| Objective: Breast cancer is the second leading cause of cancer death inwomen and its incidence is increasing in many countries. In China, it isestimated40，000people die from breast cancer every year. Even though earlydetection methods and treatment options greatly improved, the resistance ofcancer cells to chemotherapeutic agents or radiation therapy frequently resultsin the subsequent recurrence and metastasis of cancer. About30%of all breastcancer patients who are successfully treated at early stages are suffering arelapse accompanied by metastasis and chemoresistance. Hence, anadvancement of the treatment by avoiding drug resistance and a betterprediction of chemotherapy efficacy would improve the clinical outcome forbreast cancer patients.MicroRNAs (miRNAs) are endogenous, short (about19~24nucleotideslong) strands of non-coding RNA that regulate the expression of multiplegenes. It is reported that miRNAs dysregulated in many cancers, such asbreast, prostate, colon and lung. Thereby, microRNAs can function asoncomiRs or tumor-suppressor-miRs depending on their respective targetgenes. MiR-155is a kind of miRNA with multifunction and produced fromthe processing of the B-Cell Integration Cluster (BIC), which is located atchromosome21q21.3. Early studies found that miR-155plays a critical role invarious physiological and pathological processes such as hematopoieticlineage differentiation, immunity, inflammation, viral infections, cancer,cardiovascular disease, and Down syndrome. As an important miRNA,miR-155overexpressed in many human tumors and is associated withproliferation, apoptosis, metastasis and poor prognosis. In normal murinemammary gland (NMuMG) epithelial cells model, miR-155plays animportant role in TGF-beta-induced EMT and cell migration and invasion by targeting RhoA. Furthermore, miR-155also plays a critical role in promotingproliferation or anti-apoptosis in cancer. Ectopic overexpression of miR-155in BT-474induces cell survival and chemoresistance to multiple agents,whereas knockdown of miR-155in HS578T renders cells to apoptosis andenhances chemosensitivity. The same results were also conducted inMDA-MB-231and MCF-7. Furthermore, other studies have shown thatmiR-155also play important role in prognosis and chemoresistance orradioresistance. For instance, miR-155is overexpressed in lung cancer, whichcorrelates with poor patient prognosis. Researchers found that increased levelsof miR-155radioprotects lung cancer cell lines such as A549or H460, whileinhibition of miR-155radiosensitizes these cells.Although a number of studies show that miR-155acts as oncomiRpromoting the development of tumor, however, in other tumors, miR-155hasthe opposite effect. It is reported that miR-155overexpression suppressedEGF-induced EMT, decreased migration/invasion capacities, inhibited cellproliferation and increased the chemosensitivity to DDP in human Caskicervical cancer cells. Xiang X et.al reported that stable overexpression ofmiR-155in4T1breast tumor cells reduces significantly the aggressiveness oftumor cell dissemination as a result of preventing epithelial-to-mesenchymaltransition (EMT) of tumor cells in vivo. While when tumor cells are injecteddirectly into the bloodstream, miR-155remarkably promotes macroscopictumor formation in the lung. Another studies also found that miR-155wassignificantly downregulated in gastric cancer cell lines compared with animmortalized gastric epithelial cell line (GES-1). Overexpression of miR-155in SGC-7901and MKN-45gastric cancer cells dramatically suppressed cellmigration, invasion and adhesion in vitro. Taken together, these data suggestthat miR-155may function as a tumor suppressor. What is the reason that therole of miR-155varied according to different tumors? It is to be our furtherresearch.To investigate the effects of microRNA-155on the proliferation,metastasis, invasion and apoptosis of4T1cells, lentiviral vector containing miR-155precusor was used to get the ectopic overexpression miR-155in4T1cells. Chemotherapy is a common treatment in breast cancer. In this study,doxorubicin was used to explore the relationship between miR-155and thechemotherapeutics.Methods：1The Lentiviral carrying miR-155precusor was packaged with themethod of transfecting293T cell lines in vitro. Then,4T1cells were infectedwith the fresh virus according to kit instructions. Then4T1-miR155cell linethat stably overexpression miR155were selected.4T-miR155-inh cell linewith transiently