Regulatory Effects Of MiR-20b-5p On The Proliferation And Apoptosis Of Breast Cancer Stem Cells And The Underlying Mechanisms

BackgroundBreast cancer is one of the major malignancies that threaten women's health.Its incidence and mortality rank the second and first among all the malignancies of women,respectively.Despite the improved overall survival(OS)of breast cancer patients in recent years by early diagnosis and surgical treatment combined with radiochemotherapy,endocrine therapy and targeted therapy,drug resistance and tumor relapse occur inevitably,leading to the failure of breast cancer treatment.Therefore,revealing novel targets is essential and critical for the improvement of therapeutic effects.Based on the hypothesis that breast cancer stem cells(BCSCs)are the origin of drug resistance and tumor relapse of breast cancer,we considered it of great significance to investigate the regulatory mechanisms of BCSCs and thus reveal potential new targets for the treatment of breast cancer.In our previous studies,screening in the gene chip dataset demonstrated that mi R-20b-5p(hsa-miR-20b-5p)played a key role in regulating BCSCs,and bioinformatic analysis found that mi R-20b-5p was possibly associated with the proliferation and apoptosis of BCSCs.ObjectiveBased on our previous findings,the current study aimed to determine the regulatory effects of mi R-20b-5p on the proliferation and apoptosis of BCSCs and to explore the mechanisms underlying these effects.MethodsFirst of all,T47 D and MCF-7 cells were cultured in serum-free medium to induce mammosphere formation.The differential expressions of stemness-specific genes between BCSC-like and non-stem breast cancer cells were detected by q RT-PCR,and the percentage of CD44+CD24-/low cells was analyzed by flow cytometry.Then the expressions of mi R-20b-5p were detected by q RT-PCR.in breast cancer cells(T47D and MCF-7)and BCSCs(T47D and MCF-7 cancer stem cells).T47 D cancer stem cells(CSCs)were infected with lentiviruses to establish overexpression and suppression systems of mi R-20b-5p.These systems,Ed U cell proliferation assay,and cell apoptosis assay were used to determine the effects of mi R-20b-5p on the proliferation and apoptosis of T47D-CSCs.Next,the folding free energy of RNAs at the 3?-UTR of E2F1 and the minimum free energy(MFE)for the binding of mi R-20b-5p to the 3?-UTR of E2F1 were compared using RNAfold and Bibiserv2 online databases.The direct interaction between mi R-20b-5p and the 3?-UTR of potential target E2F1 was then confirmed by dual luciferase assay.Furthermore,the effects of miR-20b-5p on the protein expression of E2F1 gene were analyzed by western blot.Finally,a xenografted mouse model was established to validate the effects of mi R-20b-5p on the growth of T47 D cells and on the protein expression of E2F1 in vivo.Results1.Previous bioinformatic analysis has screened out the miRNAs essential for the regulation of BCSCs,of which mi R-20b-5p has the highest regulatory degree.In the current study,breast cancer cell lines T47 D and MCF-7 were cultured in serum-free medium and mammospheres were formed.Flow cytometry demonstrated that the proportion of CD44+CD24-/low cells increased in BCSCs compared with non-stem cancer cells.The expressions of stemness-specific genes,OCT4,NANOG and SOX2,increased by 3.00,10.76 and 13.35 times in MCF-7-CSCs,and by 7.00,96.73 and 45.97 times in T47D-CSCs.These results suggested that BCSCs had been successfully induced.Analysis of qRT-PCR showed that the expression of miR-20b-5p increased by 3.91 times(P=0.004)and 2.19 times(P=0.006)in MCF-7-CSCs and T47D-CSCs,respectively.2.The mi R-20b-5p overexpression and suppression systems were established by lentiviral infection of T47D-CSCs.The EdU proliferation experiment showed that the cell proliferation ratio was significantly higher in the mi R-20b-5p overexpression group than in the empty vector group(40.9% vs 35.7%,P=0.011),and was significantly lower in the mi R-20b-5p suppression group than in the empty vector group(36.9% vs 41.0%,P=0.012).The apoptosis experiment showed that the percentage of apoptotic cells was significantly lower in the mi R-20b-5p overexpression group than in the empty vector group(8.15% vs 4.82%,P=0.004),and was significantly higher in the miR-20b-5p suppression group than in the empty vector group(13.34% vs 7.82%,P=0.011).3.Our previous study predicted E2F1 as a potential target gene of mi R-20b-5p to promote proliferation.In the current study,western blot showed that miR-20b-5p overexpression downregulated the protein expression level of E2F1,whereas inhibited mi R-20b-5p expression upregulated the protein expression level of E2F1.Moreover,the protein expression level of E2F1 was downregulated in T47D-CSCs compared with T47 D cells.Dual luciferase reporter assay demonstrated that mi R-20b-5p could directly bind to the 3?-UTR of E2F1 and downregulated dual luciferase activity by 18.60%(P=0.003).4.Also,vivo experiments confirmed that miR-20b-5p remarkably promoted tumor growth in a xenografted mouse model.Immunohistochemical analysis revealed that the protein expression of E2F1 was greatly decreased in the mi R-20b-5p overexpression group.ConclusionThe current study has successfully induced and identified BCSCs.Based on our previous findings of mi R-20b-5p as a BCSC-characteristic mi RNA,the current study has demonstrated that mi R-20b-5p expression is upregulated in BCSCs and miR-20b-5p can promote the proliferation and inhibit the apoptosis of BCSCs at functional level,and confirmed that E2F1 is a real target gene of mi R-20b-5p at protein and molecular levels and mi R-20b-5p can downregulate the expression of E2F1 in vivo,thus promoting the growth of breast cancer xenografts.