| Background and PurposeChina is the high incidence country of esophageal cancer in the word.Although the esophageal cancer patients can be treated by Surgery, radiotherapy and chemotherapy treatments,but the survival rate of the patients with esophageal cancer is only about 30% in 5 years.The invasion and metastasis of esophageal cancer is the main reason for low survival rate of the patients,therefore,it has become the hotspot in this field.EMT(epithelial- mesenchymal transition, EMT) plays an important role in the process of tumor invasion and metastasis. Epithelial cells transiting into mesenchymal cells is a key step in the embryonic development of multicellular organisms and it determines the structure characteristics of organ development, And now the more research focused on the invasion and metastasis of tumor.Elizabeth had done a detailed description about EMT and clarified the main differences of the mechanism of EMT in embryonic development and tumor formation for the first time at the EMT meeting in Vancouver.In the process of tumor epithelial cells invasion and metastasis,after undergo EMT, the expression of epithelial markers decreased,such asE-cadherin and cytokeratin etc, the expression of Vimentin, α muscle actin(α-smooth muscle actin, SMA) which are the interstitial markers increased,with these signs of the expression changing,it makes the epithelial cells lose cell polarity,cell adhesion capacity decreased,tumor cell invasion and migration capacity enhance,thus contributing to tumor cells fall off from the primary site,the formation of the local invasion, and metastasize to distant sites.Cancer stem cells(CSCs) are non-specialized cells have the ability of self-renewal and multiple differentiation potential, they are rare in tumor tissues.At present, CSCs Has been sorting out in a variety of solid tumors,and confirmed:they have a high tumorigenic ability and can drive tumor formation, promote the tumor metastasis and are associated with chemotherapy tolerance.So Researchers widely believe :CSCs may play an important role in tumor origin and metastasis and will become the target to cure cancer.p75 NTR also said the low affinity neurotrophin receptor p75,and confirmed:p75NTR+ cells separation from normal esophageal tissue and esophageal cancer cell line that with stem cell characteristics significantly.At present, the researchers have used p75 NTR as a marker for sorting normal esophageal epithelial stem cell / progenitor cell.Hypoxia is a ubiquitous phenomenon in solid tumors.Previous studies showed that: the characteristics of cancer cell itself are the main reason resulting in the invasion and metastasis of tumor, while ignoring the important role played by the tumor microenvironment.Modern research shows that tumor invasion and metastasis are the final result of the interactions between the tumor cells and the tumor microenvironment.HIF-1(Hypoxia-inducible factor-1) is a heterodimer which is composed of HIF-1α and HIF-β.it is considered the primary transcription factor which regulates cells responsing to hypoxia.Under hypoxia the combination of HIF-1α and HIF-β within the cell nucleus can form a heterodimer,then,with the hypoxic response elements of the promoter or enhancer in the target gene began to combine and start the expression of downstream gene.Researchs show that: hypoxia and hypoxia signaling pathway can promote the occurrence and development of tumor via regulating the ability of surviving,the invasion and metastasis of tumor cells,the angiogenesis in tumor tissue and the chemotherapy drug tolerance.Researches also show that: in the process of tumor metastasis,after EMT,the epithelial tumor cells not only increase the migration and invasion ability,but also change in the metabolism, epigenetics and differentiation etc.Differentiated epithelial tumour cells can be reversed by EMT as an undifferentiated state tumour cells which can not only express stem cell markers,but also can obtain the characteristics of stem cell.More important,hypoxia and hypoxia signaling pathway play a crucial role in the regulation of CSCs and EMT.,further influence the progression and prognosis of tumor patients.Therefore, this is an important research topic to explore the influence of hypoxia microenvironment and hypoxia signaling pathway on tumor invasion and metastasis.This study was to study the role of hypoxia on the formation of cancer stem cells and the epithelial mesenchymal transition in the esophageal squamous carcinoma cells,and further elucidate the mechanism and the possible factors of EMT in esophageal squamous cell carcinoma.Part I Establishment of cellular hypoxia model for esophageal squamous carcinoma cells and the effect of the hypoxia on HIF-1α expression.MethodsHuman ESCC cell line Eca-109 was incubated in medium with a final concentration of 150μmol/L Co Cl2 to simulate hypoxic environment, and cultured for different period of time(4h,8h,12 h,24h,36h). Eca-109 cell line was incubated in medium without Co Cl2 as the normoxia control.1. The changes in morphology and growth status of cultured cells were observed under an inverted microscope to select the optimal time for Co Cl2 action. 