Role of microRNA-155 in Brain Metastasis of Hypoxic Lung Cancer

Abstract

To clarify the relationship between hypoxia stress and the microRNA-155 released from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer.  Methods  The hypoxia model of A549 lung cancer cells was established. Lung cancer cells were cultured under the hypoxia condition or normal oxygen condition as control for 0.5, 2, 4, 8, 12 and 24 h immunofluorescence and Western blot methods were used to determine the expression level of heat shock protein 70 (hsp70) in lung cancer cells. Hsp70 overexpressed lung cancer cell line was established, the levels of microRNA-155 in A549 and hsp70 overexpressed A549 cell culture medium were determined by qRT-PCR.An in vitro blood-brain barrier model was established, and was treated with A549 cell culture medium collected at different hypoxia time points. Horseradish peroxidase (HRP) was used to detect the changes of permeability of the in vitro blood-brain barrier model, automatic cell technical instrument was used to count A549 lung cancer cells in the culture medium in under Transwell room. Culture mediums of A549 lung cancer cells collected at different hypoxia time points were injected into rats via tail vein, Western blot was used to analyze the expression of occludin in brain tissue, Evans blue was used to detect the change of blood-brain barrier permeability in animals.  Results  When lung cancer cells were hypoxic cultured for 8 h, both the expression level of hsp70 in lung cancer cells and microRNA-155 in culture medium reached the highest level (P < 0.05). Compared with A549 cells, the enhancement of microRNA-155 level in culture medium of hsp70 overexpressed cell was more notably under hypoxia condition. At the same time, the permeability of blood-brain barrier was the highest, and the number of lung cancer cells crossed the blood-brain barrier model was the most. In animal experiment, after injection the lung cancer cell culture fluid with hypoxia 8 h, the tight junction protein occludin expression in blood-brain barrier was lowest, and the permeability of blood-brain barrier was the largest.  Conclusion  Hypoxia can cause the increase of hsp70 production in lung cancer cells. Increased hsp70 promotes the synthesis and release of microRNA-155, which in turn leads to reduced expression of occludin protein in the blood-brain barrier, resulting in increased permeability of the blood-brain barrier and eventually causing lung cancer cells to metastasize into the brain.

Keywords: Brain metastasis of lung cancer, Hsp70, microRNA-155, Tight junction protein