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Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion.

Yang H, Zhou H, Feng P, Zhou X, Wen H, Xie X, Shen H, Zhu X - J. Exp. Clin. Cancer Res. (2010)

Bottom Line: TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells).TLR4AsiRNA mediated the strongest effect.The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Laboratory, The Second affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.

ABSTRACT

Background: Tumor cell expression of Toll-like receptors (TLRs) can promote inflammation and cell survival in the tumor microenvironment. Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, and it is regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. The expression of TLR4 in human breast cancer cell line MDA-MB-231 and its biological function in the development and progression of breast cancer have not been investigated. We sought to characterize the expression of TLR1-TLR10 in the established human breast cancer cell line MDA-MB-231, and to investigate the biological roles of TLR4 in breast cancer cells growth, survival, and its potential as a target for breast cancer therapy.

Methods: TLRs mRNA and protein expressions were detected in human breast cancer cell line MDA-MB-231 by RT-PCR, real-time PCR and flow cytometry (FCM). RNA interference was used to knockdown the expression of TLR4 in MDA-MB-231. MDA-MB-231 transfected with the vector pGenesil-1 and the vector containing a scrambled siRNA were as controls. Recombinant plasmids named TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA specific to TLR4 were transfected into human breast cancer cell line MDA-MB-231 with Lipfectamine 2000 reagent. TLR4 mRNA and protein expressions were investigated by RT-PCR, real-time PCR, FCM and immunofluorescence after silence. MTT analysis was performed to detect cell proliferation and FCM was used to detect the secretion of inflammatory cytokines in supernatant of transfected cells.

Results: The human breast cancer cell line MDA-MB-231 was found to express TLR1-TLR10 at both the mRNA and protein levels. TLR4 was found to be the highest expressed TLR in MDA-MB-231. TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells). TLR4AsiRNA mediated the strongest effect. Knockdown of TLR4 gene in MDA-MB-231 resulted in a dramatic reduction of breast cancer cell viability. The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control. No significant difference was observed in siRNA control (Recombinant plasmid named ScrambledsiRNA transfected cells) compared to vector control.

Conclusions: These studies identified the expression levels of multiple TLRs in human breast cancer cell line MDA-MB-231 and demonstrated that knockdown of TLR4 could actively inhibit proliferation and survival of breast cancer cells. Taken together, our results suggest RNAi-directed targeting of TLR4 may be a beneficial strategy for breast cancer therapy.

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mRNA and protein expression levels of TLRs (TLR1 through TLR10) in human breast cancer cell line MDA-MB-231. A, semi-quantitative RT-PCR of TLRs in MDA-MB-231. The GAPDH mRNA was amplified as control. B, real-time RT-PCR of TLRs in MDA-MB-231. The expression of TLR3 was normalized to 1.0 as it was expressed weakest among all TLRs. C, Flow cytometry of TLRs protein expression levels in MDA-MB-231. All results are representative of three separate experiments.
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Figure 1: mRNA and protein expression levels of TLRs (TLR1 through TLR10) in human breast cancer cell line MDA-MB-231. A, semi-quantitative RT-PCR of TLRs in MDA-MB-231. The GAPDH mRNA was amplified as control. B, real-time RT-PCR of TLRs in MDA-MB-231. The expression of TLR3 was normalized to 1.0 as it was expressed weakest among all TLRs. C, Flow cytometry of TLRs protein expression levels in MDA-MB-231. All results are representative of three separate experiments.

Mentions: As TLRs have been identified in some tumor cells, we sought to detect if they were expressed in the human breast cancer cell line MDA-MB-231. Qualitative RT-PCR analysis revealed that MDA-MB-231 expressed mRNA of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 (Figure 1A). Real-time PCR analysis revealed the relative expressions of each TLR examined. The expression of TLR3 was normalized to 1.0, as it was expressed the most weakly. TLR4 was 5-fold higher than TLR3, while other TLRs were expressed between 1- and 4-fold higher than TLR3 (Figure 1B). By FCM detection, we were able to examine the different protein expression levels of the TLRs, TLR4, TLR6, TLR7 and TLR5 were expressed moderately; the other TLRs were expressed weakly or unexpressed. Again, TLR4 protein level was the highest out of TLR1-TLR10 (Figure 1C). Collectively, these results demonstrated that MDA-MB-231 expressed all the TLRs examined (TLR1-TLR10) and TLR4 was expressed highest. TLR4 was strategically selected to investigate its function on the growth and progression of MDA-MB-231 in subsequent studies.


Reduced expression of Toll-like receptor 4 inhibits human breast cancer cells proliferation and inflammatory cytokines secretion.

