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. 2013 Jul 17:12:77.
doi: 10.1186/1476-4598-12-77.

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

Haiyan Guo  1 , Ying Chen, Xiaobo Hu, Guanxiang Qian, Shengfang Ge, Jianjun Zhang
Affiliations

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

Haiyan Guo et al. Mol Cancer. .

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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Figures

Figure 1
Figure 1
High TLR2 levels are associated with malignant transformation and lower overall survival in CRC patients. (A) Expression of TLR2 in 39 pairs of tumour samples and matched adjacent noncancerous tissues was measured by real-time PCR. (B) Expression of TLR2 was determined by real-time PCR in 3 normal colon epithelial cells and 4 CRC cell lines (SW480, LS174T, SW620, and HCT116). (C) Expression of TLR2 was determined in 79 CRC tissues (grade1 n = 18; grade2 n = 20; grade3 n = 41) by real-time PCR. The statistical method analysed the correlation between the expression of TLR2 and tumour pathological grade. (D) Expression of TLR2 was checked in 79 CRC tissues (N0 n = 28; N1 n = 31; N2 n = 20) by real-time PCR. The statistical method analysed the correlation between the expression of TLR2 and tumour lymph node metastasis. (E) Lower overall survival of patients with high TLR2 levels (above median; n = 37) compared to patients with low TLR2 levels (below median; n = 42). (***p < 0.001; **p < 0.01; *p < 0.05).
Figure 2
Figure 2
High TLR2 levels facilitate CRC cell migration and invasion. (A) Expression of TLR2 was measured in SW620 and HCT116 cells treated with siRNA for TLR2 (siTLR2). (B) Proliferation was detected using a Cell Counting Kit-8 in SW620 and HCT116 cells treated with siTLR2. (C) Migration of SW620 and HCT116 cells treated with siTLR2 was determined using Transwell chambers untreated with matrigel. (D) Invasion of SW620 and HCT116 cells treated with siTLR2 was determined using matrigel-treated Transwell chambers. (**p < 0.01; *p < 0.05).
Figure 3
Figure 3
Expression levels of TLR2 are inversely correlated with miR-143 in CRC tissues and cell lines. (A) Expression of miR-143 was determined using real-time PCR in 79 CRC tissue samples, and the correlation of TLR2 expression levels with miR-143 in CRC tissues was evaluated using nonparametric tests (R2 = 0.4486; p = 0.000). (B) Expression of miR-143 was determined by northern blotting in 5 normal colon epithelial cells and 4 CRC cell lines (SW480, LS174T, SW620, and HCT116). (C) Expression of miR-143 was determined in 79 CRC tissues (grade1 n = 18; grade2 n = 20; grade3 n = 41) by real-time PCR. The statistical method analysed the correlation between the expression of miR-143 and tumour pathological grade. (D) Expression of miR-143 was determined in 79 CRC tissues (N0 n = 28; N1 n = 31; N2 n = 20) by real-time PCR. The statistical method analysed the correlation between the expression of miR-143 and tumour lymph node metastasis. (E) Higher overall survival of patients with high miR-143 levels (above median; n = 38) compared to patients with low miR-143 levels (below median; n = 41). (***p < 0.001; **p < 0.01; *p < 0.05).
Figure 4
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).
Figure 5
Figure 5
TLR2 modulation accounts for the antimetastatic effect of miR-143. (A) Western blot showing TLR2 expression in SW620 cells 48 h after transfection with the artificial TLR2 expression vector (TLR2). (B) The effects of miR-143, TLR2, and miR-143 combined with TLR2 on the migration and invasion of SW620 cells. (C) Western blot showing TLR2 translation in HCT116 cells 48 hours after transfection with TLR2. (D) The effects of miR-143, TLR2, and miR-143 combined with TLR2 on the migration and invasion of HCT116 cells. (E) Aliquots of 1 ×106 stable miR-143 or miR-NC over-expressing SW620 cells were injected into immunodeficient mice and evaluated for lung colonisation capacity in tail-vein assays. Lung colonisation was measured using bioluminescence at 1, 2, 3, 4, 5 and 6 weeks after injection. (F) The bar graph represents 24 hour time-point measurements of the normalised photon flux from animals injected with SW620 cells. Representative images are shown. n = 5 for each group; error bars indicate SD.
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