downregulated miR-155was obtained by using Lipofectamine2000. miR-155expression levels were detected by Taqman probe quantitativereal-time PCR.2Cell morphology was observed by light microscope. Cell proliferationwas detected by MTS. E-cadherin, vimentin expression level were detected byWestern Blot and immunofluorescence. The growth and migration of cellswere detected by transwell, wound healing assay and colony formation.CXCR4mRNA was detected by real time PCR. The apoptosis was determinedby TUNEL and Caspase3/9activity assay. Furthermore, Tumor sizes andsurvival were observed after inoculation with4T1-miR155or4T1-NC in vivo.3To explore the role of miR-155in4T1, we detected the protein level ofp-AKT, p-ERK1/2, FOXO3a as well as p-FOXO3a by Western blot. The cellcycle distribution was analyzed by flow cytometry.44T1cells were treated with different concentration of doxorubicin(DOX) at a final concentration of0μg/mL,0.2μg/mL,0.4μg/mL,0.8μg/mLand1.6μg/mL. MTS assay was performed to determine the IC50of DOX in4T1cells. miR-155expression was detected in4T1cells after DOX treated byquantitative real time PCR.5Cell proliferation assay was detected by MTS in4T1-NC or4T1-miR155after treated with DOX or not. The apoptosis was determined byTUNEL and Caspase3/9assay. Cell cycle assay was carried out by flowcytometry analysis. Results:1A stable overexpression of miR-155(4T1-miR155) and a transientknockdown miR-155cell line4T1-miR155-inh were obtained. miR-155levelwas759±39fold increased in4T1-miR155compaerd to control (4T1-NC) anddecreased81%in4T1-miR155-inh cells, respectively.2The appearance of4T1-miR155cells was changed from spindle type tocobblestone-like epithelial morphology. The expression level of E-cadherinwas significantly higher and vimentin was lower in4T1-miR155comparedwith4T1-NC. While in4T1-miR155-inh cells, there is no statistic differencein E-cadherin expression but vimentin was higher compared with control.3Compared with4T1-NC, cell proliferation was significantly decreasedin4T1-miR-155while there is no significant change in4T1-miR155-inh cells.4To investigate whether miR-155has a direct role in cell migration,transwell assay and wound healing assay were performed. Overexpression ofmiR-155impeded the migration of4T1cells significantly. Real-time PCRresults indicate that the expression level of CXCR4is higher in4T1-controlcells than in4T1-miR-155cells.5The colony formation ability of4T1-miR155was significantlydecreased than4T1-NC.6Caspase3/9activity and TUNEL assay revealed that miR-155did notinduce4T1cell apoptosis.7Overexpression of miR-155restrains tumor growth and prolongssurvival time of tumor-bearing mice in vivo.8The results of western blot analysis indicate that the level of p-AKTand p-FOXO3a in4T1-miR155cell line increased.9Flow cytometry revealed that G0/G1phrase cell proportion (%) in4T1-miR-155cells was significant higher than that in4T1-NC (4T1-miR15566.96±1.40%vs4T1-NC36.98±1.56%).10MTS assays showed that IC50of DOX in4T1cells were（0.8±0.12）μg/mL.0.7μg/mL was used as the final concentration in experiment. Whentreated with DOX at a final concentration of0.7μg/mL, the expression of miR-155was (4.5±1.5) folds increased in4T1cells.11In proliferation experiments, after treated with DOX, the inhibition ofcell growth was more pronounced in4T1-NC cells than4T1-miR155cells.12Caspase3activity of4T1-NC cells was higher after DOX treatedwhile no difference in4T1-miR155. There is no change of caspase9in bothof the two cells after DOX treated.13After treated with DOX, most of4T1-NC cells appeared apoptosiswhile4T1-miR155did not.14Cell cycle analysis showed that the G0/G1phase proportion decreasedand the G2/M phase proportion increased in4T1-NC cells after DOX treated(P<0.01). While in4T1-miR155，the G0/G1phase proportion decreased butthe G2/M phase proportion did not change. In addition, compared with4T1-NC group, the proportion of G0/G1phase increased and G2/M phasedecreased in4T1-miR155group (P<0.01).Conclusions:1A model that overexpressing or knocking down miR-155in4T1cellswas successfully obtained.2Overexpression of miR-155dramatically suppressed cell proliferation,migration, colony formation and EMT in vitro, prolonged animal survival.While knocking down expression of miR-155play the opposite role.3miR-155suppresses proliferation and migration by AKT/FOXO3apathway and induces G0/G1arrest.4miR-155induced dormancy in4T1cells.5miR-155showed antagonism to DOX in4T1cells. |
PDF Full Text Request