Objective To study changes in synaptic plasticity in the contralesional mirror area of the cortexes of rats with cerebral infarction treated by low-frequency electrical stimulation(LFES)and to explore the therapeutic mechanism of LFES on the molecular level.Methods Forty-eight Sprague-Dawley rats were randomly allocated into a LFES group,a placebo group and a sham-operation group.Following middle cerebral artery occlusion(MCAO),rats in the LFES group were treated with LFES for 7 d(20 min/d),while the ones in placebo group were connected with the same LFES device but without electricity.Rats in the sham-operation group were subjected to a MCAO operation without occlusion and then received no special treatment.Synaptic ultra-structures and the expression levels of glia fibrillary acidic protein(CFAP)and synaptophysin in the contralesional mirror area of the cortexes of the rats in each group were measured with electron-microscopy and Western blotting.Results Compared with the placebo group or the rats before treatment,rats treated with LFES exhibited ultra-structural changes in the form of larger curvature of synaptic interfaces and narrower synaptic clefts.GFAP expression levels did not fluctuate significantly,but the expression of synaptophysin was significantly up-regulated.Conclusion LFES treatment can induce active changes in synaptic plasticity in the contralesional mirror area of the cortex of rats after cerebral infarction.

Objective To study the effects of low-frequency electrical stimulation(LFES)on motor function and the expression of glia fibrillary acidic protein(GFAP)around cerebral infarction sites in rats.Methods Fifty-four male adult Sprague-Dawley rats were randomly divided into a LFES group,a placebo group and a sham operation group(18/group).All groups were randomly divided into 3 treatment groups.A rat model of middle cerebral artery occlusion(MCAO)was established using intraluminal filament occlusion.Treatment was carried out 3 d after the operation.Rats in the LFES treatment groups were stimulated with LFES for 3,7 or 14 days (10 min/d);the placebo groups were treated in the same way without electric stimulation;the sham operation subgroups didn't receive any therapy.Scores on a beam-walking test,a rotating pole test and a screen test were assessed at each time point mentioned above.Expression of GFAP was also assessed using immunohistochemcal techniques.Results The paralysed limbs recovered motor function better in the LFES groups than in the control groups.GFAP-positive cells were more numerous at the margins of the infarction area in the treated groups than in the control groups.Conclusions LFES might increase the expression of GFAP,which might be an important mechanism in improving brain plasticity after cerebral ischemia,aiding the recovery of the central nervous system and rebuilding its functioning.

Objective:To investigate the inhibitory effect of microRNA-106b in the process of migration and invasion of human malignant pleural mesothelioma cell NCI-H2452.Methods:In April 2017, the expression level of miRNA-106b in malignant pleural mesothelioma cells (NCI-H2452, MSTO-211H, NCI-H2052) and normal mesothelial cells MeT-5A was detected and analyzed. Using NCI-H2452 cells as a model, the NCI-H2452 cell model with miRNA-106b overexpression was established by transfecting miRNA-106b mimics. The expression level of miRNA-106b in the cells was detected by real-time fluorescent quantitative PCR. The effect of miRNA-106b on the migration and invasion ability of NCI-H2452 cells was analyzed. The gene expression data of malignant mesothelioma and the downstream target gene data of miRNA-106b in public databases were analyzed to screen the downstream target genes of miRNA-106b in mesothelioma cells that affect cell migration and invasion ability, and to verify the expression of this gene in NCI-H2452 cells with miRNA-106b overexpression.Results:The expression of miRNA-106b in three MPM cells was decreased compared with MeT-5A cells ( P<0.001) . The expression level of miRNA-106b was significantly increased after transfection of miRNA-106b mimics ( P<0.001) . The scratch migration levels of the experimental group were 28.45%±4.37%, 38.12%±4.82% and 50.06%±8.92% at 24h, 31h and 48h, respectively. Compared with the control group, the migration level decreased by 37.48%±2.65%, 49.21%±3.45% and 68.14%±3.81% ( P<0.01) . The number of cell migration and invasion decreased in the experimental group compared with the control group ( P<0.001) . Public databases were used to screen and analyze the possibility that TCF21 gene, as a downstream target gene, could affect the migration and invasion ability of MPM cells. The expression level of TCF21 gene was increased after transfection of miRNA-106b mimics in NCI-H2452 cells ( P=0.009) . Conclusion:MiRNA-106b can inhibit the migration and invasion of NCI-H2452 cells and increase the expression of TCF21 gene.

