Study Of The Stem Cell Of P75^NTR-positive Human Esophageal Squamous Cell Carcinoma

Esophageal tumor stem cells （ETSC） is a small part of esophageal tumortissue with self-renewal， unlimited proliferation and multi-differentiationcapacity in tumor cells. While ETSC has small proportion in the tumor tissue,it is closly related with tumor origin, development and metastasis. Low-affinityneurotrophin receptor （p75NTR）, a member of tumor necrosis factorsuperfamily, has been shown to paradoxically mediate neuronal survival anddifferentiation or apoptotic cell death, depending on the environment of thecells. In previous studies, p75NTRwas used for screening neural crown stemcells. p75NTRwas also used for screening rat fat multipotential stem cells,and human corneal epithelial progenitor cells. Okumura et al reported thatp75^NTR+esophageal epithelial cells were stem cells due to their capacity ofproliferation, self-renewal and multidirectional differentiation.This experiment use flow cytometry to sort esophageal squamous cellcarcinoma with p75NTRas the mark. Then, study the biological characteristicsand the differences in gene expression of the p75^NTR+and p75NTR-esophagealsquamous carcinoma cells.After that,using one of the differences gene（EZH2）as the aim gene, we investigate the influence of aim gene down-regulation byRNA interference on the proliferation and invasion of stem cell of p75^NTR+human esophageal squamous cell carcinoma.Part1Biological characteristics of p75^NTR+esophagealsquamous carcinoma cellsObjective: To study the biological characteristics of p75^NTR+and p75NTR-cells that were separated by fluorescence-activated cell sorting, and toinvestigate whether p75^NTR+cells have the characteristics of cancer stem cells.Methods: To get p75^NTR+and p75NTR-of human esophageal squamouscarcinoma cells by fluorescence-activated cell sorting, to assess theirinvasiveness, tumorigenic ability, the resistance of chemotherapy drugs and toexplore whether the p75^NTR+ of human esophageal squamous carcinomacells have the tumor stem cells properties. Results: We success to separate p75^NTR+of human esophageal squamouscarcinoma cells.Comparing p75^NTR+cells with p75^NTR-cells, we find thatp75^NTR+cells possess higher capacity of cloning than p75^NTR-cells. p75^NTR+cells possess higher proliferation ability than p75^NTR-cells. Also p75^NTR+cellswere much stronger than p75^NTR-cells both in the resistance to chemotherapydrugs and tumorigenic ability in mice.Conclusions: Comparing to p75^NTR-cells, p75^NTR+cells possess higheraggressive, chemotherapy-resistant and tumorigenic capacity. So we supposethe stem cells of human esophageal squamous carcinoma cells may beenriched in p75^NTR+cells.Part2Separating the differences gene expression of p75^NTR+and p75^NTR-esophageal squamous carcinoma cellsObjective: To study the differences in gene expression of p75^NTR+andp75^NTR-esophageal squamous carcinoma cells, in order to investigate stemcell-related gene expression of p75^NTR+cells and provide clues for studying thepathogenesis of esophageal tumor and looking for new therapeutic targetsfrom the perspective of cancer stem cells theory.Methods: Using Human Genome U133Plus2.0Array （Affymatrix）,weseparate the differences gene expression of p75^NTR-and p75^NTR+esophagealsquamous carcinoma cells.Then compare stem cell-related geneexpression（Bmi-1and EZH2） of p75^NTR+and p75^NTR-esophageal squamouscarcinoma cells by RT-PCR.Results: Comparing to p75^NTR-cells, p75^NTR+cells highly express Bmi-1andEZH2. We have initially screened12genes related with TGF-β and Wntsignaling pathway which closely related with the characteristics of stem cellsaccording to the biological function classification results.Conclusions: Comparing to p75^NTR-cells, p75^NTR+cells highly express thegenes closely related with the characteristics of stem cells, such as Bmi-1andEZH2. This further confirme that p75^NTR+cells isolated from ESCC have thecharacteristics of cancer stem cells. These results provide clues for looking for targeted therapy of esophageal squamous cell carcinoma stem cells.Part3Effects of EZH2-siRNA on Proliferation and Invasiveness ofp75^NTR+Esophageal Squamous cell CarcinomalObjective: Using one of the differences gene（EZH2） as the aim gene,weinvestigate the influence of EZH2gene down-regulation by RNA interferenceon the proliferation and invasion of stem cell of p75^NTR+human esophagealsquamous cell carcinoma.Methods: Small interfering RNA （siRNA） targeting to EZH2was transfectedinto p75^NTR+human esophageal squamous cell carcinoma by the means oflipofectamin2000.The proliferation capcity of p75^NTR+cells was determinedby calculating the cell population doubling time、the cell cycle、the cellcloning and the percentage of apoptotic cells.The invasive ability of p75^NTR+cells was detemined by Transwell assay.Results: p75^NTR+human esophageal squamous cell carcinoma weresuccessful transfected with small interfering RNA （siRNA） targeting to EZH2.In vitro experiment showed that，compared with the control groups，p75^NTR+cells transfected with EZH2-siRNA had significantly longer cell populationdoubling time， lower percentage of cell cloning， higher cell apoptotic rate，and lower cell invasion rate.Conclusions: Our date shows that EZH2siRNA can reduce the proliferationand invasion of P75NTR+human esophageal squamous cell carcinoma，which suggests that EZH2siRNA might be a potential gene therapertic targetof the stem cell of human esophageal squamous carcinoma.