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The regulation of Toll-like receptor 2 by miR-143 suppresses the invasion and migration of a subset of human colorectal carcinoma cells.

Guo H, Chen Y, Hu X, Qian G, Ge S, Zhang J - Mol. Cancer (2013)

Bottom Line: We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2.Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo. miR-143 blocks the TLR2 signalling pathway in human CRC cells.This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Laboratory, Shanghai Third People's Hospital, School of medicine, Shanghai Jiao Tong University, Shanghai 200019, PR China. sxguohaiyan@126.com

ABSTRACT

Background: The Toll-like receptor 2 (TLR2)-driven tissue response may promote neoangiogenesis and tumour growth by mechanisms that are poorly understood.

Methods: We investigated the expression levels of TLR2 and associated-miRNAs in colorectal carcinoma (CRC) tissues and cell lines using real-time PCR, northern blotting and western blotting. Survival curver was generated by Log-Rank test and the role of TLR2 signalling in tumour invasion and migration was determined by transwell analysis kits.

Results: We observed that the tissues from CRC patients express relatively high levels of TLR2. Targeting TLR2 markedly reduces the invasion and migration of CRC cells. We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2. Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo.

Conclusion: miR-143 blocks the TLR2 signalling pathway in human CRC cells. This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

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miR-143 directly targets TLR2. (A) Expression of TLR2, TLR3, TLR4, and TLR5 was measured in SW620 and HCT116 cells transfected with miR-143. NC is a double-stranded RNA and represents the miRNA mimic negative control. (B) Expression of TLR2 was measured in SW620 and HCT116 cells transfected with Anti-miR-143. NC is a single-stranded RNA and represents the anti-miRNA negative control. (C) The levels of TLR2 mRNA are negatively regulated by miR-143 in SW620 and HCT116 cells. (D) The sequence alignment of miR-143 with the putative binding site in the 3'UTR of the TLR2 gene. Reporter assay in SW620 cells transfected with luciferase constructs (UTR-WT or UTR-MUT) (mean ± SD). (E) Inhibition of migration and invasion of cancer cells by miR-143 was measured using Transwell chambers untreated with matrigel and matrigel-treated Transwell chambers. (WT: wild type; MUT: mutant; **p < 0.01; ***p < 0.001).
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Figure 4: miR-143 directly targets TLR2. (A) Expression of TLR2, TLR3, TLR4, and TLR5 was measured in SW620 and HCT116 cells transfected with miR-143. NC is a double-stranded RNA and represents the miRNA mimic negative control. (B) Expression of TLR2 was measured in SW620 and HCT116 cells transfected with Anti-miR-143. NC is a single-stranded RNA and represents the anti-miRNA negative control. (C) The levels of TLR2 mRNA are negatively regulated by miR-143 in SW620 and HCT116 cells. (D) The sequence alignment of miR-143 with the putative binding site in the 3'UTR of the TLR2 gene. Reporter assay in SW620 cells transfected with luciferase constructs (UTR-WT or UTR-MUT) (mean ± SD). (E) Inhibition of migration and invasion of cancer cells by miR-143 was measured using Transwell chambers untreated with matrigel and matrigel-treated Transwell chambers. (WT: wild type; MUT: mutant; **p < 0.01; ***p < 0.001).

Mentions: To examine the down-regulatory effect of miR-143 on TLR2 expression, we performed a western blot analysis 48 h after transfecting SW620 and HCT116 cells with the miR-143 mimic (100 nM) or Anti-miR-143 (100 nM). Compared to the negative control group (miR-NC mimic), ectopic expression of miR-143 significantly decreased the expression of TLR2 protein levels (Figures 4A and Additional file 4: Figure S4A). This inhibition was abolished by treatment with Anti-miR-143, which is an antagonist for miR-143 (Figures 4B and Additional file 4: Figure S4B). Previous studies have reported that the Toll-like receptor family members TLR3 [27], TLR4 [28], and TLR5 [29] were up-regulated in colorectal carcinoma tissues. Therefore, we examined whether TLR3, TLR4, and TLR5 are also regulated by miR-143 in human CRC. The expression of TLR3, TLR4, and TLR5 protein was not affected by miR-143 in SW620 and HCT116 cells (Figures 4A and Additional file 4: Figure S4A). Using a qRT-PCR assay, we detected miR-143-induced down-regulation of TLR2 mRNA expression in SW620 and HCT116 cells, and a blockage of endogenous miR-143 up-regulated expression of TLR2 mRNA in SW620 and HCT116 cells (Figures 4C and Additional file 4: Figure S4C). These results suggest that miR-143 targets TLR2 by playing a role in mRNA cleavage.


The regulation of Toll-like receptor 2 by miR-143 suppresses the invasion and migration of a subset of human colorectal carcinoma cells.

