Authors: Liu Liling, Xu Jiangang, Ouyang Zhikun, Yang Yangfan, Wu Kaili, Yu Minbin
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To investigate the effects of transient receptor potential melastatin (TRPM) 7 on dexamethasone (Dex)-mediated cytoskeleton remodeling in human trabecular meshwork.
Human trabecular meshwork cells (HTMs) were primarily cultured and the cells of generation 3 to 6 were used in this study.The expression of TRPM7 protein in the cells was located using immunofluorescence technology.Dex at the dose of 0.2 mg was added into culture medium for 4 days with the final concentration of 1×10-5, 1×10-6 and 1×10-7 mol/L, respectively.Western blot assay was employed to detect the relative expression level of TRPM7 protein.Cultured cells were divided into non-transfected group, siRNA transfected group, TRPM7-siRNA1 transfected group and TRPM7-siRNA2 transfected group, and the expressions of TRPM7 protein and p-cofilin protein in the cells were assayed by Western blot method.Cultured cells were divided into normal control group, Dex-treated group, siRNA transfected group and TRPM7-siRNA transfected group, and the expression of phalloidin (a cytoskeletal protein) and Vinculin (focal adhesion protein) was detected by immunofluorescence staining.In addition, cultured cells were divided into normal control group, Dex-treated group, 2-APB (a Ca2+ inhibitor) treated group, ethylene glycol tetraacetic acid (EGTA) (a calcium chelator)-treated group, TRPM7-siRNA transfected group and TRPM7-siRNA+ Dex group, and the [Ca2+ ]i in the cells was observed by Fluo-3AM immunofluorescence staining.Western blot assay was used to detect the expression of p-cofilin in the cells.
TRPM7 was positively expressed on the cell membrane.The relative expression of TRPM7 was gradually reduced with an increase of Dex dose (F=4.210, P<0.05), and the expression of TRPM7 was significantly decreased in 1×10-5 mol/L Dex group compared with the normal control group (P<0.05). Western blot assay revealed that the relative expression levels of TRPM7 in the TRPM7-siRNA1 and TRPM7-siRNA2 group were significantly lower than those of non-siRNA transfected group and siRNA transfected group (all at P<0.05). In the Dex-treated group and TRPM7-siRNA transfected group, the cells were enlarged in size with the lessened processes in comparison with the normal control group.Immunofluorescence staining showed that the actin fiber and vinculin increased in the Dex-treated group, and more spread but depolymerized actin fiber was seen in the TRPM7-siRNA transfected group.Compared with the normal control group, the fluorescence intensity of [Ca2+ ]i was weak in the Dex-treated group and TRPM7-siRNA transfected group.The relative expression levels of p-confilin protein was lower in the TRPM7-siRNA transfected group than that in the siRNA transfected group (0.317±0.031 vs.0.092±0.071) (t=5.030, P=0.007).
Dex induces the downregulation of TRPM7 expression in HTMs.The downregulation of TRPM7 probably participates in Dex-induced cytoskeletal remodeling by causing the depolymerization of actin cytoskeleton and reduction of [Ca2+ ]i in HTMs.