Effects Of Casticin On Stemness Of Liver Cancer Stem-like Cells Derived From MHCC97-H Cells And The Expression Of MiR-139-5p

Objective: To illuminate the mechanism by which casticin(CAS)inhibits the stemness of MHCC97-H-derived liver cancer stem-like cells(LCSLCs)by regulating the expression of miR-139-5p.Methods:1.MHCC97-H cells were cultured in vitro.The MHCC97-H cell line-derived spheroid cells were obtained by sphere formation culture method and used as a model of MHCC97-H derived LCSLCs.Flow cytometry,sphere formation and clonogenic assays,wound healing and transwell cell invasion assays,q RT-PCR as well as Western blot were used to analyze the CD24+,CD44+,CD90+ cell percentage,self renewal ability,migration and invasion ability,stem cell surface markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein expression levels of MHCC97-H cells and spheroid cells.q RT-PCR was used to detect the expression levels of miR-139-5p in L-02 cells,MHCC97-H cells and LCSLCs.2.miR-139-5p mimic(50n M)or inhibitor(100n M)were used to transfect LCSLCs to manipulate the expression level of miR-139-5p,then determined the effect of miR-139-5p on LCSLCs stemness.3.Taking DMSO as a negative(solvent)control and Apatinib(5.0 μM)as a positive control,LCSLCs were treated with different concentrations of CAS(1.0,2.5,5.0 μM)for 24 h to analyze the effect of CAS on the LCSLCs stemness.4.miR-139-5p mimic(50n M)combined with CAS(5.0 μM)were used to treat LCSLCs,and then detected the LCSLCs stemness.Results:1.Compared with MHCC97-H cells,MHCC97-H cell line spheroid cells have higher positive rate of CD24+,CD44+,CD90+,the sphere size was larger and the number were increased by nearly 5 times,the number of soft agar colonies were 5 times higher,the migration rate 4 times higher,6.7 times higher invasion rate,higher expression levels of stem cell surface markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein,can be used as LCSLCs model derived from MHCC97-H cells;The expression level of miR-139-5p was3.2 times lower than that of its parent cells and 7 times that of L-02 cells(P<0.05).2.miR-139-5p mimic transfection increased the miR-139-5p expression level of LCSLCs by 25 times,reduced the sphere formation rate by 3.8times and soft agar colony formation rate by 4.8 times,inhibited the migration rate by 3.3 times and the invasion rate by 5.8 times,Significantly downregulated the expression levels of stem cell surface markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein(P<0.05).3.The transfection of miR-139-5p inhibitor reduced the expression level of miR-139-5p of LCSLCs by 2 times,increased the sphere formation rate by 1.4 times and soft agar colony formation rate by 1.3 times,increased the migration rate by 2.5 times and the invasion rate by 2 times,Up-regulated the expression levels of stem cell surface markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein(P<0.05).4.CAS and Apatinib preferentially inhibit the proliferation of LCSLCs derived from MHCC97-H cells in a dose-dependent manner;CAS reduced the LCSLCs sphere formation rate and soft agar colony formation rate in a dose-dependent manner,inhibited migration and invasion rates,and downregulated the surface of stem cells Markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein expression levels(P<0.05).5.CAS can regulate the miR-139-5p expression level of LCSLCs in a dose-dependent manner;CAS combined with miR-139-5p mimic transfection can further reduce sphere formation rate and soft colony formation rate,inhibit migration rate and invasion rate,downregulate the expression levels of stem cell surface markers CD24,CD44,CD90 and stem-related factors ABCG2,Nanog,Ep CAM m RNA and protein of LCSLCs(P<0.05).Conclusion:CAS can upregulate miR-139-5p expression to inhibit the stemness of LCSLCs derived from MHCC97-H cells.