| Background Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.Cancer stem cells(CSCs),a characteristic subpopulation of tumor,could maintain and promote tumor growth.In recent years,it has been found that micro RNA plays an important role in the development of tumor,cell proliferation and multidirectional differentiation,especially the let-7 family.In our previous study,let-7i was significantly elevated in liver cancer stem cells.Therefore,we will investigate the effect of let-7i on stemness maintenance of liver CSCs,and find the regulation of Chinese medicine ingredients on let-7i in the study.Purpose To investigate the effect of let-7i on stemness maintenance of liver CSCs,and explore the regualtion of Chinese medicines ingredient on let-7i.Methods 1.Detection of let-7i in HCC tissues and spheres The expression of let-7i in HCC and adjacent tissues were detected by stem-loop RTq PCR.Spheres of Hep G2,SMMC7721 cells were established with serum-free suspension culture.The expression of let-7i in adherent cells and spheres was compared.2.Effects of let-7i on stemness maintenance of liver CSCs in vitro First,let-7i mimic and let-7i inhibitor were transfected with 50 n M and 100 n M respectively.The expression of let-7i in HCC cells was verified by stem-loop RT-q PCR.Then,the effects of let-7i on stemness maintenance of liver CSCs were observed.(1)The expression of CD133 and Ep CAM protein were detected by western blot.(2)Sphere formation assay,stem cell genes,plate cloning,soft agar cloning and cell proliferation assay were used to observe the self-renewal ability of liver CSCs.(3)To investigate the differentiation,the expression of hepatic stem cell and mature hepatocytes genes were measured by RT-q PCR.(4)Drug resistance was measured by MTT assay after transfection.(5)Scratch test and transwell invasion test were used to detect the invasion and metastasis.3.Effects of let-7i on tumorigenesis in vivo Subcutaneous tumor of nude mice was used for the effect of let-7i on tumorigenesis in vivo.The expression of let-7i in the tumor tissues was detected by stem-loop RT-q PCR.Tumor growth,tumor volume and tumor weight were detected in six weeks.The expression of CD133 and Ep CAM protein was measured by western blot.4.Regulation of curcumin and matrine on let-7i IC50 concentration of curcumin and matrine were measured by MTT assay in SMMC7721 and Hep G2 cells.The expression of let-7i was tested by stem-loop RTq PCR after treated with IC50 of curcumin and matrine.Results 1.let-7i was highly expressed in HCC tissues and spheres The expression of let-7i in HCC tissues was 3.92±0.84 times higher than that of adjacent tissues(P<0.05).In sphere formation assay,the expression of let-7i in sphere were higher than that in adherent cells(P<0.05),which were 3.92±0.73 times in SMMC7721 cells and7.07±0.37 times in Hep G2 cells.2.let-7i maintained the characteristics of liver CSCs in vitro 2.1.The up-regulation and down-regulation of let-7i were established Compared with control group,the expression of let-7i wereup-regulated by 5.02±0.68 times(P<0.01)and 11.61±2.84 times(P<0.05)after transfected let-7i mimic in SMMC7721 and Hep G2 cells,respectively.Compared with control group,the expression of let-7i werere spectively decreased by 57%±11%(P<0.05)and 62%±20%(P<0.05)after transfected let-7i inhibitor in SMMC7721 and Hep G2 cells.2.2.let-7i increased the expression of CD133 and Ep CAM protein The levels of CD133 and Ep CAM in SMMC7721 and Hep G2 cells were higher than those in control group(P<0.05)after up-regulation of let-7i.While down-regulation of let-7i could significantly reduce the expression of CD133 and Ep CAM(P<0.05).2.3.let-7i enhanced the self-renewal of liver cancer(1)In SMMC7721 and Hep G2 cells,there were more spheres formation after increasing let-7i.In down-regulation of let-7i,the number of tumor spheres were significantly reduced(P<0.01).Especially in SMMC7721 cells,there were almost no spheres formation.(2)The expression of stem cell-related genes,such as Nanog,SOX2,Oct4 and Bmi1,were promoted after up-regulating let-7i(P<0.05).In contrast,stem cell-related genes were obviously decreased after down-regulating let-7i(P<0.05).(3)Compared with control group,larger and more clones were formed after increasing let-7i in SMMC7721 and Hep G2 cells.The clone numbers were respectively 1.44±0.02 and 1.58±0.10 times than those in control group.Compared with control group,smaller and fewer clones were established after decreasing let-7i in SMMC7721 and Hep G2 cells.The clone numbers were reduced by 58%±19% and 65%±7.0% than those in control group,respectively.(4)Compared with control group,larger and more soft agar clones were founded after increasing let-7i in SMMC7721 and Hep G2 cells.The clone numbers were respectively 1.56±0.07 and 1.74±0.29 times than those in control group.Compared with control group,smaller and fewer soft agar clones were appearedafter decreasing let-7i in SMMC7721 and Hep G2 cells.The clone numbers were respectively reduced by 44% ± 4.5% and 43% ± 6.7% than those in control group.(5)let-7i could increase the proliferation of SMMC7721 and Hep G2 cells.Especially on the 6th day,the number significantly increased in SMMC7721 and Hep G2 cells(P<0.05).Meanwhile,the growth of SMMC7721 and Hep G2 cells were significantly restrained on the 6th day after reducing let-7i(P<0.05).2.4.let-7i promoted differentiation of liver cancer cells let-7i significantly increased the expression of hepatic stem cell genes,including CK-18,MYC and AFP(P<0.05).let-7i significantly decreased the expression of mature hepatocytes genes,such as CYP3A4,G6 P and ALB(P<0.05).Hepatic stem cell genes were reduced,and mature hepatocytes genes were increased after inhibiting let-7i.2.5.let-7i induced the drug resistance of liver cancer cells Compared with control group,SMMC7721 and Hep G2 cells showed higher drug resistance to 5-FU and DOX after up-regulation of let-7i.However,down-regulation of let-7i had significantly weaker drug resistance(P<0.05).2.6.let-7i enhanced the migration and invasion of liver cancer cells Compared with the control group,let-7i increased the migration distance and the cell number through membrane.Compared with the control group,the migration distance and the cell number through membrane were significantly reduced by down-regulation of let-7i(P<0.05).3.let-7i strengthened the tumorigenic ability in vivo Using stem-loop RT-q PCR,the expression of let-7i in tumors was significantly increased after transfection of let-7i agomir(P<0.05),which was 3.57±0.87 times higher than that in control group.Compared with the control group,let-7i promoted tumor growth,increased tumor volume and tumor weight(P<0.05).let-7i significantly increased the expression of CD133 and Ep CAM(P<0.05).Among them,CD133 increased 1.40±0.11 times,and Ep CAM rose 2.88 ± 0.21 times remarkably.4.Effect of curcumin and matrine on let-7i The expression of let-7i was significantly decreased in curcumin-treated cells compared with control group(P<0.05).While let-7i was significantly increased in matrinetreated cells(P<0.05).Conclusion let-7i was highly expressed in HCC tissues and spheres.It maintained stemness of liver CSCs in vitro and in vivo.In addition,curcumin downregulated let-7i,and matrine upregulated let-7i.These results will help to reveal the mechanism of the stemness maintenance of liver CSCs,and discover new drugs targeting CSCs. |
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