Yang H, Zhou H, Feng P, Zhou X, Wen H, Xie X, Shen H, Zhu X - J. Exp. Clin. Cancer Res. (2010)

mRNA and protein expression levels of TLRs (TLR1 through TLR10) in human breast cancer cell line MDA-MB-231. A, semi-quantitative RT-PCR of TLRs in MDA-MB-231. The GAPDH mRNA was amplified as control. B, real-time RT-PCR of TLRs in MDA-MB-231. The expression of TLR3 was normalized to 1.0 as it was expressed weakest among all TLRs. C, Flow cytometry of TLRs protein expression levels in MDA-MB-231. All results are representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913950&req=5

Figure 1: mRNA and protein expression levels of TLRs (TLR1 through TLR10) in human breast cancer cell line MDA-MB-231. A, semi-quantitative RT-PCR of TLRs in MDA-MB-231. The GAPDH mRNA was amplified as control. B, real-time RT-PCR of TLRs in MDA-MB-231. The expression of TLR3 was normalized to 1.0 as it was expressed weakest among all TLRs. C, Flow cytometry of TLRs protein expression levels in MDA-MB-231. All results are representative of three separate experiments.
Mentions: As TLRs have been identified in some tumor cells, we sought to detect if they were expressed in the human breast cancer cell line MDA-MB-231. Qualitative RT-PCR analysis revealed that MDA-MB-231 expressed mRNA of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 (Figure 1A). Real-time PCR analysis revealed the relative expressions of each TLR examined. The expression of TLR3 was normalized to 1.0, as it was expressed the most weakly. TLR4 was 5-fold higher than TLR3, while other TLRs were expressed between 1- and 4-fold higher than TLR3 (Figure 1B). By FCM detection, we were able to examine the different protein expression levels of the TLRs, TLR4, TLR6, TLR7 and TLR5 were expressed moderately; the other TLRs were expressed weakly or unexpressed. Again, TLR4 protein level was the highest out of TLR1-TLR10 (Figure 1C). Collectively, these results demonstrated that MDA-MB-231 expressed all the TLRs examined (TLR1-TLR10) and TLR4 was expressed highest. TLR4 was strategically selected to investigate its function on the growth and progression of MDA-MB-231 in subsequent studies.

Bottom Line: TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells).TLR4AsiRNA mediated the strongest effect.The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Laboratory, The Second affiliated Hospital of Soochow University, Suzhou, Jiangsu, China.

ABSTRACT

Background: Tumor cell expression of Toll-like receptors (TLRs) can promote inflammation and cell survival in the tumor microenvironment. Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, and it is regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. The expression of TLR4 in human breast cancer cell line MDA-MB-231 and its biological function in the development and progression of breast cancer have not been investigated. We sought to characterize the expression of TLR1-TLR10 in the established human breast cancer cell line MDA-MB-231, and to investigate the biological roles of TLR4 in breast cancer cells growth, survival, and its potential as a target for breast cancer therapy.

Methods: TLRs mRNA and protein expressions were detected in human breast cancer cell line MDA-MB-231 by RT-PCR, real-time PCR and flow cytometry (FCM). RNA interference was used to knockdown the expression of TLR4 in MDA-MB-231. MDA-MB-231 transfected with the vector pGenesil-1 and the vector containing a scrambled siRNA were as controls. Recombinant plasmids named TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA specific to TLR4 were transfected into human breast cancer cell line MDA-MB-231 with Lipfectamine 2000 reagent. TLR4 mRNA and protein expressions were investigated by RT-PCR, real-time PCR, FCM and immunofluorescence after silence. MTT analysis was performed to detect cell proliferation and FCM was used to detect the secretion of inflammatory cytokines in supernatant of transfected cells.

Results: The human breast cancer cell line MDA-MB-231 was found to express TLR1-TLR10 at both the mRNA and protein levels. TLR4 was found to be the highest expressed TLR in MDA-MB-231. TLR4AsiRNA, TLR4BsiRNA and TLR4CsiRNA were found to significantly inhibit TLR4 expression in MDA-MB-231 at both mRNA and protein levels as compared to vector control(vector transfected cells). TLR4AsiRNA mediated the strongest effect. Knockdown of TLR4 gene in MDA-MB-231 resulted in a dramatic reduction of breast cancer cell viability. The cytokines which were secreted by the TLR4 silenced cells, such as IL-6 and IL-8, also decreased significantly as compared with vector control. No significant difference was observed in siRNA control (Recombinant plasmid named ScrambledsiRNA transfected cells) compared to vector control.

Conclusions: These studies identified the expression levels of multiple TLRs in human breast cancer cell line MDA-MB-231 and demonstrated that knockdown of TLR4 could actively inhibit proliferation and survival of breast cancer cells. Taken together, our results suggest RNAi-directed targeting of TLR4 may be a beneficial strategy for breast cancer therapy.

Show MeSH
Related in: MedlinePlus