Role Of MiR-19 And Cancer Cell Stemness In Butyl Benzyl Phthalate-mediated Breast Cancer Cell Promotion

Objective:Breast cancer is the most common cancer in women.Growing evidence suggests that Butyl benzyl phthalate(BBP)can promote the growth,invasion,metastasis,and drug resistance of breast cancer cells.MicroRNAs(miRNAs)plays an important role in the regulation of target mRNAs.Cancer stem cell(CSCs)play central role in the formation,development,invasion,metastasis and drug resistance of breast cancer cells.To date,no studies have been conducted yet to reveal the actions of miRNAs in BBP-mediated breast cancer promotion and the effects of BBP on breast CSCs.Findings from this research will provide important new insights into the molecular mechanisms by which BBP exerts its breast cancer-promoting effect as well as its target intervention.Methods:MTT was used to observe the proliferation of human breast cancer cell line ER(+)MCF-7 and ER(-)MDA-MB-231 after different dose of BBP treatment.The distribution of MCF-7 and MDA-MB-231 cells at different stages in the cell cycle was determined by flow cytometric analysis.The expression of cell proliferation related protein Cyclin D1,PCNA and p21 were observed by western blotting.qRT-PCR analysis was served to measure the expression of miR-19 after BBP treatment.The effect of BBP on PTEN/p-AKT/p21 axis in breast cancer MCF-7 and MDA-MB-231 cell lines were observed by Western blotting and Real-time PCR.Using miR-19 mimics or inhibitors to disturb the expression of miR-19 in MCF-7 and MDA-MB-231 cells,MTT observed cell viability and western blotting determinated the expression change of Cyclin D1,PCNA and PTEN/p-AKT/p21 axis signaling.Using miR-19 inhibitors to downregulate the expression of miR-19 in MCF-7 and MDA-MB-231 cells combination with BBP treatment,and then cell viability were observed by MTT and the expression change of Cyclin D1,PCNA,p21 and PTEN/AKT/p21 axis were detected by western blotting.There are three miR-19 binding sites in the PTEN 3’-UTR.Luciferase vectors containing only one miR-19 binding site of the PTEN 3’-UTR were transfected into MCF-7 and MDA-MB-231 cells before BBP treatment and then luciferase activity were detected.Four luciferase vectors were constructed which contained the second wild-type binding site,the second mutant binding site,the third wild-type binding site and the third mutant binding site respectively.The breast cancer stem cell from MCF-7 and MDA-MB-231 cells were enriched through suspension culture in serum-free medium,and then the sphere growth were observed under a microscope and the cancer stem cell markers CD44,ALDH1A1,Oct-4 and Nanog were detected by Western blotting to test whether breast cancer stem cells can be enriched.The MCF-7 and MDA-MB-231 cell spheres obtained through suspension culture were treated with different doses of BBP,then the size of sphere were photographed and the protein levels of cancer stem cell markers CD44,ALDH1A1,Oct-4 and Nanog were detected by Western blotting.Meanwhile,the changes of signaling molecules in Wnt/β-catenin and Sonic Hedgehog pathway after BBP treatment were also be observed by Western blotting,including GSK-3 β,β-catenin,C-myc,Cyclin D1,and Shh,Smo,Gli1,Gli2 as well.Results:Part Ⅰ:Role of miR-19 in Butyl Benzyl Phthalate-mediated Breast Cancer Cell Proliferation1.BBP triggered the proliferation of breast cancer cells independent of ERTreatment of cells with 10-6-10-5 M BBP significantly increased cell viability,and the numbers of survival cells were significantly increased following BBP treatment.Western blotting also demonstrated that BBP not only elevated the expression level of Cyclin D1,but also the level of proliferating cell nuclear antigen PCNA.Flow cytometry was showed that 10-5 M of BBP significantly accelerated G1 to S phase progression.These results illustrated the proliferative effects of BBP on ER(+)MCF-7 breast cancer cells.Treatment of cells with 10-9-10-6 M BBP significantly increased cell viability following BBP treatment.Western blotting also demonstrated that BBP not only elevated the expression level of Cyclin D1,but also the level of proliferating cell nuclear antigen PCNA.Flow cytometry was showed that 10-7 M of BBP significantly accelerated G1 to S phase progression.These results illustrated the proliferative effects of BBP on ER(-)MDA-MB-231 breast cancer cells.2.BBP induced the expression of miR-19miR-19 is a key oncogenic component of mir-17-92.Real-time PCR analysis revealed that 10-6-10-5 M BBP significantly induced the expression of miR-19a and miR-19b in MCF-7 cells.Similarly,10-8-10-7M BBP also induced the sharp upregulation of miR-19a and miR-19b in MDA-MB-231 cells.These data hinted that miR-19 upregulation may be in connection with the BBP-mediated proliferation of breast cancer cells.3.BBP modulated PTEN/p-AKT/p21 axisBBP decreased the level of PTEN and p21,increased the expression of p-AKT in both MCF-7 and MDA-MB-231 cells.These data suggested that BBP modulatedPTEN/p-AKT/p21 to promote breast carcinogenesis.4.Overexpression of miR-19 promoted the growth and influenced PTEN/p-AKT/p21 axis of breast cancer cellsmiR-19a or miR-19b mimic were able to induce the growth of MCF-7 breast cancer cell.Meanwhile,the protein