Genistein Inhibits Cell Growth In Human Breast MCF-7 Cells Via Modulating The Expression Of MiR-34a

Background: Breast cancer is one of the most common malignant tumors in women,with high morbidity and mortality,is liable to relapse and metastasize.In recent years,great achievements has been made in the field of treatment for breast cancer,but it's still not enough to overcome the escape and resistance mechanism of cancer cells,we need further research the mechanism of formation and development of neoplasm and find out more effective and non-toxic antitumor drugs.Genistein,the predominant isoflavone found in soy products,has been shown anti-cancer properties to inhibit various types of tumors and has low toxicity to normal cells in recent researches.It has been reported that genistein could also influence the formation and development of tumors via modulating the expression of miRNAs and its target genes.Nevertheless,the relationship between genistein and miRNAs in breast cancer cells has not yet been reported.MicroRNAs,non-protein coding RNAs,21-25 nucleotides in length generated from single-stranded,regulate the expression of its target genes.We generally found the expression level of miR-34 a is relatively low in human cancer cells.Up-regulating the expression level of miR-34 a could induce cell apoptosis,cell cycle arrest and cell senescence,inhibit cell proliferation,migration and invasion and so on in various types of tumors.High mobility protein A2,one of non-histone chromatin protein,works as architectural transcription factor and oncofetal protein.HMGA2 expression in almost all kinds of malignant neoplasms and the expression level of it is higher in high degree of malignant tumors,has not been found in normal tissues,it was closely related to the formation and development and poor prognosis of the neoplasms.Object:Investigate the effect of genistein on the expression of miR-34 a and breast cancer cells proliferation,migration and invasion to provide theoretical foundation and practical basis of genistein applying to clinical treatment of breast cancer;Methods: In human breast cancer MCF-7 cells,investigate the effect of genistein treatment on expression of miR-34 a by q RT-PCR,detect the IC50 of genistein at 48 hours by CCK-8 assays,the IC50 was applied in the following assays;Mi R-34 a mimics were transfected into MCF-7 cells,and then CCK-8 assays,clony formation assays,wound healing assays and invasion assays were used to verify the effect of genistein and miR-34 a and the combination on MCF-7 cells proliferation,clony formation,migration and invasion;Western blotting and q RT-PCR were utilized to study the influence of genistein and miR-34 a and the combination to the expression level of HMGA2 protein and m RNA;Si RNA-HMGA2 were transfected into MCF-7 cells to knockdown HMGA2,CCK-8 assays and clony formation assays were used to explore the role of HMGA2 in MCF-7 cells proliferation and clony formation.Results: 1.The IC50 of genistein at 48 hours is 8.05?M;2.Genistein can up-regulate the expression of miR-34 a in MCF-7 cells obviously;3.CCK-8 assays results show that the living cells ratio of genistein treatment group was 0.50 + /-0.03,the living cells ratio of miR-34 a treatment group was 0.46 + /-0.01,the living cells ratio of the combined treatment group was 0.27 + /-0.01,(P<0.01),either genistein or miR-34 a could inhibit MCF-7 cells proliferation and the combination is more significant;the living cells ratio of si RNA-NC group was 0.91+ /-0.02,the living cells ratio of si RNA-HMGA2 was 0.37+ /-0.02,knockdowning HMGA2 can inhibit MCF-7 cells proliferation significantly;4.Clony formation assays results show that: the clony number of the four groups(the blank control group,the genistein treatment group,miR-34 a treatment group,combined treatment group)was 204.67 + /-9.07,104.33 + /-11.93,95.33 + /-10.11,54.67 + /-8.33,(P<0.01),either genistein or miR-34 a could inhibit MCF-7 cells clony formation and the combination is more significant;the clony number of the control group and the si RNA-NC group and the si RNA-HMGA2 group was 344+ /-9.85?325.7+ /-14.05?235+ /-12.53,knockdowning HMGA2 can inhibit MCF-7 cells clone formation significantly;5.Wound healing assays results show that: the cell migration rate of the four group at 24 hours was 50%,37.5%,27.8%,16.7%,(P<0.01),either genistein or miR-34 a could inhibit MCF-7 cells migration and the combination is more significant;6.Invasion assays results show that: the cell number passed through the membrane of the four group was 135.00 + /-9.85,53.67 + /-7.02,47.33 + /-4.93,was 32.33 + /-5.86,(P<0.01),either genistein or miR-34 a could inhibit MCF-7 cells invasion and the combination is more significant;7.QRT-PCR and western blotting results show that either genistein or miR-34 a could down-regulate the expression level of HMGA2 m RNA and protein,compared to the blank control group;HMGA2 si RNA-1 could knockdown HMGA2 and inhibit the expression of HMGA2 m RNA and protein effectively.Conclusion:Either genistein or miR-34 a could inhibit breast cancer cells' proliferation,clone formation,migration and invasion,furthermore,genistein may inhibit the bio-function of MCF-7 cells through up-regulating the expression of miR-34 a.Either genistein or miR-34 could down-regulate the expression levels of HMGA2 mRNA and protein,and genistein may down-regulate the expression of HMGA2 through up-regulate the expression of miR-34a;Knockdowning HMGA2 inhibit breast cancer MCF-7 cells' proliferation and clone formation ability.