MicroRNA-154 Inhibits Growth And Invasion Of Breast Cancer Cells Through Targeting E2F5

Breast cancer was a major health problem that affects women worldwide.As such,detecting breast cancer at an early stage and studying its mechanism anticipated better disease outcome and prolonged patient survival.Extensive research has shown that microRNA(miRNA)are disorder at all stages of breast cancer,exhibiting both tumor suppressive and oncogenic roles.The miR-154 was downregulated and exerts suppressive role in several types of cancers including hepatocellular carcinoma,prostate cancer,osteosarcoma,colorectal cancer,non-small lung cancer,and thyroid cancer.However,the biological function and underling molecular mechanism of miR-154 in breast cancer has not been defined.Therefore,the aims of this study were to investigate the clinical significance of miR-154 in human breast cancer tissues and to clarify biological roles and the underlying mechanisms by which it is involved in tumorigenic processes of breast cancer.Objectives:To investigate the role of miR-154 in the development of breast cancer,suggesting miR-154 as a potential therapeutic target for the treatment of breast cancer.Method:miR-154 expression level was measured via quantitative real-time RT-PCR(q RT-PCR)in 36 pairs of human breast cancer tissues and adjacent normal breast tissues and in a panel of human breast cancer cell lines.Cell proliferation,cycle,migration,and invasion were assessed by CCK8 assay,flow cytometer assay,wound healing assay and transwell invasion assay,respectively.Luciferase reporter assay and Western blot was used to verify E2 F transcription factor 5 protein(E2F5)as a novel target gene of miR-154.The results are as follows:(1)miR-154 is downregulated in breast cancer tissues and cell linesTo determine the role of miR-154 in breast cancer progression,we investigated miR-154 expression in breast cancer samples and the corresponding adjacent normal tissue from 36 patients with breast cancer by real time quantitative RT-PCR(q RT-PCR).Our results showed that miR-154 expression was significantly lower in breast cancer tissue than in adjacent normal breast tissues(P<0.01).We extend our investigations to four breast cancer cell lines(MCF-7,MDA-MB-231,BT-549 and MDA-MB-453)and found that their miR-154 expression levels were significantly lower than that of human mammary epithelial cell line(MCF-10A).(2)miR-154 inhibits proliferation and colony formation of breast cancer cellsWe transfected miR-154 mimic or miR-Ctrl into MCF-7 cells and determined their miR-154 levels 48 hours after transfection.Results of q RT-PCR showed increased miR-154 levels in MCF-7 cells transfected with miR-154 mimic compared to miR-Ctrl transfected cells.We then investigated the effect of miR-154 on MCF-7 cell proliferation and colony formation.As shown in results,restoration of miR-154 expression in MCF-7 cells significantly inhibited proliferation and colony formation.It was well known that proliferation directly links to cell cycle distribution.We therefore tested cell cycle effect of miR-154 in breast cancer cells by flow cytometer assay.Our results showed that the percentage of G0/G1 phase cells increased,and the percentage of S phase cells decreased in miR-154 transfected cells compared to miR-Ctrl transfected cells.(3)miR-154 inhibits migration and invasion of breast cancer cellsIt was found that overexpression of miR-154 in MCF-7 cells led to significantly inhibited cell migration and cell invasion capability.Collectively,these results indicated that miR-154 suppressed breast cancer migration,and invasion.(4)miR-154 inhibits growth of nude mouse breast cancer cells in vivoWe transplant miR-154 transfected MCF-7 breast cancer cells into nude mice,and then test the expression of miR-154 in nude mice by real time quantitative RT-PCR(q RT-PCR).The results shows that: compare with the miR-Ctrl nude mice,the miR-154 expression was significantly higher in miR-154 transfected nude mice(P<0.01).miR-154 can inhibits the volum of breast cancer in nude mice(P<0.05).(5)E2F5 is a direct target of miR-154We used Targetscan6.2,microRNA.org,and miRWalk databases to predict potential miR-154 targets.We selected E2F5 for further analysis.We inserted wide-type or mutant-type 3'UTR into luciferase reporter vectors and cotransfected with miR-154 mimic or miR-Ctrl into MCF-7 cells.Forty-eight hours after transfection,luciferase activity was determined,and found that transfected with miR-154 mimic repressed wild-type 3'UTR-E2F5 reporter activity(P<0.01),while had no inhibition effect on the mutant 3'UTR-E2F5 reporter activity,suggesting that E2F5 may be a target of miR-154 in breast cancer.Our results demonstrated that overexpression of miR-154 in MCF-7 cells obviously decrease E2F5 expression on mRNA level and protein level.(6)E2F5 was up-regulated in breast cancer tissues and cell lines,and inversely correlated with miR-154 levels in breast cancer tissuesWe next detected expression in breast cancer samples and the corresponding adjacent normal tissues.q RT-PCR and Western blot assay showed that the expression of E2F5 on mRNA and protein level was significantly higher in breast cancer tissues than those of adjacent normal tissues.In addition,Pearson's correlation of E2F5 on mRNA and protein level was significantly higher in breast cancer tissues than those of adjacent normal tissues.In addition,Pearson's correlation assay showed that E2F5 mRNA expression level was inverse correlated with miR-154 expression in breast cancer tissue(r =-0.451,P =0.006).We also investigated E2F5 protein expression in four breast cancer cell lines(MCF-7,MDA-MB-231,BT-549 and MDA-MB-453).The Western blot showed that E2F5 protein expression level was obviously increased in four breast cancer cell lines compared with human mammary epithelial cell line(MCF-10A).(7)Knockdown E2F5 inhibited cell proliferation,colony formation,migration,and invasion in breast cellsMCF-7 cells were transfected with si-E2F5 or the si-Ctrl,then E2F5 expression was determined by western blot after transfection.The western blot showed the E2F5 protein expression levels was downregulated in si-E2F5 transfected cells compared with cells si-Ctrl transfected cells.Significantly,we also found that downregulation of E2F5 in MCF-7 cells significantly inhibited cell proliferation and colony formation,increased cell arrest at G1/G0 stage,suppressed cell migration and invasion,which mimicked the effect of miR-154 overexpression on breast cancer cells.Conclusion and innovation pointThe result of our study firstly showed that miR-154 expression was downregulated in breast cancer tissues and cell lines.Overexpression of miR-154 in breast cancer cells inhibited cell growth.Furthermore,we showed that E2F5 was a direct targetof miR-154 in breast cancer.These findings indicate that miR-154 acts as a tumor suppressor by targeting E2F5,suggesting miR-154 as a potential therapeutic target for the treatment of breast cancer.