| Background:Breast cancer seriously threatens the health of female worldwide,however,up to now,there are still no effective treatments.MicroRNA(miRNA)is a kind of small non-coding single stranded RNA that is commonly found in living organisms and contains approximately 19 to 22 nucleotides,can target the specific gene 3’un-translated region(UTR)by complementary pairing,suppress the translation of target gene,and regulate gene expression and function.Phosphatase and tensin homolog deleted from chromosome 10(PTEN)could inhibit the activity of phosphatidylinositol 3-kinase(PI3K)/Protein Kinase B(AKT)through regulation of the phosphorylation of PI3K/AKT.As significant signaling pathway in promoting cancer progression,the activation of PI3K/AKT would regulate cell proliferation,cell migration,and cell apoptosis of cancer cell lines,etc.PTEN is widely expressed in the human normal tissue cells,especially in the heart,brain,lung,liver and kidney,and plays a pivotal role in theprogressionof breast cancer.Objective:1.How miRNA-142-5p and PTEN Express in Breast Cancer?2.What are the roles of miRNA-142-5p and PTEN in the proliferation and apoptosis of breast cancer?3.How miRNA-142-5p and PTEN participate in breast cancer proliferation and apoptosis?Methods:1.We first collected tumor tissues and the adjacent tissue from female patients,and also the breast cancer cell lines.Detection of miR-142-5p expression and PTEN mRNA levels in patient tissues and breast cancer cell lines were performed using real time quantitative polymerase chain reaction(RT-qPCR).We analyzed the relationship between miR-142-5p/PTEN and pathological information of breast cancer.2.Western blot was carried out to detect the protein expression levels of PTEN,p-PI3Kand p-AKT.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,Thiazolyl Blue Tetrazolium Bromide(MTT)was used for the examination of cell proliferation.Flow Cytometry(FCM)was applied for the detection of cell apoptosis.Online tool TargetScan was used to predict the binding sites and sequences between miR-142-5p and PTEN.Dual luciferase reporter assay was used for the detection of interaction between miR-142-5p and PTEN.3.BALB/c Nude Mice(4 weeks old,all females)were purchased to establish the xenograph tumor models,mice were divided into miR-NC and miR-142-5p inhibitor groups,and the expressions of miR-142-5p and PTEN in tumors were examined.Results:1.1 Theaverageexpression level of miR-142-5p in tumor samples was about 2.5 times as it was in the adjacent tissue,the differences have statistical significance(p<0.01);The levels of average PTEN in tumor samples was about 1/2 of that in adjacent tissue,the differences have statistical significance(p<0.01).1.2miR-142-5p and PTEN has no correlation with the age of the patients(p>0.01);the expression of miR-142-5p was positively correlated with the tumor size and the TNM stage,the differences has statistical significance(p<0.01);The expression of PTEN was negatively correlated with the tumor size and the TNM stage,the differences have statistical significance(p<0.01).2.1 The activity of the luciferase was significantly increased in miR-142-5p and WT PTEN co-transfected group was about 3 times as much as themiRNA-NC and WT PTEN co-transfected group,the differences have statistical significance(p<0.01).2.2 The expression ofmiRNA-142-5p was slightly increased in SK-BR-3 cells compared with the MCF10A cell,but has no statistical significance(p>0.05);The expression ofmiRNA-142-5p in MDA-MB-231 was as twice as much as it in MCF10A cells,the differences have statistical significance(p<0.01).After transfection of miR-142-5p inhibitor in MDA-MB-231 cells,the expression of miR-142-5p decreased to about 1/5 of the control and miR-142-5p inhibitor NC group,the differences have statistical significance(p<0.01);the expression of PTEN was significantly increased after transfection of miR-142-5p inhibitor,and the levels of PTEN was about 3 times as mush as it in the other two groups,the differences have statistical significance(p<0.01).2.3 Results of MTT assay indicated that the OD value at 490nm showed no significantly difference among the three groups,suggesting that the viability of the cells has no significantly difference;after transfection for 24 and 48h,the OD value for the control and miR-142-5p NC group is about 0.9 and 1.3,while the OD value for the miR-142-5p inhibitor group is about 0.5,the differences have statistical significance(p<0.01).2.4 At 72h,the apoptosis rate of cells in the control and miR-142-5p NC group is about 20%,and the the apoptosis rate of cells in the miR-142-5p inhibitor group is about 40%,the differences have statistical significance(p<0.01).2.5 Transfection of miR-142-5p inhibitor increased the expression of PTEN to about 3 times as much as it in the other two groups,the differences have statistical significance(p<0.01);,and decreased the expression of p-PI3K、p-AKT to about 1/3 and 1/4 as much as it in the other two groups,the differences have statistical significance(p<0.05).3.The size of the tumors in the miR-142-5p inhibitor group was smaller,and the tumor grows slow,the volume of the tumor is always about 50mm3 during the whole experiment;the volume of the tumors in the miR-NC groups is positively correlated with the time of the experiments;compared with the miR-142-5p inhibitor group,the size of the tumors in the miR-NC was significantly bigger;moreover,the expression of PTEN in the tumors of the miR-142-5p inhibitor group was about 2 times as much as it in the miR-NC grooup,the differences have statistical significance(p<0.05).Conclusion:1.The level of miR-142-5p is highly expressed in breast cancer tissues and the level of PTEN is low;and both are closely related to tumor size and metastasis.2.Inhibition of miRNA-142-5p can reduce breast cancer cell proliferation,promote its apoptosis,and inhibit tumor growth in vitro.3.The inhibition of miRNA-142-5p on breast cancer proliferation and apoptosis may be through the PTEN/PI3K/Akt signaling pathway. |
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