| TNF-α plays a paradoxical role in the progression of cancer. It can act as a tumornecrosis factor as well as a tumor promoting factor. Breast cancer is the commonest cancerin women all over the world. Clinical researches have revealed overexpression of TNF-αexists in breast cancer tissue and correlates with the invasive behavior and prognosis oftumor. Many preclinical studies have also suggested that TNF-α plays a crucial role inbreast cancer progression. Tumor associated macrophages (TAM) has long been consideredas the sources of TNF-α in breast cancer tissue, and mediates the promotion ofinflammation to cancer. However, several researches have found that expression ofTNF-αwas only minimally detected in infiltrating leukocytes of all groups of patients, itwas clearly observed in breast tumor cells, with mainly a cytoplasmic staining pattern. It isobvious that sources of TNF-αin breast cancer tissue are still controversial.Our previous research data as well as other researcher have identified that MDA-MB-231expresses high levels of TNF-α.In this study, we generate single stable transfectionclones of malignant MDA-MB-231breast cancer cells with a construct expressing a shRNAspecifically targeted to human TNF-αu n dr eth e co nr to l o f a U6p romoter aswell as the empty vector (p GFP-V-RS vector)transfected clone, and compare the malignantbehavior, cell cycle distribution and sensitivity to the chemotherapeutic drug Docetaxel inthese cells, so as to explore the complicated mechanisms of action for TNF-α in cancer andimprove the therapeutic strategy based on TNF-α modulation.Part Ⅰ Generation and identification of single cell stable clone inMDA-MB-231with RNAi interference of endogenous TNF-αexpression1. Basal expression level of tm TNF-αin MDA-MB-231：Using the monoclonal mouseanti-human antibody against tmTNF-α made in our laboratory, we tested the expression level of tmTNF-α on MDA-MB-231cells was61.6%by flow cytometry, and provedthe high expression level of TNF-αon MDA-MB-231.2. Transformation, exaction and sequencing of target plasmid:We transformed theshRNA plasmid specifically targeted to human TNF-αgene as well as empty vectorplasmid designed and constructed by Origene into competent DH5αbacteria, choosepositive clones, exact plasmids, and sequencing. Known target sequence existed in theplasmid with correct sequence.3. Transient transfection of TNF-αshRNA plasmid as well as empty vector plasmidinto MDA-MB-231cells:We transiently transfected TNF-αshRNA as well as itsrespective empty vector plasmid into MDA-MB-231cells and identified the efficiencyof transfection by FCM: GFP positive cells were over80%, suggesting successfultransfection of plasmids into cells.4. Monitoring the GFP expression in single cell stable cloning with fluorescentmicroscopy: We identified and mark the GFP positive single cell clones in96wellplate. Monitor the stable expression of functional plasmid under fluorescentmicroscopy by GFP positivity.5. FCM identification of single cell stable clones of MDA-MB-231：We eventuallygenerated3stable single cell clones of MDA-MB-231cells transfected with TNF-αshRNA plasmid (namely MDA-MB-231shTNF-αClone A,B,C) and two clones withempty vector plasmid.Positive rate of GFP tested in these clones were all above99%,suggesting successful screening of single cell stable cloning.6. Expression level of tmTNF-α in the stably transfected cell clones: Western blot wasused to test the expression level of tmTNF-α in the three clones MDA-MB-231shTNF-α cells compared with parental MDA-MB-231cells and the MDA-MB-231Vector. We found that tmTNF-α were successfully downregulated with the lowestexpression in MDA-MB-231shTNF-αin Clone C which is chosen for the followingexperiments. 7. mRNA level of tmTNF-αin TNF-αshRNA cells: Using realtime PCR, we found thattranscription level of tmTNF-α were downregulated by70%in MDA-MB-231shTNF-αcompared with normal and vector control cells, further verifying stabletransfection of TNF-αshRNA successfully downregulation of transcription of TNF-αgene.Part Ⅱ In vitro study of the influence of stable TNF-αsilencing onmalignant behavior and chemosensitivity to Docetaxel in MDA-MB-231cell1. Influence of cell proliferation in MDA-MB-231by stable knockdown of TNF-α:Weuse viable cell counting and Cell counting kit-8to draw the proliferation curve of the3groups of cells and found the cell growth was significantly inhibited in MDA-MB-231shTNF-α. This result is further confirmed in colony formation assay, suggestingexpression of endogenous TNF-α promotes cell growth.2. Influence of cell cycle distribution in MDA-MB-231by stable knockdown ofTNF-α:Percentage cells of shTNF-α in G0/G1phase was significantly higher thanthose of MDA-MB-231and MDA-MB-231Vector cells, with S phage correspondentlylower. These results suggested that stable knockdown of TNF-α arrested MDA-MB-231cells in G0/G1phase.3. Influence of cell migration in MDA-MB-231by stable knockdown of TNF-α:Inscratch assay, we found that migration distance of MDA-MB-231shTNF-α of only43%of that of parental MDA-MB-231cells. It suggested that stable knockdown of TNF-αreduced migration of MDA-MB-231cells.4. Influence of cell invision in MDA-MB-231by stable knockdown of TNF-α:Numbers of cell trans-migrated through the matrigel-coated membrane in transwell invasion assay was significantly reduced in MDA-MB-231shTNF-α cells, suggestingstable knockdown of TNF-α significantly reduced invasion of MDA-MB-231cells.5. Influence of anchorage independent growth in MDA-MB-231by stable knockdownof TNF-α:The colonies formed in soft agar were significantly smaller and lesser inshTNF-α group compared with MDA-MB-231and MDA-MB-231Vector group,suggesting that stable knockdown of TNF-α significantly reduced the ability ofanchorage independent growth in MDA-MB-231cells.6. Influence of chemosensitivity to Docetaxel in MDA-MB-231by stable knockdownof TNF-α:The inhibition rate were significantly higher in MDA-MB-231shTNF-α atcorresponding drug concentration compared with that of MDA-MB-231and Vectorgroup. It is clear that stable knockdown of endogenous TNF-α expression increasedsensitivity of these cells to chemotherapeutic drug Docetaxel.7. Influence of constitutive NF-κB activation in MDA-MB-231by stable knockdownof TNF-α:We evaluated the constitutive activation of NF-κB by assessing IκB andphosphorylated p65level in MDA-MB-231shTNF-α by Western blot, and found thatthe constitutive activation of NF-κB was reduced by stable knockdown of TNF-α ascompared with MDA-MB-231and MDA-MB-231Vector, indicated by increased IκBand reduced phosphorylated p65level.8. Influence on Cyclin D1expression in MDA-MB-231by stable knockdown ofTNF-α:We found that Cyclin D1expression was down-regulated in MDA-MB-231shTNF-α cells, indicating that stable knockdown of TNF-α arrest MDA-MB-231cellsin G0/G1phase by down-regulating Cyclin D1expression.In summary, we found that stable knockdown of endogenous TNF-α significantlyreduced proliferation, migration, invasion and anchorage independent growth in MDA-MB-231cells, block these cells in G0/G1phase; meanwhile increased their sensitivity tothe first line chemotherapeutic drugs Docetaxel. These results strongly suggests that endogenous TNF-α plays a significant role in the growth and progression of breast cancer. |
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