Expressions Of Toll-like Receptors And Toll-like Receptor 4 Gene Silence Of Human Breast Cancer Cells

Objective: To study the expression and significances of TLRs, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10, in the human breast cancer cell lines MDA-MB-231 and MCF-7. TLR4 was chosen as target gene for its apparently different expression in the two cell lines. RNA interference (RNAi) technology was used to silence human TLR4 gene in breast cancer MDA-MB-231 cells, and then biological function changes such as mRNA and protein expression of TLR4, cell proliferation, cell apoptosis and secretion of cell inflammatory factors were determined before and after silence. Investigation of TLR4 biology effects in the development and progress of breast cancer will provide an experimental foundation for further investigation of breast cancer gene therapy targeting TLR4 gene.Methods: (1) TLRs mRNA expressions were detected in breast cancer cell lines MDA-MB-231 and MCF-7 by RT-PCR and Real-time PCR. TLRs protein expressions were detected by flow cytometry (FCM) and Immunofluorescence methods. (2) TLR4-siRNA-pGenesil-1 was constructed, including A,B,C three recombinant plasmids, named TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA. (3) TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA specific to TLR4 were transfected into human breast cancer MDA-MB-231 cells respectively through LipfectamineTM2000 reagent. Meanwhile, plasmid vector pGenesil-1 (pGsil-1) group and untranfected cell group were as controls. TLR4 mRNA changes were investigated by RT-PCR and Real-time PCR methods after silence. (4) After TLR4AsiRNA, TLR4BsiRNA, TLR4CsiRNA and pGsil-1 transfection, TLR4 protein expression in MDA-MB-231 cells was detected by FCM (5) To research the biological function changes of MDA-MB-231 cells transfected with TLR4AsiRNA and pGsil-1 respectively, Immunofluorescence method was used to detect TLR4 protein expression, DAPI staining was adopted to detect cell apoptosis. MTT analysis was performed to detect cell proliferation and FCM was used to detect the secretion of cytokines (IL-6, IL-8).Results: (1) Human breast cancer cell lines MDA-MB-231 and MCF-7 expressed TLRs including TLR1-TLR10 at both mRNA and protein levels. Comparing the TLR/GAPDH mRNA relative amount between the two cell lines, the expression of TLR4, TLR6, TLR8 and TLR9 in MDA-MB-231 cells was obviously higher than that in MCF-7 cells respectively, and the expression in MDA-MB-231 cells was 39.4±3.2, 10.8±2.4, 5.8±2.6 and 3.2±2.5 times as much as that in MCF-7 cells respectively (P<0.01). No significant difference of TLR1, TLR2, TLR3, TLR5, TLR7 and TLR10 was observed in two cell lines (P>0.05). Protein expression of TLR4, TLR6, TLR7 and TLR5 in MDA-MB-231 cells was significantly higher than that in MCF-7 cells respectively, and the expression in MDA-MB-231 cells was 6.4±1.6, 4.1±0.9, 3.7±0.8 and 2.8±0.4 times as much as that in MCF-7 cells respectively(P<0.01). (2) The recombinant plasmids of TLR4-siRNA-pGenesil-1:TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were successfully constructed. (3) Compared with negative control, TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA dramatically depressed TLR4 expression in MDA-MB-231 cells at mRNA level. The inhibition ratio was 75.1±3.8%, 57.3±3.5% and 62.3±4.3% respectively (P<0.01).However, no significant difference was observed in pGsil vector transfected cells (P>0.05). (4) The results of FCM showed that the protein expression inhibition ratio was 53.7%±2.9%, 38.8%±3.7% and 46.3%±3.5% in TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA group when compared with negative control respectively(P<0.01),but no obvious difference was seen in pGsil vector group (P>0.05). (5) TLR4AsiRNA had the strongest effect for silencing TLR gene. (6) At 48 hours transfection by TLR4AsiRNA, red fluorescence on MDA-MB-231 cells stained with TLR4 was drastically reduced, which meant the deduction of TLR4 protein. DAPI staining results showed that there were many apoptotic bodies in MDA-MB-231 cells transfected by TLR4AsiRNA. MTT results indicated that the proliferation rate of MDA-MB-231 cells transfected with TLR4AsiRNA was significantly reduced. The FCM results indicated that the inhibition ration of cytokine IL-6 and IL-8 was 46.5±3.5% and 48.9±2.8% respectively when compared with negative control (P<0.01), no significant difference was seen in pGsil vector group. Conclusions: These studies demonstrated that TLRs expressed in human breast cancer cell lines MDA-MB-231 and MCF-7. RNAi specific to TLR4 could have the ability to inhibit cell proliferation, to induce cell apoptosis and to deduce secretion of IL-6 and IL-8 in human breast cancer MDA-MB-231 cells. Thus, TLR4 may provide new basement for breast cancer target gene therapy.