Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

Overexpressed GATA-4 bone marrow-derived mesenchymal stem cell exosomes improve myocardial infarction via miRNA-673-5p/Tsc-1 molecular axis / 医学研究生学报

ObjectiveExosomes secreted by BMSC overexpressing GATA-4 gene (BMSCGATA-4-exosome) can promote the differentiation of BMSC into cardiomyocyte-like cells, thereby improve cardiac function after myocardial infarction. However, the molecular mechanism of BMSCGATA-4-exosome in cardiomyocyte-like cell differentiation is unknown. The effect of the secretion of BMSCGATA-4 exosome from bone marrow mesenchymal stem cells (BMSC) in the differentiation of stem cells into cardiomyocytes was determined in miRNA-673-5p/Tsc-1 axis dependent manner.MethodsMouse models of myocardial infarction were established and divided into seven groups. Simulation group (BMSCmiR-673-5p-mimic exosome), inhibition group (BMSCmiR-673-5p-inhibitor exosome), GATA-4 group (BMSCGATA-4 exosome), empty vector group (BMSCempty vector exosome）, and BMSC group (BMSC exosome) were injected into the tail vein for 48 h, and the untreated and normal mice were used as the control group. Cardiac ultrasound was used to detect cardiac function in each group. miRNA-673-5p expression in myocardial infarction was detected using real-time polymerase chain reaction (RT-PCR). The myocardial tissues were extracted from the same myocardial infarction site. Myocardial-specific molecules, such as α-actin, Desmin, cTnT, and Cx43, were detected using RT-PCR. Western blot was used to determine the expression of the corresponding target gene of miRNA-673-5p, Tsc-1, Erk1/2, and Mef2c proteins.ResultsThe simulation group wan shown the most significantly improved myocardial function (P<0.05) with an expression peak of miRNA-673-5p in cardiomyocytes (P<0.05). The highest content of myocardial-specific molecules including α-actin, Desmin, cTnT, and Cx43 was found in the simulation group. The simulation group had the lowest expression of Tsc-1 in cardiomyocytes (P<0.05).ConclusionOverexpressed BMSCGATA-4 exosomes inhibit Tsc-1 expression through miRNA-673-5p to improve cardiac function during myocardial infarction.

Gata-4-overexpressed bone marrow mesenchymal stem cell secreted exosomes promote bone marrow mesenchymal stem cell differentiation into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND: Preliminary study has shown that overexpression of GATA-4-overexpressing bone marrow mesenchymal stem cell (BMSCs) exosomes (BMSCsGATA-4-exosome) can promote BMSCs differentiate into cardiomyocytes, indicating it can repair myocardial infarction. Additionally, high expression of miRNA-673-5p is observed in miRNA-673-5p BMSCsGATA-4-exosome and focal myocardium of myocardial infarction, which involve in cell differentiation, suggesting that miRNA-673-5p may be a key molecular of BMSCsGATA-4-exosome for repairing myocardial infarction. OBJECTIVE: To investigate the molecular regulatory network of BMSCsGATA-4-exosomes that promotes BMSCs differentiation into cardiomyocyte-like cells. METHODS: miR-673-5p-mimic was added to the BMSCs culture system as an experimental group (BMSCsmiR-330-3p-mimic ). BMSCsGATA-4, BMSCsGATA-4-empty vector, BMSCs and BMSCsGATA-4-miR-673-5p-inhibitor groups were set as confounding factor control groups. The exosomes and myocardial cells secreted by each group were co-cultured for 24 hours. The expression levels of myocardium specific molecules α-actin, Desmin, cTnT and Cx43 were detected by immunofluorescence and RT-PCR. The expression levels of the corresponding miRNA-673-5p target genes TSC-1, ERK1/2 and Mef2c were detected through western blot assay based on the prediction results of the microRNA target gene. RESULTS AND CONCLUSION: The BMSCsmiR-673-5p-mimic-exosome+BMSCs culture group had the highest α-actin, Desmin, cTnT and Cx43 levels (P < 0.05), and the lowest TSC-1 expression (P < 0.05). In summary, BMSCsGATA-4-exosome inhibits the expression of TSC-1 via miRNA-673-5p to promote BMSCs differentiation into cardiomyocyte-like cells.

Genetic analysis of a pedigree affected with tuberous sclerosis complex caused by a novel mutation of TSC1 / 中华医学遗传学杂志

Objective@#To analyze the clinical phenotype of a Chinese pedigree affected with Tuberous sclerosis complex(TSC) and explore pathogenic mutations of TSC1 and TSC2 gene.@*Methods@#Unique clinical phenotypes, the results of imaging, examination of the proband and special family history, collectively, made the constellation of features of TSC.Genomic DNA was obtained from six affected and eight unaffected members of the family and potential mutations of the TSC1 and TSC2 genes were detected by PCR-amplification of the exons and exon-intron boundaries and direct sequencing.A total of 150 normal unrelated individuals were used as controls.@*Results@#Genetic analysis documented the presence of a heterozygous mutation, c. 1781_1782delTG (p.Val594GlyfsX11), in the exon 15 of TSC1 gene within all the patients of the family. This mutation was not observed in the eight unaffected family members or in the 150 unrelated control subjects from the same population , or the Human Gene Mutation Database(HGMD)and had completely co-segregated with the disease phenotype in the family.@*Conclusions@#The c. 1781_1782delTG mutation of TSC1 gene may be responsible for the tuberous sclerosis complex in this family. The data presented in the present study are of significance to clinicians, as well as genetic counselors, and may provide new clues for molecular diagnosis of this disease.