Guo H, Chen Y, Hu X, Qian G, Ge S, Zhang J - Mol. Cancer (2013)

miR-143 directly targets TLR2. (A) Expression of TLR2, TLR3, TLR4, and TLR5 was measured in SW620 and HCT116 cells transfected with miR-143. NC is a double-stranded RNA and represents the miRNA mimic negative control. (B) Expression of TLR2 was measured in SW620 and HCT116 cells transfected with Anti-miR-143. NC is a single-stranded RNA and represents the anti-miRNA negative control. (C) The levels of TLR2 mRNA are negatively regulated by miR-143 in SW620 and HCT116 cells. (D) The sequence alignment of miR-143 with the putative binding site in the 3'UTR of the TLR2 gene. Reporter assay in SW620 cells transfected with luciferase constructs (UTR-WT or UTR-MUT) (mean ± SD). (E) Inhibition of migration and invasion of cancer cells by miR-143 was measured using Transwell chambers untreated with matrigel and matrigel-treated Transwell chambers. (WT: wild type; MUT: mutant; **p < 0.01; ***p < 0.001).
© Copyright Policy - open-access
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Figure 4: miR-143 directly targets TLR2. (A) Expression of TLR2, TLR3, TLR4, and TLR5 was measured in SW620 and HCT116 cells transfected with miR-143. NC is a double-stranded RNA and represents the miRNA mimic negative control. (B) Expression of TLR2 was measured in SW620 and HCT116 cells transfected with Anti-miR-143. NC is a single-stranded RNA and represents the anti-miRNA negative control. (C) The levels of TLR2 mRNA are negatively regulated by miR-143 in SW620 and HCT116 cells. (D) The sequence alignment of miR-143 with the putative binding site in the 3'UTR of the TLR2 gene. Reporter assay in SW620 cells transfected with luciferase constructs (UTR-WT or UTR-MUT) (mean ± SD). (E) Inhibition of migration and invasion of cancer cells by miR-143 was measured using Transwell chambers untreated with matrigel and matrigel-treated Transwell chambers. (WT: wild type; MUT: mutant; **p < 0.01; ***p < 0.001).
Mentions: To examine the down-regulatory effect of miR-143 on TLR2 expression, we performed a western blot analysis 48 h after transfecting SW620 and HCT116 cells with the miR-143 mimic (100 nM) or Anti-miR-143 (100 nM). Compared to the negative control group (miR-NC mimic), ectopic expression of miR-143 significantly decreased the expression of TLR2 protein levels (Figures 4A and Additional file 4: Figure S4A). This inhibition was abolished by treatment with Anti-miR-143, which is an antagonist for miR-143 (Figures 4B and Additional file 4: Figure S4B). Previous studies have reported that the Toll-like receptor family members TLR3 [27], TLR4 [28], and TLR5 [29] were up-regulated in colorectal carcinoma tissues. Therefore, we examined whether TLR3, TLR4, and TLR5 are also regulated by miR-143 in human CRC. The expression of TLR3, TLR4, and TLR5 protein was not affected by miR-143 in SW620 and HCT116 cells (Figures 4A and Additional file 4: Figure S4A). Using a qRT-PCR assay, we detected miR-143-induced down-regulation of TLR2 mRNA expression in SW620 and HCT116 cells, and a blockage of endogenous miR-143 up-regulated expression of TLR2 mRNA in SW620 and HCT116 cells (Figures 4C and Additional file 4: Figure S4C). These results suggest that miR-143 targets TLR2 by playing a role in mRNA cleavage.

Bottom Line: We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2.Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo. miR-143 blocks the TLR2 signalling pathway in human CRC cells.This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Laboratory, Shanghai Third People's Hospital, School of medicine, Shanghai Jiao Tong University, Shanghai 200019, PR China. sxguohaiyan@126.com

ABSTRACT

Background: The Toll-like receptor 2 (TLR2)-driven tissue response may promote neoangiogenesis and tumour growth by mechanisms that are poorly understood.

Methods: We investigated the expression levels of TLR2 and associated-miRNAs in colorectal carcinoma (CRC) tissues and cell lines using real-time PCR, northern blotting and western blotting. Survival curver was generated by Log-Rank test and the role of TLR2 signalling in tumour invasion and migration was determined by transwell analysis kits.

Results: We observed that the tissues from CRC patients express relatively high levels of TLR2. Targeting TLR2 markedly reduces the invasion and migration of CRC cells. We also found that miR-143, a putative tumour suppressor that is down-regulated in CRC tissues, reduces the invasion and migration of CRC cells primarily via TLR2. Utilising a xenograft mouse model, we demonstrated that re-expression of miR-143 inhibits CRC cell colonisation in vivo.

Conclusion: miR-143 blocks the TLR2 signalling pathway in human CRC cells. This knowledge may pave the way for new clinical applications utilising miR-143 mimics in the treatment of patients with CRC.

Show MeSH
Related in: MedlinePlus