Extracellular vesicle-carried GTF2I from mesenchymal stem cells promotes the expression of tumor-suppressive FAT1 and inhibits stemness maintenance in thyroid carcinoma / 医学前沿

Through bioinformatics predictions, we identified that GTF2I and FAT1 were downregulated in thyroid carcinoma (TC). Further, Pearson's correlation coefficient revealed a positive correlation between GTF2I expression and FAT1 expression. Therefore, we selected them for this present study, where the effects of bone marrow mesenchymal stem cell-derived EVs (BMSDs-EVs) enriched with GTF2I were evaluated on the epithelial-to-mesenchymal transition (EMT) and stemness maintenance in TC. The under-expression of GTF2I and FAT1 was validated in TC cell lines. Ectopically expressed GTF2I and FAT1 were found to augment malignant phenotypes of TC cells, EMT, and stemness maintenance. Mechanistic studies revealed that GTF2I bound to the promoter region of FAT1 and consequently upregulated its expression. MSC-EVs could shuttle GTF2I into TPC-1 cells, where GTF2I inhibited TC malignant phenotypes, EMT, and stemness maintenance by increasing the expression of FAT1 and facilitating the FAT1-mediated CDK4/FOXM1 downregulation. In vivo experiments confirmed that silencing of GTF2I accelerated tumor growth in nude mice. Taken together, our work suggests that GTF2I transferred by MSC-EVs confer antioncogenic effects through the FAT1/CDK4/FOXM1 axis and may be used as a promising biomarker for TC treatment.

FAT1 Gene Alteration in Facioscapulohumeral Muscular Dystrophy Type 1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array. Recent studies revealed that the FAT1 expression is associated with disease activity of FSHD, and the FAT1 alterations result in myopathy with a FSHD-like phenotype. We describe a 59-year-old woman with both contracted D4Z4 repeat units and a FAT1 mutation. Shoulder girdle muscle weakness developed at the age of 56 years, and was followed by proximal leg weakness. When we examined her at 59 years of age, she displayed asymmetric and predominant weakness of facial and proximal muscles. Muscle biopsy showed increased variation in fiber size and multifocal degenerating fibers with lymphocytic infiltration. Southern blot analysis revealed 8 D4Z4 repeat units, and targeted sequencing of modifier genes demonstrated the c.10331 A>G variant in the FAT1 gene. This FAT1 variant has previously been reported as pathogenic variant in a patient with FSHD-like phenotype. Our study is the first report of a FAT1 mutation in a FSHD1 patient, and suggests that FAT1 alterations might work as a genetic modifier.

FAT1 inhibits cell proliferation of esophageal squamous cell carcinoma through regulating the expression of CDK4/CDK6/CCND1 complex / 中华肿瘤杂志

Objective@#To explore the expression of FAT1 in esophageal squamous cell carcinoma (ESCC) tissues, and its effect on cell proliferation.@*Methods@#The expression levels of FAT1 protein in human ESCC tissues and matched adjacent normal tissues were determined by immunohistochemistry (IHC). Lentivirus based knockdown of FAT1 was carried out in YSE2 and Colo680N cell lines and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assays was performed to examine the effect of FAT1 on the proliferation of these ESCC cells. Colony formation assay was used to detect the colony formation ability. Flow cytometry was performed to analyze the cell cycle and apoptosis. The expression levels of cell cycle markers in FAT1 knock out ESCC cell lines were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot.@*Results@#The relative expression of FAT1 in ESCC tissues was 66.97±21.53, significantly lower than 78.13±16.76 of adjacent normal tissues(P<0.05). Knockdown of FAT1 promoted cell proliferation and colony formation. In YSE2 cell, the division time in negative control (NC) group was (1 570±51) min, significantly longer than (1 356±31) min in shFAT1 group. In Colo680N cell, division time in NC group was (1 532±53) min, significantly longer than (1 290±30) min in shFAT1 group (P<0.05). Knockdown of FAT1 promoted G1-to S-phase transition and resulted in the upregulation of CDK4/CDK6/CCND1.@*Conclusion@#FAT1 inhibits the proliferation and G1-to S-phase transition of ESCC cells through regulating the protein expression of CDK4/CDK6/CCND1 complex.