level of PTEN and p21 were decreased,and the expression of PCNA,Cyclin D1 and p-AKT were increased.In MDA-MB-231 breast cancer cell,miR-19a or miR-19b mimic also lead to the reduction of PTEN and p21 and the increment of PCNA,Cyclin D1 and p-AKT.These data illustrated that miR-19 influenced the growth and PTEN/p-AKT/p21 axis of breast cancer cells.5.Downregulation of miR-19 suppressed the growth and influenced PTEN/p-AKT/p21 axis of breast cancer cellsmiR-19a or miR-19b inhibitor were able to restrain the growth of MCF-7 breast cancer cell.Meanwhile,the protein level of PTEN and p21 were increased,and the expression of PCNA,Cyclin D1 and p-AKT were decreased.In MDA-MB-231 breast cancer cell,miR-19a or miR-19b mimic also lead to the increment of PTEN and p21 and the reduction of PCNA,Cyclin D1 and p-AKT.These data illustrated that miR-19 influenced the growth and PTEN/p-AKT/p21 axis of breast cancer cells.6.miR-19 mediated BBP-triggered breast cancer cell proliferationAfter reducing the expression of miR-19a or miR-19b,BBP did not exhibit the potential of promoting cell proliferation and upregulation of PCNA and Cyclin D1 in both MCF-7 and MDA-MB-231 breast cancer cells.At the same time,miR-19a or miR-19b inhibitor rescued the down regulation of PTEN and p21 and upregulation of p-AKT caused by BBP treatment in both cells.These results indicated the role of miR-19 in the promotive effect of BBP on the proliferation of breast cancer cells.7.BBP suppressed PTEN translational level through miR-19 targeting of PTEN3’-UTRUsing bioinformatics methodology,we discovered that PTEN 3’-UTR contains binding sites of miR-19.After transfected PTEN 3’_UTR luciferase reportor gene plasmid,which contains only one wild-type or mutant miR-19 binding sites,MCF-7 and MDA-MB-231 cells were treated with BBP and then luciferase activity were detected.Dual luciferase reporter gene assay showed that BBP reduced wild-type PTEN 3’-UTR fluorescence intensity,while mutant PTEN 3’-UTR fluorescence intensity could not be lessened by BBP treatment.These results illustrated that BBP suppressed PTEN translational level through miR-19 targeting of PTEN 3’-UTR.Part Ⅱ:Low concentrations of Butyl Benzyl Phthalate promoted the activity of breast cancer stem cells8.Breast cancer stem cells were enriched through suspension culture in serum-free medium.To investigate the effect of BBP on breast cancer stem cells,MCF-7 and MDA-MB-231 cells were suspension cultured in serum-free medium to enrich the breast cancer stem cell.Western blotting analysis showed that the protein level of breast cancer stem cell markers were significantly increased,including CD44,ALDH1A1,Oct-4 and Nanog in suspension cell sphere compared with adherent cell.These results reveal that suspension culture in serum-free medium can enrich the breast cancer stem cell.9.BBP promoted the growth of breast cancer stem cellsSuspension cultured human breast cancer MCF-7 and MDA-MB-231 cells were treated with different concentrations of BBP and the growth of cells were observed.Compared with control group,the size of mammosphere were observed enlarged after treating with 10-10-10-8M significantly.Western blotting results showed that 10-10-10-8M of BBP markedly up the expression of PCNA and down the p21 protein level.These data indicated that BBP can promote the growth of breast cancer stem cell.10.BBP upregulated the markers of breast cancer stem cellsMCF-7 and MDA-MB-231 stem cells were treated with 10-10-10-8M BBP,and the breast cancer stem cell markers were detected by Western blotting.We found that CD44,ALDH1A1,Oct-4,Nanog and ΔNp63 were strikingly increased after BBP treatment in comparison with DMSO control.These hinted that BBP can promote the stemness of breast cancer stem cells.11.BBP activated Wnt/β-catenin pathway of breast cancer stem cellsBreast cancer stem cell were given different dose of BBP,and then Wnt/β-catenin pathway signaling were tested in both MCF-7 and MDA-MB-231 cells.Western blotting showed that the protein level of p-GSK-3β,β-catenin,C-myc and Cyclin D1 were sigficantly upregulated after BBP treatment.12.BBP activated Sonic Hedgehog pathway of breast cancer stem cellsBreast cancer stem cells were treated with different doses of BBP,and Sonic Hedgehog signaling pathway were then tested in both MCF-7 and MDA-MB-231 cells.Western blotting showed that the protein level of Shh,Smo,Gli1 和 Gli2 were markedly increased following BBP treatment.Conclusions:BBP induced the proliferation of both ER(+)MCF-7 and ER(-)MDA-MB-231 breast cancer cells.BBP upregulated the expression of oncogenic miR-19 and affected PTEN/p-AKT/p21 axis.Notably,we for the first time revealed that miR-19 played crucial role in the breast cancer-promoting action of BBP,which targeted PTEN 3’-UTR and suppressed its translational activity.On the other hand,low concentration of BBP enhanced the stemness of breast cancer stem cells through increased the size of mammosphere,upregulatied breast cancer stem cell markers CD44,ALDH1A1,Oct-4 and Nanog,and activated Wnt/β-catenin and Sonic Hedgehog pathways.Findings from this research could provide important new insights into the molecular mechanisms by which BBP exerts its breast cancer-promoting effect as well as its target intervention.