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Objective:To explore the role of transgelin(TAGLN) in the occurrence and development of papillary thyroid carcinoma (PTC) and its possible signal pathway.Methods:One hundred cases of PTC tissues and corresponding paracancerous normal thyroid tissues were collected. Realtime quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry were used to analyze the expression of TAGLN in PTC tissues and corresponding paracancerous normal thyroid tissues. PTC cells were transfected with plasmid and shRNA lentivirus vector respectively to up-regulate or down-regulate the expression of TAGLN in order to detect the effects of them on the proliferation, invasion, and migration by cell proliferation assay(cell counting kit-8, CCK-8)and cell invasion and migration assays (Transwell). The effects of TAGLN on mitogen-activated protein kinase (MAPK)/extracellular-signal regulating kinase (ERK) signal pathway was detected with Western blotting.Results:RT-qPCR showed that there was no difference in the expression of TAGLN mRNA between PTC and corresponding paracancerous normal thyroid tissues ( P>0.05); Western blotting demonstrated that the expression of TAGLN protein in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues ( P<0.01). Immunohistochemical results revealed that the expression of TAGLN in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues. Overexpression of TAGLN inhibited the proliferation, invasion, and migration of PTC cells ( P<0.01), but knockdown of TAGLN promoted the proliferation, invasion, and migration of PTC cells ( P<0.01). Overexpression of TAGLN decreased the expression of phosphorylated ERK ( P<0.05), whereas silencing TAGLN increased phosphorylated ERK level in PTC cells( P<0.01). Conclusion:The expression of TAGLN in PTC is significantly decreased. It is related to the occurrence and development of PTC, and its mechanism may be related to MAPK/ERK signal pathway.
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Objective:To evaluate the effects of human umbilical cord mesenchymal stem cells-derived exosomes (hUCMSCs-ex) injection on cardiac function and myocardial fibrosis in dilated cardiomyopathy (DCM) rats induced by Adriamycin(ADR).Methods:One hundred male SD rats were randomly divided into the normal group (20 rats) and the DCM group (80 rats). The rats in DCM group were treated with ADR by intravenous injection to induce DCM.DCM rats were randomly divided equally into DCM group, low-dose group, medium-dose group and high-dose group which were received intravenous injection 1 mL/kg Dulbecco′s modified eagle medium(DMEM), 20 μg/kg, 100 μg/kg and 250 μg/kg exosomes.After modeling, 10 rats in normal group and 30 rats in DCM group were randomly selected to receive echocardiography to evaluate the cardiac function.After exosomes treatment, 10 rats were randomly selected form each group for echocardiography to evaluate the cardiac function.The morphological changes in myocardial cells were observed by using Masson staining in each group; Western blot detection between groups of rats was used to analyze the expression of myocardial collagen Ⅰ type(COLⅠ), Smad2 and alpha smooth muscle actin (α-SMA).Results:Left ventricular ejection fraction(LVEF) and left ventricular fraction shortening (LVFS)in the DCM group [(64.30±3.51)% and (38.70±2.85)%] were significantly lower than those of the normal group [(78.80±1.52)% and (50.60±1.50)%], and the differences were statistically significant ( t=20.518, 22.311, all P<0.01). The left ventricular end-diastolic diameter(LVEDD) and left ventricular end-systolic diameter (LVESD) [(4.62±0.13) mm and (3.40±0.12) mm] of the DCM group were significantly higher than those of the normal group[(3.29±0.24) mm and (3.16±0.33) mm], and the differences were statistically significant( t=2.854, 3.800, all P<0.01). After exosomes treatment, LVEF[(84.3±2.6)% and (83.4±3.2)%] in the medium-dose and high-dose groups were significantly higher than that in the DCM group [(79.2±2.4)%], and the diffe-rences were statistically significant(all P<0.01). Masson staining found that collagen fibers were less in exosomes treating group than those in the DCM group; Western blot test showed that high-dose exosomes can reduce the expression of α-SMA and Smad2, high-dose and low-dose exosomes can both significantly reduce the expression of COLⅠ. Conclusions:It suggests that exosomes intravenous injection from hUCMSCs-ex can significantly improve myocardial fibrosis in DCM rats induced by ADR and cardiac function.
