokine release in A549 cells such as TNF-α and IL-1β. The transcriptional activity of NF-κB is dampened by SIGIRR transfection，implying that the anti-inflammatory effects of SIGIRR may be involved in the regulation of NF-κB.
ObjectiveTo explore the diagnostic value of ultrasound elastography (USE) combined with long non-coding RNA actin filament associated protein 1 anti-sense RNA 1 (AFAP1-AS1) mRNA in thyroid fine-needle aspiration (FNA) wash-out fluid for distinguishing benign from malignant thyroid nodules. MethodsThe patients with thyroid nodules who were treated in the Shenzhen Futian District Second People’s Hospital from January 2020 to June 2022 were collected. Before operation, the patients’ thyroid nodules were evaluated by the USE score and the AFAP1-AS1 mRNA in the thyroid FNA wash-out fluid was detected. The pathological result of the thyroid nodule after operation was as a gold standard for diagnosis of malignant thyroid nodules. The clinical diagnostic value of USE score combined with AFAP1-AS1 mRNA in the FNA wash-out fluid of the benign and malignant thyroid nodules were analyzed. ResultsA total of 174 thyroid nodules (124 patients) were detected in this study, of which 62 (45 patients) were histologically diagnosed as malignant. There was a statistical difference in the comparison of the composition ratio of USE score grading between the benign and malignant thyroid nodules (Z=8.82, P<0.001). The point of USE of the benign thyroid nodules was statistically lower than that of the malignant thyroid nodules [2.28±1.16 vs. 4.26±1.01, mean difference (MD) and 95% confidence interval (95%CI)=2.98 (2.76, 3.20), t=30.85, P<0.001]. The AFAP1-AS1 mRNA in the FNA wash-out fluid of the malignant thyroid nodules was statistically higher than that of the benign thyroid nodules [1.45±0.27 vs. 1.13±0.16, MD (95%CI)=1.45(1.39, 1.50), t=10.69, P<0.001]. Pearson correlation analysis showed that there was a positive correlation between the USE score of thyroid nodules and the expression of AFAP1-AS1 mRNA in the FNA wash-out fluid (r=0.58, P<0.001). The sensitivity and specificity of USE score in combination with expression of AFAP1-AS1 mRNA in the FNA wash-out fluid for diagnosing the malignant thyroid nodules by receiver operating characteristic (ROC) curve was 93.5% and 88.4% respectively. The area under the ROC curve (95%CI) was 0.91 (0.86, 0.96). Conclusion According to preliminary results of this study, USE score combined with AFAP1-AS1 mRNA in the thyroid FNA wash-out fluid is more sensitive and shows a potential diagnostic performance than USE score or AFAP1-AS1 mRNA detection alone for distinguishing benign from malignant thyroid nodules.
ObjectiveTo investagte the effects of single immunoglobin IL-1 receptor related protein (SIGIRR) on inflammation induced by cigarette smoke extract (CSE) in A549 cells derived from mouse alveolar epithelial cells. MethodsA549 cells were divided into a control group and an over-expressed SIGIRR group. Eukaryotic expression vectors pcDNA3.1(+) constructed with SIGIRR cDNA were transiently transfected into A549 cells, in which SIGIRR was forced to be over-expressed. The expression level of SIGIRR after transfection was detected with Western blot and RT-PCR method. After stimulated by CSE in both groups, the protein level of IL-6 was detected by ELISA, the transcriptional activity of cyclooxygenase-2(COX-2) was detected by dual-luciferase reporter assay system, and the release of reactive oxygen species (ROS) was measured by chemiluminescence method. ResultsThe expression level of SIGIRR increased significantly in A549 cells transfected with SIGIRR vectors. The COX-2 expression and the levels of ROS and IL-6 were significantly increased in the control group after CSE stimulation. Nevertheless, in the over-expressed SIGIRR group, the COX-2 expression and the release of ROS was reduced while the protein level of IL-6 was down-regulated compared with the control group(P < 0.05). ConclusionsUp-regulated SIGIRR expression can suppress the levels of ROS, COX-2 and IL-6 in A549 cells stimulated by CSE. It suggests that SIGIRR can inhibit airway inflammation caused by smoking.
Objective To investigate the effect of mitochondrial fission mediated by mitochondrial dynamics related protein 1 (DRP1) on glucose metabolism reprogramming in lung cancer cells, and the regulatory mechanism on phosphatidylinositol-3-kinases (PI3K)/protein kinase B (Akt) signaling pathway. Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression of DRP1 in lung cancer tissue and lung cancer cells. Mitochondrial fission inhibitor (Mdivi-1) and Mdivi-1+PI3K/Akt signaling pathway activator 740Y-P were used to treat H1299 cells. Mitochondrial fission agonist (WY14643) + signal inhibitor LY294002 were used to intervene PC14 cells. The reagent kit was used to detect the glucose consumption, lactate release, and ATP production of each group of cells. 5-ethynyl-2-deoxyuridine (EdU) labeling experiment was used to detect the proliferation of cells in each group, and acridine orange/ethidium bromide (AO/EB) staining was used to detect the apoptosis of cells in each group. MitoTracker Red CMXRos was used to detect the mitochondrial morphology of each group of cells. Tetramethylrhodamine ethyl ester (TMRE) staining was used to detect the mitochondrial membrane potential of cells. Dihydroethidium (DHE) staining was used to detect the level of reactive oxygen species (ROS) in cells. Western blot was used to detect was used to detect the expression of pyruvate kinase M2 (PKM2), hexokinase 2 (HK2), phosphofructokinase-1 (PFK1), DRP1, phosphorylated DRP1 (p-DRP1), PI3K, Akt, phosphorylated PI3K (p-PI3K), and phosphorylated Ak t(p-Akt) in each group of cells. Results The mRNA expression of DRP1 was significantly increased in lung cancer tissue and lung cancer cells. Mdivi-1 promoted the development of lung cancer and exerts anticancer effects, while activating PI3K/Akt signaling could partially reverse the anticancer effects of Mdivi-1. WY14643 exerted a pro-cancer effect, and inhibiting PI3K/Akt signaling could partially reverse the pro-cancer effect of WY14643, and the differences were statistically significant (all P<0.05). Conclusions In lung cancer, the expression of DRP1 is significantly increased, and DRP1 affects the glycolysis process and proliferation performance of lung cancer cells by regulating the activation of PI3K/Akt signaling.