TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis - PubMed (original) (raw)

TLR3 expression correlates with apoptosis, proliferation and angiogenesis in hepatocellular carcinoma and predicts prognosis

Ming-Ming Yuan et al. BMC Cancer. 2015.

Abstract

Background: Toll-like receptor 3 (TLR3) plays a key role in innate immunity. In the present study, we analyzed tissues of patients with human hepatocellular carcinoma (HCC) to determine the significance of the relationship between TLR3 expression and cell proliferation, apoptosis, hepatitis B virus infections, angiogenesis and prognosis.

Methods: We collected paraffin-embedded tissues from 85 patients with HCC who had complete histories and were followed for >5 years. The expression and intracellular localization of TLR3 and downstream proteins (TRIF, NF-κB, and IRF3) were detected using immunohistochemistry. Further, we determined the expression of proteins that mediate cell proliferation (Ki67, cyclin D1), apoptosis (survivin, bcl-2, caspases 3, 8, and 9), and angiogenesis (CD34, MMP-2) as well as the HBV proteins HBsAg and HBcAg. Apoptosis in HCC tissues was detected using TUNEL. We conducted dual-labeling immunohistochemical analyses of TLR3 expression and TUNEL activity.

Results: TLR3 expression was significantly lower in HCC tissues compared with adjacent tissues. TRIF, NF-κB, and IRF3 correlated positively with TLR3 expression. Survivin and Bcl-2 expression correlated negatively with TLR3. The frequencies of caspases 3, 8, and 9 expression correlated positively with TLR3 signaling proteins. Cytoplasmic TLR3 and serum levels of HBsAg correlated positively. The apoptotic index determined using the TUNEL method and correlated positively with TLR3 expression. TLR3 expression in the cytoplasm correlated positively with TUNEL-positive cells and HBsAg. Ki67 and cyclin D1 correlated negatively with TLR3 expression. MMP-2 expression, microvessel density (CD34(+)) and endothelial progenitor cells (EPCs) correlated negatively with TLR3 expression. Kaplan-Meier survival analysis shows that TLR3 expression correlated with longer survival.

Conclusions: The expression of TLR3 in HCC tissues may exert a synergistic effect on apoptosis and inhibit the proliferation of HCC cells, MMP-2 expression, generation of EPCs, and angiogenesis. Moreover, TLR3 expression may serve as a prognostic marker of HCC.

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Figures

Figure 1

Figure 1

Immunohistochemical analysis of TLR3 expression and localization in HCC tissues. TLR3 was detected in the cytoplasm (a) and membrane (b). Magnification × 200.

Figure 2

Figure 2

Immunohistochemical analysis of TRIF, NF-κB, and IRF3 expression and localization in HCC tissues. TRIF was detected in the cytoplasm (a), NF-κB was detected in the cytoplasm or nucleus (b, c), and IRF3 was detected in the nucleus (d). Magnification × 200.

Figure 3

Figure 3

Immunohistochemical analysis of survivin, Bcl-2, and caspase expression and TUNEL analysis of apoptosis in HCC tissues. Survivin (a), Bcl-2 (b), and caspase 3, 8 and 9 were detected in the cytoplasm (c, d and e). Apoptotic nuclei detected using the TUNEL assay are brownish-yellow (f). Magnification × 200.

Figure 4

Figure 4

The relationship between AI and the expression of TLR3 (a), TRIF (b), IRF3 (c), and NF-κB (d).

Figure 5

Figure 5

Dual immunohistochemical analysis of apoptosis and TLR3 expression in HCC tissue. (a) In well-differentiated HCC tissue, the nuclei were TUNEL-positive and TLR3 was overexpressed at equal levels in the cytoplasm and membrane. (b) The nuclei of poorly differentiated HCC cells were TUNEL-positive, and these cells expressed relatively lower levels of cytoplasmic TLR3. Magnification × 400.

Figure 6

Figure 6

Analysis of Ki67 and Cyclin D1 expression and localization in HCC cells. (a) Ki67 was detected in the nucleus. CyclinD1 exhibited nucleus staining (b). Magnification × 200.

Figure 7

Figure 7

MMP-2 and CD34 expression and location in HCC cells. (a) MMP-2 was detected in the cytoplasm. (b) CD34 was detected in the membrane or cytoplasm of vascular endothelial cells. (c) CD34 was detected in the membrane or cytoplasm of in EPCs (red arrows). Magnification × 200.

Figure 8

Figure 8

The relationship between MVD and the expression of TLR3 (a), TRIF (b), IRF3 (c), and NF-κB (d).

Figure 9

Figure 9

The relationship between the number of EPCs and the expression of TLR3 (a), TRIF (b), IRF3 (c), and NF-κB (d).

Figure 10

Figure 10

Kaplan–Meier survival curves stratified according to the expression of TLR3 (a), TRIF (b), IRF3 (c), and NF-κB (d) (n = 76).

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