Objective:To investigate the inhibitory effect of microRNA-106b in the process of migration and invasion of human malignant pleural mesothelioma cell NCI-H2452.Methods:In April 2017, the expression level of miRNA-106b in malignant pleural mesothelioma cells (NCI-H2452, MSTO-211H, NCI-H2052) and normal mesothelial cells MeT-5A was detected and analyzed. Using NCI-H2452 cells as a model, the NCI-H2452 cell model with miRNA-106b overexpression was established by transfecting miRNA-106b mimics. The expression level of miRNA-106b in the cells was detected by real-time fluorescent quantitative PCR. The effect of miRNA-106b on the migration and invasion ability of NCI-H2452 cells was analyzed. The gene expression data of malignant mesothelioma and the downstream target gene data of miRNA-106b in public databases were analyzed to screen the downstream target genes of miRNA-106b in mesothelioma cells that affect cell migration and invasion ability, and to verify the expression of this gene in NCI-H2452 cells with miRNA-106b overexpression.Results:The expression of miRNA-106b in three MPM cells was decreased compared with MeT-5A cells ( P<0.001) . The expression level of miRNA-106b was significantly increased after transfection of miRNA-106b mimics ( P<0.001) . The scratch migration levels of the experimental group were 28.45%±4.37%, 38.12%±4.82% and 50.06%±8.92% at 24h, 31h and 48h, respectively. Compared with the control group, the migration level decreased by 37.48%±2.65%, 49.21%±3.45% and 68.14%±3.81% ( P<0.01) . The number of cell migration and invasion decreased in the experimental group compared with the control group ( P<0.001) . Public databases were used to screen and analyze the possibility that TCF21 gene, as a downstream target gene, could affect the migration and invasion ability of MPM cells. The expression level of TCF21 gene was increased after transfection of miRNA-106b mimics in NCI-H2452 cells ( P=0.009) . Conclusion:MiRNA-106b can inhibit the migration and invasion of NCI-H2452 cells and increase the expression of TCF21 gene.

Objective:To investigate the survival and death risk factors of mesothelioma cases stratified by the expression levels of CD8 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) , providing new clue to evaluate disease progression and clinical outcome.Methods:This was a retrospective case report, which included 47 clinically and pathologically confirmed mesothelioma cases on November 2016. Their clinical and pathological information, asbestos exposure history and survival data were collected. Infiltrated lymphocyte, 5-methylcytosine (5-mC) , CTLA-4, CD8 and Ki-67 antigen were detected using hematoxylin-eosin staining and immunohistochemistry. Survival time and death risk factors of mesothelioma patients with different CD8 and CTLA-4 protein expression characteristics were analyzed. And analyze the influence of Ki-67 expression on the survival of patients with different CD8 and CTLA-4 protein and gene expression characteristics.Results:Among the 47 cases, 63.8% (30/47) had low/medium level of infiltrated lymphocyte. The immunohistochemistry scores of CTLA-4, CD8, 5-mC and Ki-67 were 92.97 (54.95, 120.65) , 72.41 (36.62, 89.82) , 11.09 (3.40, 52.89) and 5.88 (2.41, 11.48) , respectively. Patients with CD8 high CTLA-4 high had higher 5-mC level than those with CD8 high CTLA-4 low ( P<0.01) . The median survival time of 27 cases was 0.83±0.29 year. The median survival times of those with CD8 high CTLA-4 high and CD8 high CTLA-4 low were 0.58±0.51 year and 0.83±0.30 year, respectively ( P=0.521) . The immunohistochemistry score of Ki-67 ≥5.88 was an independent death risk factor for patients with CD8 high CTLA-4 low ( HR=8.40, P=0.01) . Under different CD8 and CTLA-4 protein expression characteristics, in the patients with CD8 high CTLA-4 low, the median survival times of those with high and low Ki-67 expression were 0.57±0.11 years and 2.31±0.46 years, respectively ( P<0.01) . Under different CD8 and CTLA-4 mRNA expression characteristics, in the patients with CD8 high CTLA-4 low, the median survival times of those with high and low Ki-67 mRNA expression were 1.20±0.36 years and 3.38±0.43 years, respectively ( P=0.018) . Conclusion:Mesothelioma case with high CD8 but low CTLA-4 content might coexist DNA hypomethylation. In the presence of high Ki-67 expression, their survival time appears to be shortened with increased death risk.