Tsc1/Tsc2 complex: A molecular target of capsaicin for protection against testicular torsion induced injury in rats / 中草药·英文版

Objective: The detailed knowledge about protective effects of capsaicin (cap)and involved mechanisms against testicular torsion (TT)is still not available completely. Methods: Male Wistar rats were assigned into four major cohorts: (i)sham, (ii)TT, (iii)three subgroups subjected to TT and different doses of cap (100, 500, and 1000 µg/mL), and (iv)three subgroups of healthy animals subjected to various concentrations of cap. The animals were decapitated at 24 h after reperfusion, and the evaluation of protein expression was performed by Western blotting assay. At 72 h after reperfusion, apoptotic cell death and tissue injury were evaluated by TUNEL nuclear and H&E staining, respectively. Results: The results showed that cap administration following TT significantly increased the expression of tuberous sclerosis proteins 1 and 2 (Tsc1/Tsc2)in a dose-dependent manner (P < 0.05). Cap decreased cell apoptosis at highest dose. Likewise, cap contributed to the preservation of tubular morphology and decreased tissue injury at the highest tested concentration (1000 µg/mL). Conclusion: Collectively, our findings demonstrate the validity of cap as a therapeutic agent against TT through targeting Tsc1/Tsc2 in a dose-dependent manner.

Striatal Inhibition of MeCP2 or TSC1 Produces Sociability Deficits and Repetitive Behaviors

Autism spectrum disorder (ASD) is a heterogeneous group of neurobehavioral disorders characterized by the two core domains of behavioral deficits, including sociability deficits and stereotyped repetitive behaviors. It is not clear whether the core symptoms of ASD are produced by dysfunction of the overall neural network of the brain or that of a limited brain region. Recent studies reported that excessive glutamatergic or dopaminergic inputs in the dorsal striatum induced sociability deficits and repetitive behaviors. These findings suggest that the dorsal striatum plays a crucial role in autistic-like behaviors. The present study addresses whether functional deficits of well-known ASD-related genes in the dorsal striatum also produce ASD core symptoms. This study also examines whether these behavioral changes can be modulated by rebalancing glutamate and/or dopamine receptor activity in the dorsal striatum. First, we found that the siRNA-mediated inhibition of Shank3, Nlgn3, Fmr1, Mecp2, or Tsc1 in the dorsal striatum produced mild to severe behavioral changes in sociability, cognition, and/or repetitive behaviors. The knockdown effects of Mecp2 and Tsc1 on behavioral changes were the most prominent. Next, we demonstrated that behavioral changes induced by striatal inhibition of MeCP2 and TSC1 were rescued by D-cycloserine (an NMDA agonist), fenobam (an mGluR5 antagonist), SCH23390 (a D1 antagonist), and/or ecopipam (a D1 partial antagonist), pharmacological drugs that are known to regulate ASD-like symptoms in animal models. Collectively, these results suggest that the dorsal striatum is a critical brain region that, when dysfunctional, produces the core symptoms of ASD.

TSC1 and TSC2 gene mutations and their implications for treatment in Tuberous Sclerosis Complex: a review

Abstract Tuberous sclerosis complex is an autosomal dominant disorder characterized by skin manifestations and formation of multiple tumors in different organs, mainly in the central nervous system. Tuberous sclerosis is caused by the mutation of one of two tumor suppressor genes, TSC1 or TSC2. Currently, the development of novel techniques and great advances in high-throughput genetic analysis made mutation screening of the TSC1 and TSC2 genes more widely available. Extensive studies of the TSC1 and TSC2 genes in patients with TSC worldwide have revealed a wide spectrum of mutations. Consequently, the discovery of the underlying genetic defects in TSC has furthered our understanding of this complex genetic disorder, and genotype-phenotype correlations are becoming possible, although there are still only a few clearly established correlations. This review focuses on the main symptoms and genetic alterations described in TSC patients from 13 countries in three continents, as well as on genotype-phenotype correlations established to date. The determination of genotype-phenotype correlations may contribute to the establishment of successful personalized treatment for TSC.