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Objective@#To detect the methylation status of ribosomal S6 kinase 4 (RSK4)in papillary thyroid carcinoma (PTC)and to study its correlation with mRNA expression and clinical features.@*Methods@#134 cases PTC tissues and corresponding paracancerous thyroid tissues were collected. DNA methylation status of RSK4 gene in PTC tissues and corresponding paracancerous tissues were analyzed by methylation specific PCR and bisulfite genomic sequencing, and mRNA expression was detected by quantitative realtime PCR. The relationship of DNA methylation status with mRNA expression and clinical features was analyzed.@*Results@#The methylation rate of RSK4 in PTC tissues was significantly higher than that in paracancerous tissues by methylation specific PCR (P<0.05)and bisulfite genomic sequencing (P<0.01). The RSK4 mRNA level in PTC tissues was significantly lower than that in paracancerous tissues (P<0.01). In PTC tissues, RSK4 mRNA expression in methylation group was lower than that in unmethylation group (P<0.01). The RSK4 mRNA level in PTC of methylation was lower than that in paracancerous tissues of methylation (P<0.01); the RSK4 mRNA level in PTC of unmethylation was also lower than that in paracancerous tissues of unmethylated ones (P<0.01). There was relationship of RSK4 hypermethylation with lymphatic metastasis and TNM grade (P<0.05). Serum concentrations of thyroid stimulating hormone, thyroid peroxidase antibody, and thyroglobulin antibody in methylation group were higher than those in unmethylation group (P<0.05).@*Conclusion@#The hypermethylation of RSK4 promoter′s CpG islands might be one of the mechanisms for poor expression of RSK4 in PTC, which may serve as a molecular target of early diagnosis and treatment.
ABSTRACT
Objective To detect the methylation status of ribosomal S6 kinase 4 ( RSK4 ) in papillary thyroid carcinoma ( PTC) and to study its correlation with mRNA expression and clinical features. Methods 134 cases PTC tissues and corresponding paracancerous thyroid tissues were collected. DNA methylation status of RSK4 gene in PTC tissues and corresponding paracancerous tissues were analyzed by methylation specific PCR and bisulfite genomic sequencing, and mRNA expression was detected by quantitative realtime PCR. The relationship of DNA methylation status with mRNA expression and clinical features was analyzed. Results The methylation rate of RSK4 in PTC tissues was significantly higher than that in paracancerous tissues by methylation specific PCR ( P<0.05) and bisulfite genomic sequencing ( P<0. 01 ) . The RSK4 mRNA level in PTC tissues was significantly lower than that in paracancerous tissues ( P<0.01) . In PTC tissues, RSK4 mRNA expression in methylation group was lower than that in unmethylation group ( P<0.01) . The RSK4 mRNA level in PTC of methylation was lower than that in paracancerous tissues of methylation ( P<0. 01);the RSK4 mRNA level in PTC of unmethylation was also lower than that in paracancerous tissues of unmethylated ones ( P<0. 01 ) . There was relationship of RSK4 hypermethylation with lymphatic metastasis and TNM grade ( P<0. 05 ) . Serum concentration, of thyroid stimulating hormone, thyroid peroxidase antibody, and thyroglobulin antibody in methylation group were higher than those in unmethylation group ( P<0.05) . Conclusion The hypermethylation of RSK4 promoter's CpG islands might be one of the mechanisms for poor expression of RSK4 in PTC, which may serve as a molecular target of early diagnosis and treatment.
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Objective The purpose of this study is to investigate the molecular mechanisms of p.C310R(c.T928C) and p.E396V(c.A1187T) lipoprotein lipase(LPL) gene mutations in vitro, which may help to construct the spectrum of LPL gene mutations and phenotype. It also can provide accurate early diagnosis for high-risk population of familial hypertriglyceridemia and provide the basis for the development of gene targeted therapy. Methods Genomic DNA was extracted from proband′s family members′ peripheral blood cells and screened by whole-exome sequencing to verify candidate gene variations. PCR products were afterwards directly sequenced again to confirm corresponding LPL variants. At the cellular level, lentiviruses containing LPL mutations were constructed and then transfected into COS-1 cells. Functional significance of the mutants was corroborated by analyzing LPL activity and mass in the cell medium and lysates via ELISA and enzyme-fluorescent method. mRNA was assayed by RT-PCR to confirm the effect on gene transcription. Results DNA sequence analysis revealed that the proband was a heterozygote for a novel c.T928C mutation in exon 6 of LPL gene, while his nephew was a compound heterozygote for the c.T928C mutation in exon 6 and a novel c.A1187T mutation in exon 8. In vitro studies, these two mutations can cause decreased activity and mass of extracellular LPL(P<0.05). Moreover, further investigation indicated that LPL C310R mutation tremendously affected post-transcriptional modification of LPL gene, whereas LPL E396V mutation dampened intracellular LPL trafficking. Conclusion Both the mutations are pathogenic by reducing the activity and mass of LPL in the plasma, which affected normal metabolism of triglycerides.