| Objectives:The present study aimed to investigate the alterations and effects of TP63α and Sonic Hedgehog pathway in NDMA-induced liver cancer stem cells activity.Furthermore,we illustrate the suppression impact of DATS on the stemness of liver CSCs.The discovery and exploration could make a significant contribution to expand the strategies targeting liver CSCs.Methods:The effect of NDMA on cell viability of LO2 cell was examined by MTT assay.To establish the neoplastic transformed model in vitro,LO2 cells were exposed to different concentrations of NDMA for 30 generations.Turmorsphere formation assy in serum-free(SFM)was utilized to isolate and enrich liver CSCs.We observed the morphology change under microcope.The growth rate was detected with the CCK-8 assay.The malignant degree of LO2 cells were detected by soft agar clone formation assay and plate clone formation assay.The Transwell and wound-healing experiment were applied to detect the ability of invasion and metastasis.Western blot analysis was used to measure the expression levels of EMT related proteins,liver CSC markers,TP63α,and Sonic Hedgehog pathway regulators.To ensure the effects of TP63α in liver CSCs,recombinant plasmid and siRNA approach were conducted to upregulate or downregulate TP63α.To clarify the impact of Sonic Hedgehog pathway on liver CSCs,the Sonic Hedgehog pathway inhibit was utilized to supress the activation of Sonic Hedgehog.Liver CSCs were treated with different concentrations of DATS to explore the DATS interventional effect.The size and number of turmorspheres were observed under microcope.Meanwhile,the expression levels of liver CSC markers,P63α,and Sonic Hedgehog pathway were examined by Western blot.After transfection with TAP63α plasimid or ΔNp63α-siRNA,tumorspheres were treated with DATS and then Western blot analyse the levels of liver CSC markers,TP63α,and Sonic Hedgehog pathway,aimed to determin the TP63α/Sonic Hedgehog axis mediation DATS suppression of liver CSCs.Results:1.Chronic NMDN exposure induces LO2 cells malignant transformationAccording to the results of MTT assay,we chose three adequate concentrations of NDMA(0,0.1 μg/mL,1 μg/mL)to expose to LO2 cells for 30 generations,which were respectively named CLO2,TLO2-Ⅰ,TLO2-Ⅱ.We obviously observed the morphology change of TLO2-Ⅱ cells compared to other groups.The morphology transform of TLO2-Ⅱ cells from circular,pebble shaped to more spindle,funicular like.Additionally,TLO2-Ⅱ cells proliferated faster than other groups.Furthermore,TLO2-Ⅱ cells could form clone spheres like Huh7,while CLO2,TLO2-Ⅰ cells nearly form few clone spheres.The Transwell experiment also demonstrated TLO2-Ⅱ cells owned stronger invasion and metastasis ability,which were paralled with the results of Western blot analysis.In general,TLO2-Ⅱ cells were successfully malignant transformated by chronic NDMA exposure,and obtained tumor-like characteristics.2.Isolation and enrichment of liver CSCs from TLO2-Ⅱ cellsIt was shown that TLO2-Ⅱ cells could significantly form tumorspheres compared with CLO2 cells in serum-free medium(SFM)culture.Western blot analysis also illustrated TLO2-Ⅱ cells overexpressed liver CSCs markers(CD133,ALDH1A1,Oct4,Nanog).The results informed we successfully isolated and enriched liver CSCs from TLO2-Ⅱcells.3.TP63α mediates NMDN-induced malignant transformation of LO2 cells to liver CSCsWestern blot analysis showed TAp63α was upregulated and ΔNp63α was downregulated both in adherent and tumorspheres cells.TAp63α overexpression orΔNp63α lowexpression could enhance the size and number of turmorspheres as well as the expression of liver CSCs markers.These results sμggested the role of Tp63α in NDMA-induced malignant transformation of LO2 cells to liver CSCs,in which TAp63α promote the progress of malignant transformation from LO2 cells to liver CSCs while ΔNp63α prevent the progress.4.Sonic Hedgehog pathway maintains the stemness of TLO2-Ⅱ liver CSCsWe measured the levels of shh,Smo,Glil and Gli2 in TLO2-Ⅱ liver CSCs by Western blot.The results showed the activation of Sonic Hedgehog pathway.After treatment with Sonic Hedgehog pathway inhibitor GDC-0449,the ability of tumorspheres formation and the expression levels of liver CSCs markers were also inhibited,which indicated Sonic Hedgehog pathway maintains the stemness of TLO2-Ⅱ liver CSCs5.TAp63α regulates Sonic Hedgehog pathway in TLO2-Ⅱ liver CSCsOverexpression of TAp63α elevated the levels of Sonic Hedgehog pathway related moleculars(shh,Smo,Glil and Gli2).In line with this,when the expression of TAp63α was inhibited,the associated proteins in Sonic Hedgehog pathway were suppressed.By contrast,upregulation or downregulation of ΔNp63α,the variety of Sonic Hedgehog pathway was opposite.These results illustrated that Tp63α regulates Sonic Hedgehog pathway in TLO2-Ⅱ liver CSCs.6.The inhibitory effects of DATS on TLO2-Ⅱ liver CSCs via TP63α/Sonic Hedgehog aixsTLO2-Ⅱ liver CSCs were handled with different concentrations of DATS(0,25μM,50 pM,75 μM).We observed that the size and number of turmorspheres were effectively reduced and the liver CSCs markers were markedly suppressed.These results revealed that DATS diminished the activity of TLO2-Ⅱ liver CSCs.It was found that the level of TAp63α was increased in TLO2-Ⅱ liver CSCs after treatment with DATS,while ΔNp63α was decreased.Tumorspheres were treated with DATS after respective transfection with TAp63α plasimid or ΔNp63α-siRNA,and then it was observed that the inhibition effects of DATS on TLO2-Ⅱ liver CSCs markers were significantly diminished.These results sgggested that Tp63α mediated the inhibitory effects of DATS on TLO2-Ⅱ liver CSCs.After DATS treatment,the inactivation of Sonic Hedgehog pathway was observed.PWY,a Sonic Hedgehog pathway activator,was used to co-treatment with DATS,and then the suppression effect of DATS on TLO2-Ⅱ liver CSC markers were cut down,which indicated that DATS inhibited the activity of TLO2-Ⅱ liver CSCs thro μ gh downregulation of sonic hedgehog pathway.After further research,the downregulation of sonic hedgehog pathway could be alleviated if TAp63α was overexpressed orΔNp63α was lowexpressed.These results spggested that p63αregulates Sonic Hedgehog pathway and mediates the DATS interventional effect on TLO2-Ⅱ liver CSCsConclusions:In conclusion,the present data showed that TP63α/Sonic Hedgehog aixs played an essential role in the activity of NDMA-induced liver CSCs and mediated the inhibitory effects of DATS on TLO2-Ⅱ liver CSCs.The results implicated that targetting TP63α/Sonic Hedgehog aixs could be a potential approach for the treatment of liver cancer CSCs. |
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