Objective:To analyze the gene mutation profile in malignant pleural mesothelioma (MPM) and investigate the expression of high-frequency mutant genes and its relationship with clinicopathological parameters. To screen out key genes and clinicopathologic factors related to the prognosis of MPM patients.Methods:The second generation sequencing data, somatic mutation data and clinical pathological data of 86 MPM cases and gene chip expression data of 89 MPM cases were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) in March 2020. Summarize the gene mutation profile of tissue samples in the TCGA database and analyze the relationship between the expression level of high-frequency mutation genes and the clinicopathological characteristics, asbestos exposure history and prognosis of MPM patients. The genes significantly related to MPM prognosis were screened out for gene set enrichment analysis (GSEA) . Survival analysis and GSEA were performed for the selected key genes and clinicopathological features verification using the microarray expression data from the GEO database.Results:The top 10 genes with highest single nucleotide variations frequencies were BAP1, NF2, TP53, TTN, SETD2, LATS2, CCDC168, FAT4, PTCH1 and ZNF469. The high expression rates of NF2, TP53, SETD2 and CCDC168 genes in wild type were higher than those of mutated type, and the differences were statistically significant ( P<0.05) . Cox multivariate analysis of TCGA data showed that MPM patients with epithelial type ( HR=0.425, 95% CI: 0.235-0.767, P<0.01) and SETD2 low expression ( HR=0.516, 95% CI: 0.307-0.868, P=0.011) had lower risk of death. The survival analysis of GEO data verified that patients with epithelial type MPM had longer survival time, while patients with sarcoma type MPM had shortest survival time ( P<0.01) . GSEA showed that SETD2 was involved in G2M checkpoint, E2F targets, MYC signaling pathways, protein secretion, mitotic spindle, MTORC1 pathway, TGF-β pathway, androgen response and uv response. Conclusion:MPM is accompanied with higher frequency of gene mutations represented by BAP1, NF2, TP53, TTN, SETD2, LATS2 and so on. SETD2 expression level and epithelia type of MPM may be influential factors for MPM prognosis.

Objective:To investigate the survival and death risk factors of mesothelioma cases stratified by the expression levels of CD8 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) , providing new clue to evaluate disease progression and clinical outcome.Methods:This was a retrospective case report, which included 47 clinically and pathologically confirmed mesothelioma cases on November 2016. Their clinical and pathological information, asbestos exposure history and survival data were collected. Infiltrated lymphocyte, 5-methylcytosine (5-mC) , CTLA-4, CD8 and Ki-67 antigen were detected using hematoxylin-eosin staining and immunohistochemistry. Survival time and death risk factors of mesothelioma patients with different CD8 and CTLA-4 protein expression characteristics were analyzed. And analyze the influence of Ki-67 expression on the survival of patients with different CD8 and CTLA-4 protein and gene expression characteristics.Results:Among the 47 cases, 63.8% (30/47) had low/medium level of infiltrated lymphocyte. The immunohistochemistry scores of CTLA-4, CD8, 5-mC and Ki-67 were 92.97 (54.95, 120.65) , 72.41 (36.62, 89.82) , 11.09 (3.40, 52.89) and 5.88 (2.41, 11.48) , respectively. Patients with CD8 high CTLA-4 high had higher 5-mC level than those with CD8 high CTLA-4 low ( P<0.01) . The median survival time of 27 cases was 0.83±0.29 year. The median survival times of those with CD8 high CTLA-4 high and CD8 high CTLA-4 low were 0.58±0.51 year and 0.83±0.30 year, respectively ( P=0.521) . The immunohistochemistry score of Ki-67 ≥5.88 was an independent death risk factor for patients with CD8 high CTLA-4 low ( HR=8.40, P=0.01) . Under different CD8 and CTLA-4 protein expression characteristics, in the patients with CD8 high CTLA-4 low, the median survival times of those with high and low Ki-67 expression were 0.57±0.11 years and 2.31±0.46 years, respectively ( P<0.01) . Under different CD8 and CTLA-4 mRNA expression characteristics, in the patients with CD8 high CTLA-4 low, the median survival times of those with high and low Ki-67 mRNA expression were 1.20±0.36 years and 3.38±0.43 years, respectively ( P=0.018) . Conclusion:Mesothelioma case with high CD8 but low CTLA-4 content might coexist DNA hypomethylation. In the presence of high Ki-67 expression, their survival time appears to be shortened with increased death risk.

