PTEN Regulates MiR-182 And AKT To Induce SKBR3 Cell Apoptosis

[Backgrounds]Breast cancer is one of the most common malignancies in women,developing from breast ductal epithelium.In recent years,the morbidity and mortality of breast cancer have been increasing in young people.There are many types of breast cancer,each of which has different pathological histomorphology,clinical manifestation and prognosis.The diagnosis of breast cancer staging also has very important significance on its right and timely therapy.With the development of molecular biology,breast cancer therapy has entered the age of molecular target.Rational drug therapies should be given for different types and staging of breast cancer.Comprehensive therapy methods on breast cancer include not only local surgical operation and radiotherapy,but also systemic treatment such as chemotherapy,endocrine therapy,molecular targeting drugs and so on.Recently,molecular targeting therapy in breast cancer has developed rapidly and its relevant studies become active.While,some molecules closely relating to the tumorigenesis and development have become the hotspot.Phosphatase and tensin homolog(PTEN)is very vital tumor suppressor gene.At present,cell signaling pathways are also one crucial way to regulate tumor with more and more studies,such as PTEN/Phosphotidylinositol 3,4,5-trisphosphate(PIP3)/Protein Kinase B(AKT).Many microRNAs(miRNA)also play a key role in regulating tumorigenesis and development.In brief,it is imperative to study those molecular mechanism relating to breast cancer pathogenesis.[Objects]In this study,breast cancer cells SKBR3 were transfected with overexpressed PTEN and overexpressed miR-182 vectors.Then expressing level of p-AKT,miR-182,miR-183 and miR-96,and cell apoptosis were measured.The study aimed to explore pathogenesis of breast cancer,excepting to provide valuable molecular target for the breast cancer therapy.[Methods]Part Ⅰ.To investigate the effects of overexpressed PTEN on PI3K/AKT signaling pathway in breast cancer cells,SKBR3 cells were purchased from American type culture collection(ATCC),and they were defrosted by water bath at 37℃,collected by centrifugation and put in Dulbecco’s modified eagle medium(DMEM)with 10%fetal bovine serum(FBS)supplemented.The cells were incubated in humid air with 5%CO2 at 37℃.Medium was changed every 3 days.Overexpressed Flag-PTEN vectors were transfected into SKBR3 cells.AKT and p-AKT protein expression levels were measured by Western blot and cell apoptosis was measured by flow cytometry.Part Ⅱ.To investigate the effects of overexpressed PTEN on miR-183,miR-182 and miR-96 in breast cancer cells,miR-183,miR-182 and miR-96 expression in the transfected SKBR3 cells were measured by qRT-PCR.Part Ⅲ.To investigate the effects of overexpressed PTEN and overexpressed miR-182 on PI3K/AKT signaling pathway and cell apoptosis,SKBR3 cells were co-transfected with Flag-PTEN and miR-183 mimic vectors.AKT and p-AKT protein expressions were measured by Western blot and cell apoptosis was tested by flow cytometry.[Results]Part Ⅰ.The effects of overexpressed PTEN on PI3K/AKT signaling pathway and cell apoptosis in breast cancer cellsWestern blot results showed an enhanced PTEN expression compared to the control group,indicating successful cell transfection concerning PTEN overexpression.Overexpressed PTEN significantly lowered p-AKT protein level,but had no impact on AKT.Flow cytometry results showed that overexpressed PTEN increased SKBR3 cell apoptosis and quantitative analysis also displayed an increased cell apoptosis rate.Part Ⅱ.The effects of overexpressed PTEN on miR-182,miR-183 and miR-96 expressionOverexpressed PTEN significantly down-regulated miR-182 expression(P<0.001).As controls,miR-183 level was also markedly inhibited(P<0.001),but miR-96 was barely changed(P>0.05).Part III.The effects of overexpressed PTEN and miR-182 on PI3K/AKT signaling pathway and cell apoptosisAfter SKBR3 cells were co-transfected with overexpressed PTEN and miR-182 mimic vectors,miR-182 expression was suppressed by overexpressed PTEN(P<0.01),and then elevated by miR-182 mimic(P<0.05),indicating a successful co-transfection.p-AKT expression level in Flag-PTEN + miR-182 group was higher than that in Flag-PTEN group,showing that overexpressed miR-182 could increase p-AKT expression,and rescue the promotion of AKT activation by PTEN.Meanwhile,overexpressed PTEN promoted cell apoptosis,but overexpressed miR-182 rescued this effects(P<0.01 or P<0.05).[Conclusions]Overexpressed PTEN in breast cancer cells can inhibit the activation of PI3K/AKT signaling pathway and promote cancer cell apoptosis.Overexpressed PTEN reduces the expression of miR-182 and miR-183,suggesting that PTEN may participate in the cancer cell function by regulating miR-182 and miR-183.Additionally,miR-182 rescues the inhibition on p-AKT and promotion on cancer cell apoptosis by PTEN.Our study may provide more reference for the breast cancer therapy concerning molecular targeting drugs.