| BackgoundThe incidence of breast cancer has a sustainable and rapid growth inthe past20years in China. The annual growth rate is about3%to4%,much higher than the world average of0.5%. Breast cancer is still one ofthe important reasons in the world cause of death for women today. Clearevidence of more than90percent of breast cancer patients eventually diedof tumor metastasis or recurrence of breast cancer recurrence andmetastasis is the main factor affecting its efficacy. The reason for researchand looking for leads to tumor invasion and metastasis of tumor treatmentis imperative. It is very difficult to predict the occurrence of tumor distantmetastasis rate.Because of this, looking for new prognostic factors ofbreast cancer proliferation, migration movements and the transfer thatrelated to the molecular biological markers of positive significance, whichhelp to take more effective and timely treatment.Many studies have confirmed that inflammation and cancer areclosely related and persistent inflammation can induce the generation ofcancer. Cytokines and chemical factors in the increased vascular play a decisive role in the transfer and the downward adjustment of specificimmune stimulation of chronic inflammation leads to tumor releasefactors that directly promote its own growth, which constitutes aninflammatory microenvironment conducive to tumor the occurrence anddevelopment. Inflammatory stimuli, tumor cells can release a variety ofimmune factors, and after that, it is difficult to re-stimulate the tumor cellsor induce their apoptosis. In other words, the inflammation induced tumorcell resistance to apoptosis.The discovery of the Toll-like receptors (Toll like receptors, TLRs) isthe most important advances in the innate immune research in recent years.Usually TLRs is activated by agonist, must be start by the pathwaydownstream signal transduction of MyD88-dependent and-independentTRIF that the formation of the cascade reaction. Another study alsosuggested showing a strong relationship of TLRs with breast cancer.Breast cancer or any other cancer, the pathophysiological mechanisms ofTLRs-mediated invasion is not clear. TLRs expression levels that relativeto normal breast epithelial cells in breast cancer is increased. Somereacher found that the estrogen receptor negative correlation with TLRs inbreast cancer. The level of expression of TLRs plays an important role inthe clinical diagnosis of invasion and metastasis of breast cancer. Newideas can provide anti-tumor through the induction of high expression. Research purposesThis research is collecting clinical data, including patient age, tumorsize, lymph node metastasis, the TNM pathological type data, and by theslice, the detection of genes, proteins, and morphology, observed TLRreceptor expression of downstream signal changes, as well as theexpression pattern of change, understanding the relationship betweenclinical data and the innate immune downstream signals. Further studies toexplore the molecular mechanisms of MyD88and TRIF expression withbreast cancer biological characteristics,and explore if it is the the newtarget of molecular biology markers predictive of human breast cancermetastasis and molecular therapy points.Methods:1. Clinical dataInformed consent of patients, randomly collected from January2010to September201135cases of breast cancer, modified radical surgery andclinical data of patients with complete specimens, and confirmed bypathology. All patients had not received radiotherapy and chemotherapy.Were female, average age52(3872) years of age, premenopausal20cases,15cases of post-menopausal.2. Immunohistochemical stainingSlice conventional dewaxing and hydration, sliced into citrate bufferantigens hot fixes, natural cooling to room temperature, dropping3%hydrogen peroxide, at room temperature incubated; dropping goat serum closed for20min. adding rabbit anti-MyD88and TRIF antibody4°Cincubation overnight. PBS washed dropping goat anti-rabbit secondaryantibody.37℃for40min, after the PBS wash, horseradish peroxidase(HRP) labeled biotin-avidin complex. Color, dehydration, transparent, andwere mounted after microscopic examination. Each slice was randomlyselected three high-power field (×400) image analysis.3Immunohistochemical results to determinePositive staining brown granules positive signal in the nucleus orcytoplasm. Immunohistochemical interpretation of positive results,according to the following two aspects:(1) the percentage of positive cells:<1%=0;1%10%=1;>10%≤30%=2;>30%≤60%3;>60%=4.â‘¡positive cells, strong or weak staining: negative=0; light yellow=1;yellow=2; brown=3.â‘ +â‘¡is the specimen Immunohistochemicalscore:0to2-(negative),3to7+(positive,++strongly positive).4total RNA extraction and RT-PCRExtracted total RNA using Trizol kit (days root Corporation)according to the instructions. Reverse transcription reagent instructions.The PCR products were1.5%agarose gel electrophoresis, gold viewdyeing. The use of UV gel camera, gray determination by the Imagesoftware, the experiment was repeated three times. Last valve the grayvalue of GAPDH ratio between of MyD88and TRIF. 5. Western blot expression detectionRemove the collection of clinical specimens of the set stored at-80℃, the use of proteolytic cleavage reagent for cracking, then theprotein concentration to quantitatively determine the concentration of thesample. Glue, add on samples energized electrophoresis. Transfermembrane PVDF membrane by adding rabbit anti-MyD88and TRIF ananti-4°C overnight, after two anti-hybrid color cameras.6. Cell culture and transfectionDMEM medium containing10%fresh fetal calf serum, cultured at37°C,5%C02under the conditions of human breast cancer cell line wastransfected to dye siRNA MyD88and TRIF adenovirus, specifictransfection step by liposomes LipofectamineTM reagent instructions.Untransfected cells and transfected with empty vector cells served ascontrols.7. MTT assay cell proliferationAfter transfection of a single cell suspension was inoculated in96-Lculture plates, each well containing an expansion of cells cultured for24hours, each hole by adding MTT solution to continue to train for fourhours, adding of DMSO, were