| Objective: Culturing of human Colorectal cancer(CRC)HCT116 suspension sphere Cells was used to identify its feasibility as a research model for cancer Stem Cells(CSCs),and to observe the effect of autophagy on its tumorigenicity and other biological characteristics.Based on colorectal cancer HCT116 suspension sphere cells,and to explore the relationship between oxaliplatin(OXA)and the stem expression,tumorigenicity and apoptosis of colorectal cancer stem cells(CRC-CSCs),and to discuss the role of autophagy in this process,so as to provide a more novel theoretical basis for the treatment of CRC.Methods:(1)To explore the stem characteristics of HCT116 sphere cells and their relationship with autophagy.1)HCT116 cells in CRC were selected for in vitro culture,and serum-free culture method was used to culture HCT116 suspension sphere cells.2)HCT116 cells were used as the control group,To explore the characteristics of HCT116 sphere cells and detect the occurrence of autophagy.then,The tumorigenicity of the cells was tested by clone formation and nude mice tumorigenesis experiments,and the invasiveness of the cells was detected by Transwell invasion experiment.The expression of CD133,SOX-2 and LC3 B was detected by Real-time PCR and Western Blot,respectively.(2)To explore the effect of OXA on the stem expression,tumorigenic ability and apoptosis of CRC-CSCs-Likecells and autophagy.1)HCT116 sphere cells were treated with OXA(5?g/ml?10?g/ml?20?g/ml?40?g/ml?80?g/ml)for 48 h.CCk-8 assay was used to detect the effect of OXA on the proliferation activity of its.And half inhibitory rate(IC50)was calculated and appropriate concentration was selected for the subsequent experiments.2)Three experimental groups were established on the basis of HCT116 sphere cell model,including normal group(without any drug treatment),OXA treatment group(OXA: 22 ug/ml),OXA + 3-M(3-methyladenine,3-methyladenine)group(OXA: 22 ug/ml + 3-M: 5 mm Mol/L).The tumorigenicity of tumor cells was detected by clone formation assay,and apoptosis was detected by flow cytometry.Real-time PCR and Western Blot were used to detect the expression ofSOX-2,LC3 B and Caspase-3.Results:(1)The stem-like biological characteristics and autophagy expression of HCT116 sphere cells1)HCT116 sphere cells had a strong tumorigenic ability,and the number of clonal colony formation increased significantly(154.7 4.63 VS 41 1.53,P<0.05).The tumorigenic rate(90% VS 55%)and tumor volume [(298 85)mm3 VS(92 31)mm3] of nude mice were significantly increased(P<0.05).HCT116 cells had a strong invasion ability,and the number of cells penetrating the basement membrane was significantly increased(28.50 0.56 VS 10.66 2.16,P<0.05).2)The results of RT-PCR and Western Blot showed that the expression of CSCs-specific marker CD133 and stem specific expression gene SOX-2 in HCT116 sphere cells were highly(P<0.05),Meanwhile,the expressions of autophagy specific related genes LC3 B increased simultaneously,and the difference was statistically significant(P<0.05).(2)The relationship between the effect of OXA on the stem expression,tumorigenic ability and apoptosis of HCT116 sphere cells and autophagy1)The results of CCK-8 cell proliferation experiment showed that OXA could inhibit the growth and proliferation of HCT116 sphere cells in a concentration-dependent manner.The half inhibition concentration of OXA was 21.87 ug/ml.2)The results of flow cytometry showed that,the percentage of apoptotic cells in normal group,OXA treatment group and OXA+3-M combination group were 14.79±0.92%,26.26±1.55% and 37.13±1.32%,respectively.Compared with the normal group,the apoptotic rate of OXA group was significantly higher(P<0.05),while the apoptosis rate was further increased in the OXA+ 3-M combined group(P<0.05).3)Sphere-forming experiment showed that,the number of newly formed cell spheres in normal group,OXA treatment group and OXA+3-M combination group were 3.03±0.25,7.16±0.31,3.83±0.30.Compared with the normal group,the number of newly formed cell spheres in OXA treatment group was relatively increased,while in OXA + 3-M combination group,the ability of cell sphere formation was significantly decreased(P<0.05).4)The results of colony formation experiment showed that the number of colony forming in the blank group,OXA group,and OXA+3-M group were 85.67±5.60,175.72±8.70,and 77.67±7.71,respectively.Compared with the normal group,the number of clonal colonies formed in OXA group was significantly higher,but the number of clonal colonies decreased after adding autophagy inhibitors,the difference was statistically significant(P < 0.05).5)Real-time PCR results showed that,after OXA treatment,mRNA expression levels of theSOX-2,LC3 B and caspase-3 were significantly up-regulated(P<0.05),while mRNA expression levels of SOX-2 and LC3 B were relatively inhibited after OXA+ 3-M treatment,while mRNA expression levels of Caspase-3 were further increased,with statistically significant differences(P<0.05).6)Western Blot results showed that,consistent with RT-PCR results,the protein expression levels of SOX-2,LC3 B and caspase-3 in HCT116 globule cells in the OXA group were increased(P<0.05),while the protein expression levels of SOX-2 were down-regulated after the addition of autophagy inhibitor 3-M,and the Caspase-3 protein expression continued to increase,the difference was statistically significant(P<0.05).Conclusions:(1)HCT116 suspension sphere cells have obvious CSCs-like biological characteristics and can be used as an ideal model for the study of CRC-CSCs.(2)The high stem characteristics of HCT116 sphere cells is closely related to the high activity of autophagy,suggesting that autophagy may promote the malignant degree of CRC-CSCs.suggesting that autophagy may promote the malignancy of CRC-CSCs and maintain their stem expression.(3)After the OXA treatment,it may promote the enrichment of CRC-CSCs.(4)Autophagy may enhance the drug resistance of CRC-CSCs-like cells to OXA by inhibiting OXA-induced apoptosis and promoting the maintenance of stem characteristic expression. |
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