Objective:To analyze the gene mutation profile in malignant pleural mesothelioma (MPM) and investigate the expression of high-frequency mutant genes and its relationship with clinicopathological parameters. To screen out key genes and clinicopathologic factors related to the prognosis of MPM patients.Methods:The second generation sequencing data, somatic mutation data and clinical pathological data of 86 MPM cases and gene chip expression data of 89 MPM cases were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) in March 2020. Summarize the gene mutation profile of tissue samples in the TCGA database and analyze the relationship between the expression level of high-frequency mutation genes and the clinicopathological characteristics, asbestos exposure history and prognosis of MPM patients. The genes significantly related to MPM prognosis were screened out for gene set enrichment analysis (GSEA) . Survival analysis and GSEA were performed for the selected key genes and clinicopathological features verification using the microarray expression data from the GEO database.Results:The top 10 genes with highest single nucleotide variations frequencies were BAP1, NF2, TP53, TTN, SETD2, LATS2, CCDC168, FAT4, PTCH1 and ZNF469. The high expression rates of NF2, TP53, SETD2 and CCDC168 genes in wild type were higher than those of mutated type, and the differences were statistically significant ( P<0.05) . Cox multivariate analysis of TCGA data showed that MPM patients with epithelial type ( HR=0.425, 95% CI: 0.235-0.767, P<0.01) and SETD2 low expression ( HR=0.516, 95% CI: 0.307-0.868, P=0.011) had lower risk of death. The survival analysis of GEO data verified that patients with epithelial type MPM had longer survival time, while patients with sarcoma type MPM had shortest survival time ( P<0.01) . GSEA showed that SETD2 was involved in G2M checkpoint, E2F targets, MYC signaling pathways, protein secretion, mitotic spindle, MTORC1 pathway, TGF-β pathway, androgen response and uv response. Conclusion:MPM is accompanied with higher frequency of gene mutations represented by BAP1, NF2, TP53, TTN, SETD2, LATS2 and so on. SETD2 expression level and epithelia type of MPM may be influential factors for MPM prognosis.

Objective:To investigate the inhibitory effect and molecular mechanism of microRNA-30d (miR-30d) in the process of proliferation, migration and invasion of malignant mesothelioma cell line MSTO-211H.Methods:In April 2017, the human MSTO-211H cells was used to establish miR-30d overexpressed MSTO-211H cell model by transfection of miR-30d mimics. The qRT-PCR was performed to detect the expression level of miR-30d in the cells transfected miR-30d mimics. The effects of miR-30d on the proliferation, apoptosis, migration and invasion of MSTO-211H cells were analyzed by CCK-8 experiment, flow cytometry, cell scratch experiment and Transwell method.Results:After transfection of miR-30d, the expression level of miR-30d in the MSTO-211H+miR-30d cells group was significantly higher than MSTO-211H+miR NC cells group ( P<0.01) . The cell activity of MSTO-211H+miR-30d group (105.13%±2.35%) was significantly lower than MSTO-211H+miR NC cells group (115.40%±1.35%) , and the level of apoptosis (3.97%±0.36%) was significantly higher than MSTO-211H+miR NC cells group (1.47%±0.10%) ( P<0.01) . The relative migration areas at 12 and 24 h of MSTO-211H+miR-30d cells group (9.35±3.16 μm 2 and 58.19±1.82 μm 2) were significantly lower than MSTO-211H+miR NC cells group (54.42 ±5.26 μm 2 and 88.32 ±1.96 μm 2) ( P<0.01) . Compared with the MSTO-211H+miR NC cells group, the numbers of cell migration and cell invasion were reduced in the MSTO-211H+miR-30d cells group ( P<0.01) . Conclusion:miR-30d can regulate the progression of malignant pleural mesothelioma by inhibiting the proliferation, apoptosis, migration and invasion of MSTO-211H cells.

Objective:To study the cytotoxicity and malignant transformation ability of chrysotile on MeT-5A cells.Methods:In June 2016, lactate dehydrogenase (LDH) method was used to detect the cytotoxicity of chrysotile to MeT-5A cells. MeT-5A cells were treated with 5 μg/cm 2 chrysotile intermittently for 24 h a time, once a week and a total of 28 times. After the cells showed anchorage independent growth, the cell features of malignant transformation were identified by colony forming frequency in soft agar, and the soft agar colony formation rates were calculated. The activities of key speed limiting enzymes of glycolysis metabolism including hexokinase (HK) , phosphofructokinase (PFK) and pyruvate kinase (PK) were determined by UV colorimetry. Results:Chrysotile was cytotoxic to MeT-5A cells in a concentration-dependent decline. Compared with the control group, the relative survival rates of MeT-5A cells were significantly decreased after exposed to chrysotile at 10, 20, 40 and 80 μg/cm 2 ( P<0.05) . After 28 times of exposure, the growth rate of the cells in chrysotile transformed MeT-5A cells was accelerated, the arrangement was disordered, the contact inhibition was lost, and the double layer growth appeared, which could grow on soft agar. The colony forming rate of the chrysotile transformed MeT-5A cells was 18.33‰±2.49‰. Compared with the control group (0) , the difference was statistically significant ( P<0.01) . The activities of glycolysis related kinase including PK [ (19.51±1.52) U/L], PFK[ (0.12±0.02) U/10 4 cell] and HK[ (0.26±0.01) U/10 4 cell] were increased in the chrysotile transformed MeT-5A cells compared with control group [ (25.00±1.04) U/L、(0.15±0.01) U/10 4 cell and (0.33±0.01) U/10 4 cell] ( P<0.01) . Conclusion:Chrysotile can induce malignant transformation of MeT-5A cells and increase the activities of glycolysis related kinases including PK, PFK and HK.