| BackgroundGastric cancer remains one of the most common malignant tumors that threaten the life of residents in China. In recent years, with the development of endoscopic techniques, early diagnosis rate is improved; the comprehensive treatment technology, mainly based on surgery, reduces local recurrence rate. However, the five-year survival rate of gastric cancer is still less than 30%. Invasion, metastasis, and drug resistance are still main causes of treatment failure. Against this backdrop, researchers are attaching increasing importance to stem cell theory. Characterized by self-differentiation, self-proliferation, and resistance to chemo-radiotherapy, cancer stem cells are considered as the root cause of tumor recurrence and metastasis. Many studies have shown that Hedgehog-Glil signaling pathway plays an important role in maintaining the biological characteristics of various cancer stem cells. Gli1 is the nodal gene of the signaling pathway. Investigators are exploring how to cure malignant tumor by inhibiting certain signaling pathways, but the effect is not satisfing. Therefore, in-depth study of the proliferation, invasion and metastasis, and drug mechanisms of gastric cancer stem cell-like cells is of great importance for improving the prognosis of patients with gastric cancer, enhancing the effect of chemotherapy and gene therapy,and providing some clues and ideas for clinical development of drugs targeted at cancer cells featuring clinical drug resistance and high incidence of metastasisObjectiveWe aimed to investigate the expression pattern of Glil in stem cell-like gastric cancer cells, elucidate the relationship between Gli1 and the proliferation, invasion and drug resistance of stem cell-like gastric cancer cells, discuss the mutual regulation between Glil and other signaling pathways, and explore the value of lentiviral vector-based gene therapy in treatment of gastric cancer.Methods1. Stem cell-like cancer cells were cultivated and identified in suspension cultures for sphere-forming. Flow cytometry, proliferation experiment, and in vivo tumorigenesis experimentwere conducted the markers and characterizations for cancer stem cells.2. Gli1 expression was down-regulated by siRNA in stem cell-like gastric cancer cells, and the knockdown of Glil was verified by qPCR and Western-blot.3. Before and after downregulating Glil:Inspected changes in proliferation of stem cell-like gastric cancer cells through clonony-forming efficiency experiment and CCK8 experiment; inspected the impact of downgrading Glil gene to migration of stem cell-like gastric cancer cells through Transwell experiment; inspected changes in chemotherapy tolerance of stem cell-like gastric cancer cells through CCK8 experiment; analyzed how SMO antagonist cyclopamine and sh-Glil-RNA differ from each other in their impact on the apoptosis of stem cell-like gastric cancer cells through apoptosis test by flow cytometry; inspected the impact of Glil gene knockdown on vivo tumorigenesis of stem cell-like gastric cancer cells in vivo through mice tumorigenesis experimen4. ZNRF3 expression:investigated the variation relationship between ZNRF3 and Glil through qPCR and Western-blot, and explore their mutual regulation relationship; inspected the impact of the downregulation on the proliferation and apoptosis of gastric cancer cells and normal mucosal cell through Annexin/PI; inspected the relationship between the downgrade with the apoptosis of Bcl-2, Ki-67, etc. and the mutual regulation relationship with proliferation through qPCR and Western-blot; inspected the expression of ZNRF3 and Glil in primary gastric cancer cells, and observed if the relationship is consistent with the expression in cell lines.Results1. Through suspension sphere culturing, proliferation experiment, and in vivo tumorigenesis experiment,we identified CD44+CD54+ as the marker of gastric cancer stem cells.CD44+CD54+gastric cancer cells are side population cells which are enriched by gastric cancer stem cells.2. After knockdown Gli1 in stem cell-like gastric cancer cells, the results of suspension sphere experiment, qPCR and Western-blot tests all indicate Gli1 expression is down-regulated, demonstrating high transfection efficiency.3. After knockdown Gli1:Cloning efficiency experiment and CCK8 experiment showed proliferation of stem cell-like gastric cancer cells is falling; Transwell experiment showed downgrading Glil gene can reduce migration of stem cell-like gastric cancer cells; CCK8 experiment showed downregulating Glil gene can reduce the chemotherapy tolerance of stem cell-like gastric cancer cells; Apoptosis test showed sh-Glil-RNA has a better effect than cyclopamine in lowering the apoptosis of stem cell-like gastric cancer cells; Mice tumorigenesis experiment showed downgrading Glil reduces vivo tumorigenesis of stem cell-like gastric cancer cell.4. After ZNRF3 overexpression:qPCR and Western-blot find Glil varies less, indicating that ZNFR3, the regulator gene of WNT signaling pathway, could increase the apoptosis of gastric cancer cells and mucous cells by suppression Glil gene.Annexin/PI inspection found that upgrading ZNRF3 can increase the proliferation and apoptosis of gastric cancer cells and mucosal cells. The impact to gastric cancer cells was more obvious; qPCR and Western-blot indicated ZNRF3 could lower the expression of Bcl-2 and Ki-67; in primary gastric cancer cell test, ZNRF3 was negatively related to Glil expression, which was consistent with the expression in cell lines.Conclusions1. Glil is involved in regulating such biological behaviors as the self-proliferation, drug resistance and vivo tumorigenesis of stem cell-like gastric cancer cells.2. Down-regulating Glil expression can effectively suppress the proliferation, invasion, chemotherapy resistance and animal tumorigenicity of stem cell-like gastric cancer cells.3. Hedgehog signaling pathway can be regulated by tumor suppressor gene ZNRF3. Up-regulating ZNFR3 can decrease the expression of Gli1, thereby increasing the apoptosis of stem cell-like gastric cancer cells.4. Gli1 plays an important role in the proliferation, invasion, drug resistance and vivo tumorigenesis of stem cell-like gastric cancer cells. Therefore, Gli1 can serve as a therapeutic target. Shh-Gli1-RNA lentivirus vector, as a way to treat gastric cancer, is worthy of further development.5. ZNRF3 can reduce the expression of Gli1, while changes in the expression of Gli1 gene can regulate the proliferation, invasion, migration and drug resistance of stem cell-like gastric cancer cells; Thus, we might speculate that ZNRF3 can affect the biological behaviors of stem cell-like gastric cancer cells by changing the expression of Gli1. It is worth further study. |
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