Researches On The Effects Of Omentin-1 On The Proliferation And Apoptosis Of Colon Cancer Stem Cells And Mechanism

Objectives To study the effects of omentin-1 on the proliferation and apoptosis of colon cancer stem cells,to observe the effects of omentin-1 on the PI3K/Akt signaling pathway in colon cancer stem cells and whether there is a synergistic effect between omentin-1 and PI3K/Akt signaling pathway inhibitor LY294002,and then discuss the possible mechanism of omentin-1 acts on colon cancer stem cells.Methods1.Colon cancer stem cells were separated from colon cancer cell lines SW480 by indirect immunomagnetic beads method,and then colon cancer stem cells were cultured in serum-free suspension medium.2.Identification of colon cancer stem cells:spheres formation assay to detect colon cancer stem cells into spheres,differentiation of colon cancer stem cells was detected by differentiation assay,and the surface marker CD133 of colon cancer stem cell was detected by flow cytometry.3.CCK-8 assay:(1)Colon cancer stem cells were divided into Control group(without omentin-1 and LY294002),Omentin1 group(add 1?g/ml omentin-1),Omentin2group(add 2?g/ml omentin-1),Omentin LY group(add 1?g/ml omentin-1 and 50?M LY294002),LY group(add 50?M LY294002),the OD values were detected after 24 hours of treatment.(2)To detecte the OD values after 0h,1h,6h,24 h and 48 h of the cells intervened by 1ug/ml Omentin-1.4.Colon cancer stem cells were divided into Control group(without omentin-1 and LY294002),Omentin1 group(add 1?g/ml omentin-1),Omentin2 group(add 2?g/ml omentin-1),Omentin LY group(add 1?g/ml omentin-1 and 50?M LY294002),LY group(add 50?M LY294002),after 24 hours of intervention,flow cytometry was used to detect the apoptosis in different groups.5.Colon cancer stem cells were divided into Control group(without omentin-1 and LY294002),Omentin1 group(add 1?g/ml omentin-1),Omentin2 group(add 2?g/ml omentin-1),after 24 hours of the cells intervened by different concentrations of omentin-1,the cell proteins were extracted and the expression of Akt and p Akt protein was detected by Western blot.Results:1.Colon cancer stem cells were suspended growth in serum-free medium,small stem cell spheres were observed on the first day,obvious stem cell spheres were observed on the fifth day,then on the tenth day the spheres further increase and became round.Differentiation assay show that the stem cell spheres all differentiated into colon cancer cells SW480 of spindle shape and adherent growth in serum-containing medium to ninth day.Flow cytometry analysis showed that CD133+ in colon cancer stem cells reached 80.3%.2.The OD values of Omentin1 group,Omentin2 group,Omentin LY group and LY group were all decreased compared with Control group,the differences were statistically significant(P<0.05).The OD value of Omentin2 group was lower compared with Omentin1 group,the difference was statistically significant(P<0.05).The OD value of Omentin LY group was lower than that of Omentin1 group and LY group,the difference was statistically significant(P<0.05).3.After 0h,1h,6h,24 h and 48 h of the colon cancer stem cells intervened by1?g/ml Omentin-1,with the increase of intervention time,the OD values decreased gradually,the differences were statistically significant(P<0.05).4.The apoptosis rates of Omentin1 group,Omentin2 group,Omentin LY group and LY group were elevated compared with Control group,the differences were statistically significant(P<0.05).The apoptosis rate of Omentin2 group was higher compared with Omentin1 group,the difference was statistically significant(P<0.05).The apoptosis rate of Omentin LY group was higher than that of Omentin1 group and LY group,the difference was statistically significant(P<0.05).5.The results of Western blot showed that the expression of p Akt/Akt protein decreased after intervened by omentin-1,Omentin2 group showed statistical significance compared with control group(P<0.05).Conclusion:1.Omentin-1 inhibits the growth of colon cancer stem cells,the effect is concentration-and-time-dependent.2.Omentin-1 promotes the apoptosis of colon cancer stem cells,the effect is concentration-dependent.3.The mechanism of omentin-1 acts on colon cancer stem cells may be related to the inhibition of Akt pathway.4.There is a synergistic effect between omentin-1 and LY294002 on the proliferation and apoptosis of colon cancer stem cells.