A sporadic case of tuberous sclerosis complex caused by a newly found heterozygous missense mutation of c.T1967C in TSC1 gene / 上海交通大学学报（医学版）

This paper reported a sporadic case of a 29-year-old Han female diagnosed with tuberous sclerosis complex (TSC) by next generation sequencing (NGS),one of genetic analysis techniques.She was admitted because of recurrent intractable seizure for 26 years,dizziness and headache for 3 months.Physical examination revealed angiofibromas over her face,shagreen patches in her lower back area,and hypomelanotic macules around her limbs and body.Cranial MRI manifested lesions on lateral ventricles,cerebellar vermis and left temporal lobe with abnormal signal changes on both sides of extensive cerebral cortex.A pathogenic and heterozygous missense mutation,c.T1967C,in exon 16 of her TSC1 gene was found via genetic tests,which has not yet been reported before.

Relationship between gene mutation and clinical phenotype in patients with tuberous sclerosis complex / 中华神经科杂志

Objective To study the relationship between gene mutation and clinical phenotype in patients with tuberous sclerosis complex (TSC).Methods The clinical data of 76 patients with TSC diagnosed in Guangdong 999 Brain Hospital were collected between May 2007 and May 2014 and then TSC gene mutation analysis was performed.Genotype-phenotype analyses for all the patients were also carried out.Results Fifty of the 76 (66％) patients were male,and 26 (34％) were female,in which 19 (31％) patients presented with cyst-like cortical tuber,69 (92％) with skin lesions,16 (30％) with renal lesions,50 (69％) with mental retardation and 39 still suffered seizures after a year.In this study,22 (29％) cases showed TSC1 gene mutation,31 (59％) presented TSC2 gene mutation,and 15 (20％)cases had no mutation identified.The mutation ratio of TSC1 ∶ TSC2 was approximately 3 ∶ 5,while the mutation ratio of TSC1 ∶ TSC2 was 1 ∶ 1 for familial TSC patients,and 1 ∶ 2 for sporadic TSC patients.Comparing to those with TSC1 gene mutation and no mutation identified,patients with TSC2 gene mutation exhibited statistical meaning on the aspects of the onset age of seizure (Z =1.688,P =0.007),seizure onset before l-year-old (x2 =10.584,P =0.001),epilepsy duration (x2 =4.996,P =0.025),spasms onset (x2 =10.111,P =0.001),cyst-like cortical tuber (x2 =9.182,P =0.002),skin lesions (x2 =9.016,P =0.003),as well as renal lesions (x2 =6.079,P =0.014).No apparent relation was found between genotype and intelligence outcome.Conclusions The patients with TSC2 gene mutations presented severer symptoms in seizure onset than those with TSC1 gene mutation and no mutation identified.The patients with TSC2 gene mutation were characterized by early onset of seizure,especially before 1-year-old,others like spasms onset,cyst-like cortical tuber,skin lesions,as well as renal lesions being more vulnerable.Therefore,more active treatment should be given to the patients with TSC2 gene mutation.

Association study between polymorphisms of TSC1, TSC2 ,PTEN genes and autism in Chinese Han population / 中华行为医学与脑科学杂志

Objective To explore the association between the polymorphisms of TSC1,TSC2,PTEN genes and autism in Chinese Han population.Methods 274 autism patients and 386 heahh controls were recruited,and SnaPshot technique was used to genotype the 13 tagSNPs of TSC1,TSC2 and PTEN genes.The allele,genotype and haplotype frequencies of the SNPs were compared using SHEsis and SNPStats softwares.Results Mter Bonferroni correction,the allele distribution of rs2809244 (TSC1) (x2 =9.537,P=0.002,adjusted P=0.016),rs1050700 (TSC1) (x2 =9.313,P=0.002,adjusted P=0.016),rs2072314(TSC2) (P＜0.01,adjusted P＜0.01) and rs8063461 (TSC2) (P＜0.01,adjusted P＜0.01)showed significant difference between two groups (P＜0.05).The genotype frequencies of rs2072314(TSC2)and rs8063461(TSC2) showed significant difference between two groups(P＜0.05).Moreover,the frequency of haplotype A-G (OR =14.548,95％ CI =5.450-38.830) in the haplotype block rs2809244-rs3761840 showed significant difference between two groups(P＜0.05),A-G significantly increases the risk of autism.The frequencies of haplotype A-A (OR=0.608,95％ CI =0.409-0.903,P=0.013),G-A (OR=7.812,95％ CI =5.338-11.459,P＜0.01)and G-G (OR=0.356,95％ CI =0.274-0.463,P＜0.01) in the haplotype block rs2074969-rs8063461 were identified,which were significant difference between two groups(P＜0.05),and AA and G-G significantly reduced but G-A increased the risk of autism.Conclusion The polymorphisms of TSC1 and TSC2 genes might associate with autism in Chinese Han population.

Role of the PI3K-AKT-mTOR signalling in cell differentiation / 基础医学与临床

Phosphatidylinositol-3-kinase(PI3K),AKT,TSC1/2,and the mammalian target of rapamycin(mTOR) signaling cascade involves many cellular processes including apoptosis,growth,proliferation and differentiation,This pathway has been found to be the most variable one in human tumors.As tumor is a poorly differentiated disease,this review examines the role of the PI3K-AKT signal pathway in cell differentiation and tumor development.