81641099 and No. to the ID1 promoter in ECA109 and TE1 cells. Quantification of immunoprecipitated DNA was shown by qRT-PCR. (E) Correlation of expression levels of LEF1 and ID1 in 98 patients (left panel) and total 129 patients (right panel) are shown. (PDF 238 kb) 13046_2019_1296_MOESM1_ESM.pdf (238K) GUID:?650C6AEA-877D-47B7-9E28-76BECA14459B Additional file 2: Table S1. Correlation between LEF1 expression and clinicopathological characteristics in 243 patients. Table S2. Correlation between LEF1 expression and clinicopathological characteristics in total 338 Ombitasvir (ABT-267) patients. (DOCX 21 kb) 13046_2019_1296_MOESM2_ESM.docx (25K) GUID:?918FB662-9CB6-482D-A054-4AA8400199FE Data Availability StatementAll data generated or analyzed during this study are included in this published article. Abstract Background Esophageal squamous cell carcinoma (ESCC) is the most difficult subtype of esophageal cancer to treat due to Rabbit Polyclonal to BAZ2A the paucity of effective targeted therapy. ESCC is usually believed to arise from cancer stem cells (CSCs) that contribute to metastasis and chemoresistance. Despite advances in diagnosis and treatment, the prognosis of ESCC patients remains poor. Methods In this study, we applied western blot, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, RNA-Seq analysis, luciferase reporter assay, Chip-qPCR, bioinformatics analysis, and a series of functional assays to show the potential role of LEF1 in regulating esophageal CSCs. Results We found that the overexpression of LEF1 was associated with aberrant clinicopathological characteristics and the poor prognosis of ESCC patients. In addition, the elevated expression of LEF1 and OV6 was significantly associated with aberrant clinicopathological features, and poor patient prognosis. Moreover, the overexpression of LEF1 was observed in esophageal CSCs purified by the magnetic sorting of adherent and spheroidal ESCC cells. The increased level of LEF1 in CSCs facilitated the expression of CSC markers, stem cell-like properties, resistance to chemotherapy, and tumorigenicity and increased the percentage of CSCs in ESCC samples. Conversely, the knockdown of LEF1 significantly diminished the self-renewal properties of ESCC. We showed that LEF1 played an important mechanical role in activating the TGF- signaling pathway by directly binding to the ID1 gene promoter. A positive association between LEF1 and ID1 expression was also observed in clinical ESCC samples. Conclusion Our results indicate that the overexpression of LEF1 promotes a CSC-like phenotype in and the tumorigenicity of ESCC by activating the TGF- signaling pathway. The inhibition of LEF1 might therefore be a novel therapeutic target to inactivate CSCs and inhibit tumor progression. Electronic supplementary material The online version of this article (10.1186/s13046-019-1296-7) contains supplementary material, which is available to authorized users. plasmid using lipofectamine 2000 reagent (Thermo Fisher, USA, No.11668019). Luciferase and signals were measured 48?h after transfection by a Dual-Luciferase Reporter Assay Kit (Promega, No. E1980). Data were normalized by the division of Ombitasvir (ABT-267) firefly luciferase activity with that of luciferase to eliminate transfection efficiency difference. Chromatin immunoprecipitation (ChIP) assays We identified the LEF1-bingding sites on ID1 promoter region by using JASPAR and also referred to Chip-Seq data of LEF1 on GEO. ChIP assay was conducted with SimpleChIP? Enzymatic Chromatin IP Kit (CST, 9003) following the manufacturers instructions. Briefly, ECA109 and TE1 cells (4??106) were cross-linked by using 1% formaldehyde and used for each immunoprecipitation experiment. Chromatin was digested with the micrococcal nuclease. 2% aliquots of lysates were used as an input reference. LEF1 antibody (Abcam, ab137872) or normal rabbit IgG (CST, 2729) were incubated with the other immunoprecipitation samples at 4?C for overnight. Then, the crosslink DNA was reversed by NaCl and proteinase K. Immunoprecipitated DNA was amplified by PCR using their specific primers. The primer sequences for ID1 gene were 5-CGCCCGCTTTAAATTTCGG-3 (forward), and 5- CACAGATGAGAGAAA. TTGAGGC ??3 (reverse). The signals were calculated as the percentage of input. Statistical analysis SPSS Ombitasvir (ABT-267) 22 software (SPSS, Chicago, IL, USA) was used to statistically analyse the data. The association between markers and clinical features were analysed by chi-square test, Fishers exact test or two-side t-test. Spearmans rank correlation was used to analyse the association between LEF1 and OV6 expression. Survival curves were analysed by using the Kaplan-Meier method. Multivariate analysis of survival was examined by Cox proportional hazard regression model. The experimental data were obtained in three independent experiments and analysed by ANOVA..
- Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking solution made up of 4% normal goat serum and 0
- When gated about viable immune cells in the tumor, the annexin V+ single-positive cells are distinct from caspase-3/7+ or apoptotic cells (Figure 1B)
- Miller 1995b)
- Each dot represents one patient, and bars indicate mean with SEM