The Role Of MiR-148a/TGF-β Signaling Pathway In The Anti-cancer Stem Cell-like Properties By Glabridin

Epidemiological studies indicate that consumption of phytochemicals is correlated with cancer, including cancer initiation and progression. Lately it has been attracted more and more attention owing to its higher effect and lower toxicity. Phytochemicals play an anti-caner effect probably due to inhibiting the activation of carcinogens, inducing protective enzymes, inhibiting the inflammation, immune activity, promoting cell cycle arrest and apoptosis and changing signaling pathways. As an active ingredient of licorice, Glabridin has a wide range of pharmacological activities, such as whitening, anti-inflammatory, anti-bacterial, antioxidant, cardiovascular and neuro-protective effect. Further researches found that Glabridin exhibited the effects of inhibiting tumor growth, metastasis and angiogenesis.Cancer stem cells are a small fraction of cells in the tumor, with characteristics of self-renewing, highly proliferative and differentiation capacity, which play a decisive role in tumorigenesis, invasion, metastasis, relapse and drug resistance. The existence of cancer stem cells gives a well explaination in that tumor shrinks or disappears in the early treatment, while it will quickly relapse and lead to death. Therefore, the understanding of cancer stem cells and their contributions to therapy is very important for further study.Amount of researches indicate that miRNAs can serve as oncogenes or tumor suppressor genes and participate in multiple biological processes of tumor initiation and progression. miRNAs have been found in regulating cancer stem cell-like properties by targeting many genes involved in the cancer stem cells, which ultimately affect tumorigenicity and change phenotype. Therefore, targeting miRNAs specificly provide a new strategy for cancer therapy.Based on the above, we proposed that Glabridin inhibits the anti-cancer stem cell like properties by altering the signaling pathway via miRNA, which provide a scientific basis for the applications of Glabridin in tumor therapy.Objective:In this study, human breast cancer cells (MDA-MB-231, Hs-578T) and human hepatocellucar carcinoma cells (HepG2, MHCC97H, Huh7) were used in vitro, xenograft model was used in vivo, to investigate whether glabridin inhibites the cancer stem cell-like properties by miRNA.Methods:RT-PCR and qRT-PCR were used to detect the expression of miR-148a and the cancer stem cells markers; flow cytometry, soft agar assay and mammosphere formation were utilized to detect side population, characteristic of malignancy and self-renewal capacity; qRT-PCR and Western Blot were used to examine the expression and phosphorylation of SMAD2, as well as the expression of snail; dual luciferase reporter assay was used to identifying the targeting-inhibition of SMAD2 by miR-148a; methylation specific PCR was used to examing the methylation level of miR-148a promoter, and immunohistochemistry was used to detected the SMAD2 expression and localization in tissue.Resluts:1. Effects of Glabridin on the cancer stem cell-like properties.Human breast cacner cell lines MDA-MB-231 were treated by 10 μM GLA for 0, 24,48,72 h, and the expressions of CD44, ALDH-1, Oct-4, BMI-1 were decreased in a time-dependent manner. Human hepatocellular carcimoma cell line HepG2 were treated by 0,10,20 μM GLA, and the expressions of CD44, EpCAM, CD 133, CD90, Oct-4, BMI-lwere decreased in a dose-dependent manner. Similarly, GLA inhibited the expressions of CD44 and EpCAM in Huh-7 and MHCC97H cells. Moreover, GLA suppressed the side population of MDA-MB-231, as well as the colony formation and mammosphere formation in HepG2, MHCC97H, Huh-7.2. Effects of TGF-β signaling pathway on the cancer stem cell-like properties.HepG2 were treated by 10 ng/mL TGF-P for 48 h, and the expressions of CD44 and EpCAM were significantly up-regulated, with increasing capacity of colony and mammosphere formation.SMAD2-siRNA was utilized to transfect the HepG2 cells, and TGF-P were added in. As a result, SMAD2-siRNA abrogated the improvement of CD44, EpCAM, colony and mammosphere formation induced by TGF-β.3. Effects of Glabridin on the TGF-P signaling pathway.After treated by GLA, MDA-MB-231 cells were treated by TGF-β, we found that GLA abolished the phosphorylation of SMAD2 induced by TGF-β. Similarly, HepG2 cells were treated by GLA followed by 10 ng/mL TGF-β, and the phosphorylation of SMAD2 and the expression of Snail were reduce. Simultaneously, GLA inhibited the expression SMAD2 total protein and mRNA.MDA-MB-231 cells were treated by GLA for 0,24,48 and 72 h, and we found that the levels of p-SMAD2 and total SMAD2 were decreased in a time-dependent manner. In HepG2, Huh-7 and MHCC97H cells, GLA also reduced expressions of SMAD2 and Snail.4. Effects of Glabridin on the expression of miR-148a.In MDA-MB-231 cells, over-expression of miR-148a significantly decreased the luciferase activity in wild type plasmid transfected cells, while no significant change in mutant plasmid transfected cells. In HepG2 cells, over-expression of miR-148a inhibited the expression of SMAD2.Both in MDA-MB-231 and Hs-578Tcells, GLA increased the expression of miR-148a in a time-dependent manner. Similarly, in HepG2, Huh-7 and MHCC97H cells, GLA also up-regulated the level of miR-148a.5. Effects of Glabridin on the methylation of miR-148a.In MDA-MB-231 cells, GLA decreased the average methylation level of miR-148a promoter. Meanwhile, the DNA methytransferase 1 and 3a were inhibited as well.6. miR-148a is involved in the inhibiton of TGF-β signaling by Glabridin.In MDA-MB-231, Hs-578T and HepG2 cells, knockdown of miR-148a blocked the attenuation effects of GLA on SMAD2 expression and phosrylation, as well as the downstream Snail.7. miR-148a is involved in the inhibiton of cancer stem cell-like properties by Glabridin.In MDA-MB-231 cells, knockdown of miR-148a blocked the attenuation effects of GLA on the expression of Vimentin, and the improvement of the expressions of ZO-1 and E-cadherin.8. miR-148a is involved in the inhibiton of cancer stem cell-like properties by GlabridinIn MDA-MB-231, Hs-578T, HepG2 and Huh7 cells, knockdown of miR-148a blocked the attenuation effects of GLA on cancer stem cell markers, side population, colony and mammosphere formation.9. Effects of Glabridin on the cancer stem cell-like properties in vivo.In xenograft, GLA inhibited the tumor growth and the expressions of the cancer stem cell markers. Simultaneously, miR-148a were up-regulated with demethylation in the promoter and decreasing expressions of DNMT. Moreover, the SMAD2 were suppressed, consistent with the previous result in vitro.Conclusion:In the present study, we identified that GLA suppressed the cancer stem cell-like properties by blocking the TGF-β/SMAD2 signaling via up-regulation the expression of miR-148a by demethylation, which targeted the SMAD2.