| Recently, the studies have shown that as a new stem cell marker, Lgr5 is an important component of signaling pathways common with Wnt/?-catenin, and also plays an important role in the regulation of biological activity of tumor stem cells. However, there is rare report on the research of this pathway and gastric cancer stem cells. Previous studies showed that the expression of key protein of this pathway is higher in gastric cancer cells than that in normal gastric cells. And Lgr5 was positively correlated with the malignant degree of gastric cancer and microvessel density. Therefore, whether Lgr5-Wnt/?-catenin pathway is involved in the regulation of biological activity of gastric cancer stem like cells, and whether by regulating the expression of related proteins to maintain the malignant biological behavior of gastric cancer stem like cells still need further study. This experiment wants to observe the changes of biological characteristics and expression of related target genes of gastric cancer stem like cells after interference by blocking the pathway through RNAi technology. We used the methods of CCK8, annexinV labeling, flow cytometry to comprehensive analyse proliferation, apoptosis and angiogenesis, oxygen metabolism and other biological behavior changes of gastric cancer stem like cells. We want to find a new target of gastric cancer stem like cells, and lay the foundation for the future biological treatment of gastric cancer.Due to the lack of specific molecular markers of gastric cancer stem cells, therefore, the cells each institute obtained precisely should be enriched gastric cancer stem like cells. Now, CD44 is a generally accepted molecular marker of gastric cancer stem cells. However, the stem cell characteristics of gastric cancer suspended sphere cells ssorted by CD44 alone are bad, and do not have the obvious advantage. So we urgently need a new molecular marker to improve the properties of cancer stem cells separated from normal cancer cells. Although CD24, CD 133 and Tff2 play some role, but they are all lack of specificity. Recently, CD54 is considered to be the molecular marker of gastric cancer stem cells. CD44+CD54+ gastric cancer cells have higher ability of tumor formation and cell proliferation. This provides a condition for improving the gastric cancer stem like cell enrichment.In this study, we first performed the purification of gastric cancer stem like cells. We randomly selected four kinds of human gastric cancer cell lines (AGS, BGC-823, MKN-28, MKN-45) to culture suspended sphere cells, and the experimental group CD44+CD54+ suspended sphere cells and control group CD44+CD54-suspended sphere cells were selected by flow cytometry after stable subculture. We analyze the differences between the experimental group and the control group by suspended sphere formation, plate clone, proliferation, resistance to chemotherapy drugs and tumorigenicity experiments. The results show that the CD44+CD54+ suspended sphere cells possessing the properties of cancer stem cells is stronger, and they are higher enrichment degree gastric cancer stem like cells. Next, the function of Lgr5 in gastric cancer stem like cells was studied. RT-PCR results suggest that the expression of Lgr5 in gastric cancer stem like cells was significantly higher than that of normal gastric cells. Further study found that, after Lgr5 interference lentivirus were stably transfected into gastric cancer stem like cells, the suspended sphere forming ability, plate clone ability, proliferation ability, resistance to chemotherapy drug ability and nude mice tumorigenicity, were all obviously decreased. At the same time, the expression of mRNA and protein of ?-catenin, Ki-67, EphA3, EphrinA5 and so on were detected by QT-PCR and western blotting. The results also showed that the expression level of these proteins is consistent with the expression level of Lgr5. All these show that Lgr5-Wnt/?-catenin signaling pathway plays an important role in maintenance the biological characteristics of gastric cancer stem like cells. |
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