2. Western blotting was used to examine the effect of hypoxia on expression of HIF-1α protein in Eca-109 under normoxia or hypoxia at 4h, 8h, 12 h, 24 h and 36 h. 3. Real-time PCR was used to examine the effect of hypoxia on expression ofHIF-1α m RNA in Eca-109 under normoxia or hypoxia at 4h, 8h, 12 h, 24 h and 36 h.Result1. Change in morphology: Compared with the cells in normoxia group, cells in hypoxia group showed broken connection between cells, gradually increased number of spindle cells and floating dead cells during incubation time course. The hypoxia group with 24 h incubation showed more dead cells than the group with 12 h incubation, but less than the group with 36 h incubation. In order to avoid toxic effect of Co Cl2, 24 h incubation was chosen as the optimal time for subsequent hypoxia experiments. 2. Western blotting findings: The results showed that the expression level of HIF-1α protein was 0.550 ± 0.011 in normoxia group, but increased to 0.941 ± 0.002 in hypoxia at 4 h, 1.070 ± 0.018 at 8h, 1.079 ± 0.032 at 12 h, 1.103 ± 0.025 at 24 h, and 0.984 ± 0.014 at 36 h. The difference in expression level of HIF-1α protein between normoxia group and each hypoxia group was statistically significant(P <0.05). 3. Real-time PCR finding: The results showed that the expression level of HIF-1α m RNA was 1.000 ± 0.066 in normoxia group, decreased in hypoxia groups to 0.631 ± 0.113 at 4h and 0.612 ± 0.150 at 8h, but increased in hypoxia groups 2.612 times to 2.612 ± 0.250 at 12 h, 3.948 times to 3.948 ± 0.930 at 24 h, and 3.052 to 3.052 ± 1.350 at 36 h. The difference in expression level of HIF-1α m RNA between normoxia group and each hypoxia group was statistically significant(P <0.05).Part II Hypoxia promotes the epithelial mesenchymal transition and the formation of cancer stem cells in esophageal squamous carcinoma cells.Methods1. Western blotting was used to detect protein expression level of HiF-1α,E-cadherin, Vimentin, Oct-4 and Nanog in Eca-109 cells in normoxia and hypoxia groups. 2. Immunofluorescence was used to detect of expression level of Nanog protein in Eca-109 cells in normoxia and hypoxia groups. 3. Real-time PCR was used to detect m RNA expression level of Hi F-1α, E-cadherin, Vimentin, Oct-4 and Nanog in Eca-109 cells in normoxia and hypoxia groups. 4. Transwell invasion chamber assay was used to evaluate the invasion ability of Eca-109 cells in normoxia and hypoxia groups. 5. Flow Cytometry was used to detect the proportion of esophageal cancer stem cells(p75NTR+) among Eca-109 cells in normoxia and hypoxia groups. Mous Ig G1-PE antibody was used as negative control.Result1. Western blot finding: The results showed that the protein expression in Eca-109 cells was 0.437 ± 0.022 for Hi F-1α,0.637 ± 0.087 for E-cadherin,0.823 ± 0.037 for Vimentin,0.876 ± 0.003 for Oct-4, and 0.582 ± 0.004 for Nanog in normoxia group. However, in hypoxia groups the protein expression in Eca-109 cells increased to 0.970 ± 0.060 for Hi F-1α, 1.240 ± 0.020 for Vimentin, 1.156 ± 0.034 for Oct-4, and 1.163 ± 0.042 for Nanog, but decreased to 0.168 ± 0.048 for E-cadherin. The difference in protein expression between anormoxia and hypoxia groups was statistically significant(P<0.05). 2. Immunofluorescence findings: Nanog protein expression in Eca-109 cells was markedly enhanced in hypoxia group when compared with that in normoxia group. 3. Real-time PCR findings: The results showed that the m RNA expression of Hi F-1α,Vimentin,Oct-4,and Nanog were 1 ± 0.228, 1 ± 0.214,1 ± 0.249,1 ± 0.333 in Eca-109 cells in normoxia group, respectively, but increased 2.5 times to 2.500 ± 0.150, 2.185 times to 2.185 ± 0.070, 2.674 times to 2.674 ± 0.364, and 3.305 times to 3.305 ± 0.327 in hypoxia group, respectively.The m RNA expression of E-cadherin was 1 ± 0.345 in normoxia group, but decreased to 0.123 ± 0.058 in hypoxia group. The difference in m RNA expression level between normoxia and hypoxia groups was statistically significant(P<0.05). 4. Flow Cytometry finding:In the the normoxia group and the hypoxia group, the proportion of p75NTR+ cells were 2.00 ± 0.327% and 2.74 ± 0.137%, respectively, The difference between two groups was statistically significant(P<0.05). These results suggest the presence of a small number of physical p75NTR+ cells in Eca-109, which significantly increased under hypoxia condition. 5. The results of Transwell in vitro invasion assay showed that the number of invaded cells was 51±11.769 in normoxia group, but increased to 118±17.875 in hypoxia group. The difference between two groups was statistically significant(P<0.05). The results suggest that hypoxia can dramatically enhance in vitro invasive ability of Eca-109 cells.Conclusions1. Hypoxia can induce epithelial to mesenchymal transition of ESCC cells. 2. Hypoxia can enhance in vitro invasion ability of ESCC cells 3. Hypoxia can induce the formation of cancer stem cells in ESCC cells. |
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