Inhibition Of Gene Beclin-1on Breast Cancer Cell Line BT-549under Normal And Hypoxic Conditions

Background and objection:Breast cancer has become the most popular tumor in female all over the world, and triple-negative breast cancer(TNBC) account for about15%.TNBC has the negative expressions of estrogen receptor(ER),progesterone receptor(PR) and human epidermal growth factor receptor2(HER2), which leads to high risk ofrecurrence and metastasis.TNBC is not only refractory to endocrine therapy targeting to ER and PR but also toTrastuzumab treatment targeting HER2. The current therapy for TNBC is operation and chemotherapy,yet neither is effective enough, and it is especially necessary to find new methods to treat TNBC.Autophagy is the new target in the research of tumor therapy, and the role of autophagyis very important in the tumorigenesis and the development. Autophagy is the process whereby organelles and some cell components are degraded by lysosomes, including the start-up phase、autophagosomes formation and the degradation. Autophagy occurs at basal levels inalmost all cells, and its main function is the degradationof cell components, including long-lived proteins, proteinaggregates and organelles produced in excess, aged, damagedand potentially dangerous or no longer needed.When the cells are lack of energy or under stimulation, autophagy provides the cells with molecules that can be used for biosynthetic purposes. However, autophagy could also play the role as an executioner of cell death(also known as autophagic or type Ⅱ cell death). Autophagy and apoptosis are two independent processes, but there are also evidences that show the common pathways between them. Beclin-1was found by Liang in1998, and it encodes a60-kDa coiledcoilprotein that interacts with the prototypic apoptosisinhibitor Bcl-2. The scientists have found in many researches that the expression of beclin-1is lower in the tumor cells than in the other ones.Beclin-1maps to a region approximately150kbcentromeric to BRCAlon chromosome17q21that iscommonly deleted in breast, ovarian, and prostatecancer. Beclin-1is one of the most decisive factors in the process of autophagy, and the overexpression of beclin-1could lead to more autophagy and autophagic cell death under stress conditions. Recent researches also demonstrated that belcin-1could have effect on the apoptosis by interacting with the prototypic apoptosis inhibitor Bcl-2.Futreal PA has found that almost half of the breast cancer cells have the absence of beclin-1on chromosome17q21, including cell BT-549. This experiment was designed and operated to find outinhibition of gene beclin-1on breast cancer cell line BT-549under normal and hypoxic conditions, and then provide evidence for the therapy targeting autophagy for TNBC.Methods:Cells BT-549were cultured in1640plus10%FBS;two kinds of plasmids,pDsRed-C1and pDS-RED-C1-Beclin l,were transferred to E. coli DH5acells; plasmids were extracted whilebeclin-lwas identified and sequenced for further study.BT-549cells were transfected with pDsRed-C1/pDS-RED-Cl-Beclin1by LipofectamineTM2000and cells were divided into three groups, the blank group cells received no treatment,BT-549.control vector group cells were transfected with pDsRed-C1and BT-549.beclinl group cells were transfected with pDS-RED-Cl-Beclin. RT-PCR was used to detected the beclin-1mRNA after transfection and the protein was detected by westernblot. After the transfection. the cells were cultured under normal and hypoxic conditions respectively:MTT was applied to evaluate the level of the proliferation in the next three days (24/48/72h); the incidence of autophagy was studied by fluorescence microscopy after staining with acridine orange (cytoplasm turns red or orange after autophagy, the others stay green); flow cytometry was applied to identify the induction of the apoptosis and cell scratch was operated to evaluate the cell migration ability (observe the change of the scratch under the microscopy once every12hours)All the experimental data was operated through SPSS13.0:LSD-t test was used to calculate the results of RT-PC、western-blot and proliferation; the interaction between beclin-1and hypoxic stimulation was evaluated by factorial design and the difference of autophagy and apoptosis between the blank group and experimental group was calculate with chi-square test. P<0.05is taken that the difference was meaningful.Results:1.The efficiency of the transfection:pDS-RED-C1contains red fluorescent protein and appears red under green fluorescence, and it’s obvious that72hours after the transfection, the efficiency got to the peak; the results of RT-PCR showed that the difference of the mRNA level was significant between experimental group and the other two groups; the protein detected by western-blot also proved that the level of the protein was higher in the experimental group. The above data was analysed with Image J1.44and the difference did make sense(P<0.05), which meant beclin-1was successfully transfected into the cells. 2.The influence of belcin-1on BT-549:(1).the proliferation:the level of proliferation in the experimental group was lower under both normal and hypoxic conditions(48h:P1=0.002,P2=0.000;72h:P1=0.006, P2=0.001;P1:normal condition,P2:hypoxic condition), and it proved that bellin-1might inhibit the proliferation. The results of the factorial design demonstrated that beclin-1and hypoxic stimulation could enhance the inhibition effect of each other on the proliferation, and the interaction between them is significant(P<0.05).(2).the difference of the incidence of autophagy was significant(P<0.0167):it’s obvious that in the experimental group, there were more cells that appeared red. The incidence of autophagy in100random cells was29%in the experimental group,15%in the control vector group and13%in the blank group under normal condition; under hypoxic condition, the incidence was48%,32%and25%respectively,(3). The incidence of apoptosis was9.8%in the experimental group and2.98%in the blank group under normal condition, and under hypoxic condition they were14.8%and4.38%respectively. It’s clear that the incidence of apoptosis was higher after the transfection(P<0.05).(4). In the scratch experiment, the scratch in the experimental group was still obvious after36hours, while in the blank group it almost disappeared. It indicated that beclin-1could reduce the cell migration ability.Conclusion:1.Theproliferation of cells BT-549was inhibited after the transfection of beclin-1under both normal and hypoxic conditions.2.The transfection of beclin-1leaded to more autophagy and apoptosis, which was the cause of the decline of the proliferation, and the transfection might in a way reduce the cell migration ability.