The Role And Mechanism Of Hsa＿circ＿0000670 Derived From Exosomes Obtained From Gastric Cancer Stem Cells In The Development Of Gastric Cancer Induced By Cigarette Smoke

Objective: To investigate the role and molecular mechanisms of cigarette smoke in promoting the development of gastric cancer through exosomes of gastric cancer stem cells,and to detect the biological mechanisms of hsa＿circ＿0000670 in this process.Methods: Screen the highly expressed circRNA in gastric cancer by analyzing GEO database data.q RT-PCR was used to detect the expression level of hsa＿circ＿0000670 in gastric cancer tissues of smokers.The gastric cancer stem cells culture model was constructed;the formation of gastric cancer stem cells spheres were observed under microscope.Western-blot and q RT-PCR were used to detect mRNA and protein expression of stemness genes OCT4,NANOG,SOX2 and LIN82 in gastric cancer stem cells obtained from SGC-7901 and HGC-27.Cell viability test was used to screen the concentration of cigarette suspension.The changes of the size and number of gastric cancer stem cells spheres were observed by microscope after treated with 0.5% cigarette smoke extract(CSE)and the changes of the expression of stemness genes in gastric cancer stem cells were detected by q RT-PCR and Western-blot.Exosomes secreted by gastric cancer stem cells before and after 0.5%CSE treatment were extracted,and their size,particle size,uantity,morphology and surface markers were characterized by NTA,TEM and Western-blot.Western-blot,transwell migration assay and matrigel invasion assay were used to detect the changes of stemness genes,EMT progress-related genes and the migration and invasion ability of gastric cancer cells,obtained from MKN-45 and HGC-27,treated with 0.5%CSE and exosomes secreted by two kinds of gastric cancer stem cells.The expression level of hsa＿circ＿0000670 in gastric cancer stem cells and exosomes before and after 0.5% CSE treatment were detected by q RT-PCR.Gastric cancer stem cells were transfected with overexpression lentivirus and the transfection efficiency of overexpression hsa＿circ＿0000670 was tested.The changes of the size of gastric cancer stem cell spheres after transfection were observed under microscope.Western-blot was used to detect the changes of stemness genes and EMT-related genes of gastric cancer stem cells before and after treated with the virus.Results: The expression level of hsa＿circ＿0000670 in gastric cancer tissues of smokers was upregulated.CSE can significantly promoted the self-renewal of gastric cancer stem cell balls and the mRNA and protein expression of stemness genes OCT4,NANOG,SOX2 and LIN28.Exosomes secreted by gastric cancer stem cells before and after 0.5%CSE treatment were observed by transmission microscope,all of which were round or elliptical membranous vesicles with diameter of about110 nm.Western-blot results revealed that exosomes expressed the characteristic surface markers of exosomes CD9,CD63,CD81 and there was no significant change in the number of two exosomes.Compared with the control group,the exosomes secreted by gastric cancer stem cells upregulated the expression of stemness genes and EMT process-related genes of gastric cancer stem cells and gastric cancer cells and promoted the migration and invasion ability of gastric cancer cells.q RT-PCR results showed the expression of hsa＿circ＿0000670 was upregulated in gastric cancer stem cells and exosomes secreted by them after 0.5% CSE treatment.The expression level of hsa＿circ＿0000670 was significantly upregulated and the self-renewal ability of gastric cancer stem cells was improved after transfecting lentivirus into gastric cancer stem cells.Western-blot results showed that over-expression of hsa＿circ＿0000670promoted the expression of stemness genes of gastric cancer stem cells and EMT process and CSE facilitated this process.Conclusion: CSE can upregulate the stemness of gastric cancer stem cells and promote EMT process and migration and invasion ability of gastric cancer cells through the exosomes.Hsa＿circ＿0000670 played an important role in this.