holes in the490nm wavelength absorptionvalues, control the volume of medium without cells.8. Cell invasion ability to detectRoom added to the media in transwell chambers, the upper chamberto join the pre-cooling of the Matrigel, serum-free DMEM, dilution, hours after transfection, cells were seeded into the upper chamber.37°C,5%CO2, incubator, cultured for hours after the exhaustion of the medium, thePBS washing, fixed methanol at room temperature with a cotton swab towipe the microporous membrane surface of cells, hematoxylin, dried atroom temperature overnight. Remove the microporous membrane, andplacing the slides on the microscope.9statistical analysesEach set of data using the SPSS18.0for statistical analysis. P <0.05was considered statistically significant.Results1. Of MyD88and TRIF in the nature of breast cancer tissue (1.5445±0.1833) and in the surrounding normal tissue of the breast cancerresistance gene (1.1058±0.1167) expression showed that the differencesbetween the two was statistically significant (P <0.01). MyD88and TRIFprotein expression in the nature of breast cancer tissue (1.0042±0.0523)and the surrounding normal tissue of the breast cancer resistance gene(0.7191±0.1102), the difference between the two was statisticallysignificant (P <0.01).2immunohistochemical assay results of MyD88and TRIF protein allpositive in35cases of breast cancer tissue expression, expression was100%. In the surrounding normal tissue in breast cancer, MyD88andTRIF in the surrounding normal tissue of breast cancer, some expression, some cells were weakly positive expression mainly in cells of theinflammatory cells, myoepithelial and spindle-shaped fibroblasts ductalepithelium did not express. of MyD88and TRIF protein showed stronghigh expression in breast cancer tissues and different cell types aredifferentially expressed. A stage II breast cancer tissue of cancer cells,including plasma cell expression of MyD88and TRIF protein; II B breastcancer organizations were strongly associated with inflammatory cellsstrongly express MyD88and TRIF protein; III A breast cancer tissue ofcancer cells and inflammatory cells strongly express high MyD88andTRIF protein.3. Breast cancer patients45years of age before and after the MyD88and TRIF expression levels are different, but no statistical significance(P>0.05) of MyD88and TRIF were correlated with clinical tumor size,invasion of the number of lymphocytes, clinical stage were positivelycorrelated (P <0.05), including clinical stage â…¢patients with breastcancer breast cancer tissue of MyD88expression was significantly higherâ… and Phase â…¡ (P <0.05).4MyD88and TRIF lentivirus siRNA gene and control transfectedMCF-7cells, among its morphology and had no significant effect,transfection efficiency can reach75%. Gene expression results showedthat:Slow virus with blank plasmid transfection group and the normalcontrol group (232.35±19.17and232.07±18.29) compared to cells of MyD88and TRIF lentivirus siRNA gene-transfected group (77.38士11.57and128.48士13.51), a statistically significant significance (P<0.01). Western blot results showed that transfection group and the normalcontrol group (0.986711±0.017and0.952425934±0.029) comparedwith blank plasmid lentiviral of MyD88and TRIF lentivirus siRNAgene-transfected cells (0.651553士0.057and0.678224211士0.047)There are statistically significant (P <0.01).5.From the growth curve before and after transfection can be seen,the two groups compared with control cells (0.16075士0.022), TRIF andMyD88lentivirus siRNA gene transfection transfection (0.181士0.035;0.183±0.024), little difference between the three. After transfection24hours between the three groups, significant differences between thethree groups (0.3165±0.032;0.251士0.033;0.25475士0.061) but notstatistically significant (P>0.05). But in the subsequent culture,48hoursbetween the three groups significant differences (0.5625士0.081;0.312士0.053;0.30275士0.042), statistically significant (P <0.05), the siRNAtransfected TRIF and MyD88gene, compared with the control group,human breast cancer MCF7cell growth was significantly decreased.6. From the results of the impact of interventions, in tumor cells withoutintervention control group, cell performance over time, MCF7cellscontinue to scratch the area for infiltration and proliferation.0hours afterthe intervention of siRNA of MyD88and TRIF genes, the two and there is no significant difference. However, there are different significantdifferences after12hours between the two groups. It can be seen notinterfere with the group of cells showed obvious infiltration and diffusion,the scratch gap has significantly narrowed the invasion capacity wassignificantly decreased, MyD88and TRIF genes in the intervention groupand did not see the obvious infiltration and proliferation, the scratches gapsignificantly shrink the spread. The MyD88and TRIF blocked the abilityto inhibit the transfer of genes for breast tumor cells.7. We apply the experimental methods of in vitro tanswell small room ofMyD88and TRIF genes in breast cancer cell line MCF-7metastaticability. Found in the experiment, compared to control cells and tumor cellsdid not interfere with the number of transmembrane (163.5±8.5) ofsiRNA of MyD88and TRIF genes intervene in cell-penetrating number(32.9±4.1;30.9士7.8) have decreased significantly, both The differencebetween the statistically significant (P <0.01). Our results find that theMyD88and TRIF blocked the ability to inhibit the transfer of genes forbreast tumor cells.Conclusion1. MyD88and TRIF protein is highly expressed in breast cancer, itsexpression and pathological features of the tumor tissue is closelyrelatieve with the clinical feature of breast cancer such as clinical tumorsize, the number of invaded lymphocytes, clinical stage was positively correlated,etc..2. MyD88and TRIF protein is widely expressed in human breastcancer MCF-7cells, play an important role in the incidence of breastcancer development. Found that low expression regulation of MyD88andTRIF protein by RNA interference that silence MyD88and TRIF genes,significant inhibit of MCF-7cell invasion and metastasis.
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