Fusion to Mistic improved fatty acid desaturase Fat-1 overexpression in E .coli / 重庆医学

Objective To construct a highly efficient expression plasmid of eukaryotic nuclear membrane protein Omega 3 fatty acid desaturase gene Fat‐1 in E .coli .Methods Using molecular cloning technology to construct the recombinant prokaryotic expression plasmid pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1 fused with Membrane proteins expression chaperon mistic ;the two re‐combinant plasmids were transformed into E .coli strain BL21 (DE3) ,the expression of Fat‐1 protein and M110 Fat‐1 protein in‐duced by IPTG were identified by SDS‐PAGE and gray degree analysed the amount of expression ,further identified by Western blot .Results The results of enzyme digestion and sequencing demonstrated that we successfully constructed the prokaryotic ex‐pression vectors pET32a Fat‐1 and pET32a‐Mistic‐Fat‐1;SDS‐PAGE and Western blot showed that Fat‐1 fatty acid desaturase wasn′t significantly induced ,but the overexpression of M110 Fat‐1 fusion protein was obtained in E .coli ,accounting for 15% of the total amount of whole cell proteins .Conclusion The fusion with Mistic proteins to express the Fat‐1 gene has realized the overex‐pression of eukaryotic nuclear membrane integrated protein Omega 3 fatty acid desaturase in prokaryotic host .

Omega-3 Polyunsaturated Fatty Acids May Attenuate Streptozotocin-Induced Pancreatic beta-Cell Death via Autophagy Activation in Fat1 Transgenic Mice

BACKGROUND: Inflammatory factors and beta-cell dysfunction due to high-fat diets aggravate chronic diseases and their complications. However, omega-3 dietary fats have anti-inflammatory effects, and the involvement of autophagy in the etiology of diabetes has been reported. Therefore, we examined the protective effects of autophagy on diabetes using fat-1 transgenic mice with omega-3 self-synthesis capability. METHODS: Streptozotocin (STZ) administration induced beta-cell dysfunction in mice; blood glucose levels and water consumption were subsequently measured. Using hematoxylin and eosin (H&E) and Masson's trichrome staining, we quantitatively assessed STZ-induced changes in the number, mass, and fibrosis of pancreatic islets in fat-1 and control mice. We identified the microtubule-associated protein 1A/1B light chain 3-immunoreactive puncta in beta-cells and quantified p62 levels in the pancreas of fat-1 and control mice. RESULTS: STZ-induced diabetic phenotypes, including hyperglycemia and polydipsia, were attenuated in fat-1 mice. Histological determination using H&E and Masson's trichrome staining revealed the protective effects of the fat-1 expression on cell death and the scarring of pancreatic islets after STZ injection. In the beta-cells of control mice, autophagy was abruptly activated after STZ treatment. Basal autophagy levels were elevated in fat-1 mice beta-cells, and this persisted after STZ treatment. Together with autophagosome detection, these results revealed that n-3 polyunsaturated fatty acid (PUFA) enrichment might partly prevent the STZ-related pancreatic islet damage by upregulating the basal activity of autophagy and improving autophagic flux disturbance. CONCLUSION: Fat-1 transgenic mice with a n-3 PUFA self-synthesis capability exert protective effects against STZ-induced beta-cell death by activating autophagy in beta-cells.

Inhibitory effect of fat-1 gene on the proliferation of colon cancer cell HT-29 / 解放军医学杂志

Objective To investigate the effect of fat-1 gene encoding n-3 fatty acid desaturase on the proliferation and apoptosis of colon cancer cell HT-29. Methods fat-1 gene was transfected into HT-29 cells by liposomal reagent. The expression of fat-1 gene was detected by fluorescent micrographs and RT-PCR. Gas chromatography, MTT and flow cytometry were used to examine the change in n-6/n-3 PUFAs ratio, proliferation, cell cycle and apoptosis, respectively. Results After transfection of fat-1 gene, n-6/n-3 PUFAs ratio decreased significantly. Apoptosis of HT-29 cells was induced and cell cycle was changed. Apoptosis mainly appeared in the synthesis phase. Conclusion fat-1 gene encoding n-3 fatty acid desaturase can significantly decrease n-6/n-3 ratio. Consequently, apoptosis was triggered and cell cycle was changed. Tranfection of fat-1 gene into HT-29 cells may be a new potential treatment for colon cancer.