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.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.
ObjectiveTo investigate the effect of different mechanical tensions on the expressions of RhoA/Rho associated protein kinases (ROCK) in rat tendon stem cells (TSCs). MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged, 2-3 months; weighing, 200-250 g) by enzymatic digestion method and cultured for 2-3 passages, then seeded on micro groovdishes. The 4% (4% stretch group) and 8% (8% stretch group) mechanical stretching was performed for 4 hours every day at 1 Hz. After 1, 2, and 3 days, the protein and mRNA expressions of RhoA and ROCK were measured by Western blot and real-time quantitative PCR. The cell proliferation was measured by cell counting kit 8. The cells were not stretched as control group. ResultsThe TSCs at passage 2 showed a cobble-stone shape and aggregation growth; TSCs seeded on micro groovdishes showed random growth, and the cells grew along the stretching direction after mechanical stretching. The mRNA expressions of RhoA and ROCK in control group, 4%, and 8% stretch groups showed an increasing tendency at 1, 2, and 3 days, showing significant difference between groups (P<0.05). The protein expressions of RhoA and ROCK in 4% and 8% stretch groups were similar to those in control group at 1 day (P>0.05), but the expressions in 4% and 8% stretch groups showed an increasing tendency at 2 and 3 days, which were significantly higher than those in control group (P<0.05). The cell proliferation of 8% stretch group was significantly lower than that of 4% stretch group and control group at each time point (P<0.05), but no significant difference was found between 4% stretch group and control group (P>0.05). ConclusionThe expressions of RhoA and ROCK of rat TSCs are positively correlated with stretch intensity. So RhoA/ROCK may be an important molecule in TSCs after mechanical stretching.
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.
ObjectiveTo explore the serum concentrations of complement C1q tumor necrosis factor related protein 5 (CTRP5) in patients with acute exacerbations and stable stage of chronic obstructive pulmonary disease (COPD), and analyze the correlation of CTRP5 with high sensitivity C-reactive protein (hs-CRP) and FEV1/FVC and FEV1%pred.MethodsThirty hospitalized patients with acute exacerbation of COPD and 30 outpatients with stable COPD according with diagnostic criteria and inclusive criteria were sampled successively. At the same time 30 healthy volunteers were selected as normal control. All subjects were measured the concentrations of CTRP5 and hs-CRP in serum and lung function test was performed.ResultsThe serum CTRP5 and hs-CRP concentrations of the acute exacerbation group was higher than those in the stable group and the control group. The serum CTRP5 and hs-CRP concentrations of the stable group was also higher than those of the control group. The FEV1/FVC of the acute exacerbation group was lower than those of the stable group and the control group; and the FEV1/FVC of the stable group was lower than that of the control group. The FEV1%pred of three groups by analysis indicated the difference was statistically significant. Further pairwise comparisons demonstrated that the FEV1%pred of two COPD groups were lower than that of the control group but the FEV1%pred of the acute exacerbation group and stable group was not significantly different. The correlation analysis of the acute exacerbation group and the stable group demonstrated that the levels of serum CTRP5 and hs-CRP were postively correlated and the level of serum CTRP5 was negatively correlated with FEV1/FVC and FEV1%pred.ConclusionsThe level of CTRP5 in serum of COPD patients is increased. No matter in acute exacerbation or stable phase, the level of serum CTRP5 is positively correlated with hs-CRP and negatively correlated with FEV1/FVC and FEV1%pred, which suggests that CTRP5 is involved in the pathogenesis of COPD but the exact mechanism needs further study.
ObjectiveTo explore the effect of La-related protein 6 (LARP6) gene on the survival of postoperative patients with gastric cancer, and to explore its relationship with immune cell infiltration.MethodsThe clinical survival information and gene expression information of gastric cancer patients were downloaded from The Cancer Genome Atlas (TCGA) database. The relationship between LARP6 gene expression and clinical characteristics of patients were analyzed. Cox proportion hazard regression model was used to find out the prognostic risk factors of gastric cancer patients, and then Kaplan-Meier plotter database was used to verify. Then the correlation between LARP6 gene expression and immunity was proved by Tumor IMmune Estimation Resource (TIMER) immune database.ResultsIn gastric cancer patients, the expression of LARP6 gene was related to pathological stage, T stage, and N stage (P<0.05), but not related to M stage and sex (P>0.05). Multivariate Cox proportion hazard regression analysis showed that age [HR=2.022, 95%CI was (1.287, 3.176), P=0.002] and LARP6 gene expression [HR=1.176, 95%CI was (1.070, 1.293), P<0.001] were prognostic factors. Further verified by Kaplan Meier plotter database, the results also showed that the overall survival (OS) and progression-free survival (PFS) of gastric cancer patients with high expression of LAPR6 gene were worse than those with low expression of LARP6 gene (P<0.001). TIMER database was used to explore the correlation between the expression level of LARP6 gene and immune cell infiltration in patients with gastric cancer, and the results showed that the expression level of LARP6 gene in gastric cancer patients was positively correlated with the infiltration number of CD4+ T cells and macrophage cell (P<0.001). Log-rank results showed that infiltration number of macrophage cell and LARP6 gene expression were risk factors for clinical prognosis of gastric cancer patients (P<0.05).ConclusionsMacrophage cell andcell and LARP6 gene expression are risk factors for gastric cancer patients. LARP6 may be a new target for the treatment of gastric cancer.
ObjectiveA competing endogenous RNA (ceRNA) regulatory network associated with long non-coding RNA (lncRNA) specific for lung adenocarcinoma (LUAD) was constructed based on bioinformatics methods, and the functional mechanism of actinfilament-associated protein 1-antisense RNA1 (AFAP1-AS1) in LUAD was analyzed, in order to provide a new direction for the study of LUAD therapeutic targets. MethodsThe gene chip of LUAD was downloaded from the Gene Expression Omnibus (GEO), and lncRNA and mRNA with differential expression between LUAD and normal tissues were screened using GEO2R online software, and their target genes were predicted by online databases to construct ceRNA networks and perform enrichment analysis. In cell experiments, AFAP1-AS1 was genetically knocked down and siRNA was constructed and transfected into LUAD cells A549 by cell transfection. CCK8, transwell, scratch assay and flow cytometry were used to detect the ability of cells to proliferate, invade, migrate and apoptosis. ResultsA total of 6 differentially expressed lncRNA and 494 differentially expressed mRNA were identified in the microarray of LUAD. The ceRNA network involved a total of 6 lncRNA, 22 miRNA, and 55 mRNA. Enrichment analysis revealed that mRNA was associated with cancer-related pathways. In cell assays, knockdown of AFAP1-AS1 inhibited cell proliferation, invasion, and migration, and AFAP1-AS1 promoted apoptosis. ConclusionIn this study, we construct a lncRNA-mediated ceRNA network, which may help to further investigate the mechanism of action of LUAD. In addition, through cellular experiments, AFAP1-AS1 is found to have potential as a therapeutic target for LUAD.
Objective To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats. Methods Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups A and B, the SCI model was established with modified Allen’s method and then the rats of groups A and B were administrated by subarachnoid injection of 6 μL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord. Results At different time points, the BBB score of group C was significantly higher than those of groups A and B (P lt; 0.05), and the BBB score of group A was significantly better than that of group B at 14 and 28 days after operation (P lt; 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P lt; 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P lt; 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P lt; 0.05). Conclusion The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.
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.