Influence of matrix stiffness on PKN3 expression and invasion/metastasis in hepatocellular carcinoma
Objective: To investigate the relationship between expression of PKC-related kinase N3 (PKN3) and the invasion and metastasis of hepatocellular carcinoma (HCC) cells and the mechanism. Methods: The collagen fiber content and PKN3 expression in the specimens of 72 HCC tissues with hemorrhagic/necrotic phenotype (HN-HCC) and 32 HCC tissues without hemorrhagic/necrotic phenotype (NHN-HCC) were determined. HCC HCCLM3 cells were cultured in polyacrylamide hydrophilic gels of different stiffness, and then, the migration and invasion abilities and the PKN3 gene and protein expressions as well as RhoC activity were measured. After PKN3 interference or PKN3 interference with concomitant RhoC overexpression, the changes in migration and invasion abilities and protein expressions of ROCK2, E-cadherin, Fibronectin in HCCLM3 cells were analyzed. Results: Compared with NHN-HCC tissue, collagen fiber content and PKN3 protein expression were significantly increased in HN-HCC (both P<0.05); in HCCLM3 cells cultured in firm matrix compared with HCCLM3 cells cultured in soft matrix, both gene and protein expressions of PKN3, and the migration and invasion abilities as well as the RhoC activity were significantly increased (all P<0.05), while the RhoC activity was suppressed with inhibition of PKN3 expression. Compared with control HCCLM3 cells, the migration and invasion abilities and the protein expressions of FAK, ROCK2 and Fibronectin were significantly decreased, and E-cadherin protein expression was significantly increased in HCCLM3 cells after PKN3 interference (all P<0.05), while the above changes were not obvious in HCCLM3 cells after PKN3 interference with concomitant RhoC overexpression (all P>0.05). Conclusion: Increased matrix stiffness can up-regulate the activity of PKN3-ROCK2 signaling pathway, and thereby promote the invasion and metastasis of HCC, which may be an important mechanism for the high invasion and metastasis ability of HN-HCC.