IL-6 promotes metastasis of non-small-cell lung cancer by up-regulating TIM-4 via NF-κB

Abstract

Objectives: Interleukin-6 (IL-6) is critical for the development of non-small-cell lung cancer (NSCLC). Recently, we identified T-cell immunoglobulin domain and mucin domain 4 (TIM-4) as a new pro-growth player in NSCLC progression. However, the role of TIM-4 in IL-6-promoted NSCLC migration, invasion and epithelial-to-mesenchymal transition (EMT) remains unclear.

Materials and methods: Expressions of TIM-4 and IL-6 were both evaluated by immunohistochemical staining in NSCLC tissues. Real-time quantitative PCR (qPCR), Western blot, flow cytometry and RT-PCR were performed to detect TIM-4 expression in NSCLC cells with IL-6 stimulation. The roles of TIM-4 in IL-6 promoting migration and invasion of NSCLC were detected by transwell assay. EMT-related markers were analysed by qPCR and Western blot in vitro, and metastasis was evaluated in BALB/c nude mice using lung cancer metastasis mouse model in vivo.

Results: High IL-6 expression was identified as an independent predictive factor for TIM-4 expression in NSCLC tissues. NSCLC patients with TIM-4 and IL-6 double high expression showed the worst prognosis. IL-6 promoted TIM-4 expression in NSCLC cells depending on NF-κB signal pathway. Both TIM-4 and IL-6 promoted migration, invasion and EMT of NSCLC cells. Interestingly, TIM-4 knockdown reversed the role of IL-6 in NSCLC and IL-6 promoted metastasis of NSCLC by up-regulating TIM-4 via NF-κB.

Conclusions: TIM-4 involves in IL-6 promoted migration, invasion and EMT of NSCLC.

Keywords: IL-6; NF-κB; TIM-4; metastasis; non-small-cell lung cancer.

Figure 1

The prognostic significance of TIM‐4 and IL‐6 in tumour cells in patients with NSCLC. A, Representative positive immunohistochemical (IHC) staining for TIM‐4 and IL‐6 of NSCLC. B, Overall Survival (OS) by TIM‐4 and IL‐6

Figure 2

IL‐6 promoted TIM‐4 expression via NF‐κB pathway. IL‐6 was used to stimulate A549 and H1975 cells. TIM‐4 mRNA and protein levels were detected by qPCR (A), Western blot (B) and flow cytometry (C), respectively. D, NF‐κB or STAT3 inhibitor was used to incubate with IL‐6 stimulated A549 or H1975 cells, and phosphorylation of P65 or STAT3 and TIM‐4 protein expression were detected by Western blot. E, In A549 and H1975 cells, the TIM‐4 promoter activity was measured using a dual fluorescent reporter assay after stimulation with IL‐6, and IL‐6 plus NF‐κB inhibitor, respectively. The box plots in A, C and E showed median ± SD of three independent experiments. ns: no significance, *P < .05, **P < .01, ***P < .001, ****P < .0001, by 2‐tailed Student's t test

Figure 3

TIM‐4 overexpression promoted metastasis of lung cancer cells. EMT‐related genes E‐Cad, N‐Cad, vimentin and slug were assayed in TIM‐4 overexpressed A549 and H1975 cells by qPCR (A) and Western blot (B), migration (C) and invasion (D) by transwell assay. The migrated and invasive cells were photographed (Bar, 100 μm). Representative pictures were shown. Data in A, C and D were shown as median ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001, by 2‐tailed Student's t test

Figure 4

IL‐6 promoted metastasis of NSCLC by up‐regulating TIM‐4 via NF‐κB in vitro. EMT‐related markers and transwell assay were performed to evaluate the effects of TIM‐4 knockdown on IL‐6 promoting metastasis of A549 and H1975 cells. E‐Cad, N‐Cad, vimentin and slug were assayed in shTIM‐4‐A549 and H1975 cells with or without IL‐6 stimulation for 48 h and in LV‐NC plus IL‐6‐A549 and H1975 cells with or without NF‐κB inhibitor by qPCR (A) and Western blot (B), respectively. (C) Migration and invasion abilities were assayed in shTIM‐4‐A549 and H1975 cells with or without IL‐6 stimulation for 24 h and in LV‐NC plus IL‐6‐A549 and H1975 cells with or without NF‐κB inhibitor. The migrated and invasive cells were photographed. Representative pictures were shown (Bar, 50 μm). Data in A, B and C were shown as median ± SD of three independent experiments. ns: no significance, *P < .05, **P < .01, ***P < .001, ****P < .0001, by 2‐tailed Student's t test

Figure 5

TIM‐4 knockdown attenuated IL‐6 promoting lung cancer metastasis in vivo. A, Experimental flow chart and design. B, Detection of TIM‐4 interference efficiency in lentivirus‐infected A549 cells by qPCR. C, Photographs of nude mice for in vivo imaging. D, Photographs of lung tissues. E, Scanning results of HE staining of lung tissues (left) and statistics of tumour nodules (right). Data in B and E were shown as median ± SD. *P < .05, **P < .01, ****P < .0001, by 2‐tailed Student's t test

Figure 6

Working model for IL‐6 promoting metastasis of NSCLC by up‐regulating TIM‐4 via NF‐κB