Effects of PAK4/LIMK1/Cofilin-1 signaling pathway on proliferation, invasion, and migration of human osteosarcoma cells

Abstract

Purpose: To explore the effects of PAK4/LIMK1/Cofilin-1 signaling pathway on the proliferation, invasion, and migration of human osteosarcoma cells.

Methods: The expression of PAK4/LIMK1/Cofilin-1 was detected by immunohistochemistry in osteosarcoma tissues. The osteosarcoma cell line MG63 was transfected and divided into Mock, Control siRNA, si-PAK4, LIMK1, and si-PAK4+LIMK1 groups. Then, the cellular biological features of MG63 cells were detected by CCK-8, wound-healing, Transwell, and flow cytometry methods. The relationship of PAK4 and LIMK1 was performed by co-immunoprecipitation test, and the protein expression of PAK4/LIMK1/Cofilin-1 was determined by Western blotting. Finally, the effect of PAK4 on the growth of osteosarcoma was verified by subcutaneous transplantation model of osteosarcoma in nude mice.

Results: The expression of PAK4/LIMK1/Cofilin-1 in both osteosarcoma tissues and cells was up-regulated. Positive PAK4, LIMK1, and Cofilin-1 expressions in osteosarcoma were associated with the clinical stage, distant metastasis, and tumor grade. The MG63 cell viability, migration, and invasion, as well as the expression of PAK4, p-LIMK/LIMK, and p-Cofilin-1/Cofilin-1, were restrained by the knock down of PAK4 while it promoted apoptosis. PAK4 silencing also suppressed the growth of subcutaneous transplanted tumor in nude mice. Co-immunocoprecipitation showed that LIMK and PAK4 protein can form complex in osteosarcoma cells. Besides, LIMK1 overexpression reversed the inhibition effect of PAK4 siRNA on the growth of osteosarcoma cells.

Conclusion: The expression of PAK4/LIMK1/Cofilin-1 pathway in osteosarcoma tissues was up-regulated. Thus, PAK4 inhibition may restrict the osteosarcoma cell proliferation, invasion, and migration but promote its apoptosis via decreasing the activity of LIMK1/Cofilin-1 pathway.

Keywords: Cofilin-1; LIMK1; PAK4; osteosarcoma.

FIGURE 1

Expression of PAK4/LIMK1/Cofilin‐1 in osteosarcoma tissues and cells. A, The expression of PAK4/LIMK1/Cofilin‐1 in osteosarcoma tissues and adjacent non‐tumor tissues detected by immunohistochemistry; B, The protein expression of PAK4, LIMK1, and Cofilin‐1 in osteosarcoma cell lines and osteoblasts cells determined by Western blotting, *, Compared with osteoblasts hFOB1.19, P < .05

FIGURE 2

Comparison of the proliferation and apoptosis of transfected osteosarcoma cells in each group. A, The proliferation of MG63 cells in each transfected group detected by CCK‐8 method; B, The apoptosis of MG63 cells in each transfected group measured by flow cytometry; *, Compared with Mock group; P < .05; #, Compared with si‐PAK4 group, P < .05; %, Compared with LIMK1 group, P < .05

FIGURE 3

Comparison of invasion and migration of MG63 cells in each transfected group. A,B, Detection of the migration ability of MG63 cells in each transfected group with wound‐healing test; C,D, Measurement of the invasion ability of osteosarcoma cells in each transfected group with Transwell test. *, Compared with Mock group, P < .05; #, Compared with si‐PAK4 group, P < .05; %, Compared with LIMK1 group, P < .05

FIGURE 4

The protein expression of PAK4/LIMK1/Cofilin‐1 in MG63 cells in each group. A, The relationship between PAK4 and LIMK1 detected by co‐immunoprecipitation method; B,C, Determination of the protein expression of PAK4/LIMK1/Cofilin‐1 in MG63 cells in each group by using Western blotting, *, Compared with Mock group, P < .05; #, Compared with si‐PAK4 group, P < .05; %, Compared with LIMK1 group, P < .05

FIGURE 5

Comparison of subcutaneous tumor of nude mice in each group. A, Growth curve of nude mice in each group; B, Representative images of the tumor morphology in nude mice; C, Comparison of the weight of transplanted tumor of nude mice in each group, *, Compared with Mock group, P < .05; #, Compared with si‐PAK4 group, P < .05; %, Compared with LIMK1 group, P < .05