Objective: To investigate the roles of phosphatase and tensin homolog deleted on chromosome 10(PTEN) in focal adhesion kinase(FAK) and α-smooth muscle actin(α-SMA) protein levels changes in human embryonic lung fibroblasts(HELFs) induced by silica.
Methods: The HELF cells were cultured in low serum medium containing 0, 25, 50, 100 and 200 μg/cm~2 silica for 24 hours, and the cell counting kit-8(CCK8) experiment was used to determine the appropriate dose of silica for stimulation. Meanwhile, the effect of different doses of silica on the morphology of HELFs was observed under inverted microscope. 50 μg/cm~2 silica solution was used to culture HELFs for 0, 1, 2, 4, 8, 12 and 24 hours(h), and the control group were used as the control group. In addition, Western blot was used to detect HELFs PTEN, p-PTEN, FAK, p-FAK and α-SMA protein levels at each culture time. Besides, HELFs were cultured with 2×10~(-3) mol/L PTEN inhibitor(VO-Ohpic) and/or silica for 24 h, including HELFs group, HELFs plus silica group, and HELFs plus silica plus VO-Ohpic group, and the FAK, p-FAK and α-SMA in each group were also detected by Western blot.
Results: With the increase of silica dose, HELFs viability firstly increased and then decreased, and the cell viability of 50 μg/cm~2 group(144. 91±5. 10) was significantly higher than that of 0 μg/cm~2 group(101. 23±6. 57)(P<0. 05). Compared with the control group of silica treated HELFs, the expression levels of PTEN and p-PTEN at 12 h and 24 h were significantly decreased(PTEN: 0. 44±0. 08 at 12 h, 0. 25±0. 02 at 24 h; p-PTEN: 0. 09±0. 01 at 12 h, 0. 01±0. 00 at 24 h; all P values<0. 05); whereas, FAK at 12 h(0. 92±0. 05) and 24 h(0. 89±0. 01), and p-FAK(0. 77±0. 02) and α-SMA at 24 h(1. 32±0. 01) were significantly increased(all P values<0. 05). The expression levels of FAK(0. 25±0. 03), p-FAK(0. 40±0. 02) and α-SMA(0. 36±0. 01) of HELFs plus silica plus VO-Ohpic group were significantly higher than those of HELFs plus silica group(P<0. 05).
Conclusion: While silica induces HELFs FAK, p-FAK, and α-SMA increase, PTEN may downregulate FAK, p-FAK and α-SMA expression levels.
Keywords: focal adhesion kinase; human embryonic lung fibroblasts; phosphatase and tensin homolog deleted on chromosome 10; silica; silicosis; α-smooth muscle actin.