Yangzheng Sanjie Decoction Regulates Self-renewal And Differentiation Of Gastric Cancer Stem Cells

ObjectiveGastric cancer is one of the common malignant tumors.Its distant metastasis,postoperative recurrence,chemotherapy resistance and radiotherapy resistance are believed to be related to Gastric Cancer Stem Cells(GCSCs),and targeted GCSCs is a reasonable and effective treatment for gastric cancer.Traditional Chinese medicine treatment is the characteristic in China for the treatment of gastric cancer,which has unique advantages in the prevention and treatment of gastric cancer.Our previous study confirmed that Yangzheng Sanjie Decoction(YZSJD)could inhibit the proliferation and induce the apoptosis of gastric cancer cells through let-7a.On this basis,this study further explored the regulation effect of YZSJD on the self-renewal and differentiation of GCSCs and its mechanism.Methods1.GCSCs were isolated from human gastric cancer cell line SGC-7901 by serum-free suspension culture.2.Levels of "stemness" markers were measured by Western Blot.Spheroid cells continuously subculture experiment and spheroids formation experiment were used to detect the self-renewal capacity of GCSCs.Differentiation potential of GCSCs was detected by immunofluorescence and flow cytometry.GCSCs were identified through these experiments.3.After the intervention of YZSJD in vitro,effect of YZSJD on expression of "stemness" markers was detected by Western Blot,then effect of YZSJD on self-renewal ability of GCSCs was detected by spheroids formation experiment and effect of YZSJD on differentiation potential of GCSCs was detected by immunofluorescence experiment as well as flow cytometry.4.Real-time fluorescence quantitative PCR was used to detect the expression of let-7a of parental cells and spheroid cells.After treated by YZSJD,effect of YZSJD on expression of let-7a of GCSCs was detected by real-time fluorescence quantitative PCR.Besides,Western Blot was used to detect the effect of YZSJD on expression of c-Myc and Ras,the downstream targets of let-7a.Results1.Human gastric cancer cell line SGC-7901 could grow in suspension under serum-free medium as well as low adhesion culture plate and form spheroids after about 3-5 days.With the extension of culture time,the volume of spheroids increases and the number of cells contained in a single spheroid increased.2.Spheroid cells were able to continuously subculture in serum-free medium.Then spheroid cells were separated into single cells for subculture for several times,spheroids formed again has clearer boundary,denser structure and more regular sphere shape.Compared with parental cells,sphere formation rate of GCSCs was higher(P<0.05).The expressions of CD44(P<0.01),Oct-4(P<0.01),Nanog(P<0.01)and Sox-2(P<0.05),"stemness" markers of GCSCs,were significantly increased.After adherent culture in serum-containing medium for 24 h,48 h and 72 h,the expression of CK18 of GCSCs,one of differentiation markers,gradually increased(P<0.01).3.After intervention of YZSJD,the expressions of CD44(P<0.01),Oct-4(P<0.01),Nanog(P<0.01)and Sox-2(P<0.01),"stemness" markers of GCSCs,were significantly decreased,as well as the sphere formation rate(P<0.05).Reversely,the expression of differentiation marker CK18 was significantly increased(P<0.01).4.Compared with parental cells,the expression of let-7a of GCSCs was significantly decreased(P<0.01).After treatment of YZSJD,expression of let-7a in GCSCs was significantly increased(P<0.01),and expression of its downstream targets,c-Myc(P<0.01)and Ras(P<0.05),were significantly decreased.ConclusionYZSJD can up-regulate the expression of let-7a and inhibit the expression of its target genes,c-Myc and Ras.Besides,YZSJD can inhibit the self-renewal of GCSCs and promote their differentiation,which may be one of the mechanisms for YZSJD to play an anti-cancer role.