Calpain subunit-1 induces human renal cancer cell proliferation by activating nuclear factor-κB signaling pathway through FAK phosphorylation

Abstract

Objective To study the effect of calpain subunit-1 (Capn4) expression on the proliferation, migration and invasion of renal clear cell carcinoma (ccRCC) and to explore its molecular mechanism. Methods Up-regulation of Capn4 was performed by using RNA interference (RNAi) method in the ccRCC cell line Caki-1, Down-regulation of Capn4 was performed by using RNAi method in the ccRCC cell line 786-O. The expression level of Capn4 was confirmed by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) methods. The cell counting kit-8 (CCK-8) assay, scrape assay and Transwell assay were used to investigate the contribution of Capn4 on biological behavior of ccRCC cells. The expression of Focal Adhesion Kinase (FAK), phosphorylated FAK, nuclear factor-κB (NF-κB) and phosphorylated NF-κB was confirmed by Western blotting. We tested FAK and NF-κB phosphorylation levels after using FAK inhibitors and tested FAK and NF-κB phosphorylation levels after using NF-κB inhibitors. Results The results of CCK-8 showed that the cell proliferation ability of Capn4 overexpression in overexpression group was increased more than control group (P=0.010), and the cell proliferation ability of Capn4 short hairpin RNA (shRNA) in down group was decreased more than control group (P=0.010). The scrape assay results showed that the migration distance of Caki1-Capn4 cells in overexpression group was significantly higher than control group (P=0.001), the migration distance of 786-O-shCapn4 cells in down group was significantly lower than control group (P=0.001). Transwell invasion assay showed that 24 hours of Caki1-Capn4 cell invasion cell number in overexpression group was higher than control group (P=0.001), the migration distance of 786-O-shCapn4 cells in down group was significantly lower than control group (P=0.001). Western blotting results showed that: Caki1-Capn4 group’s FAK and NF-κB phosphorylation level in overexpression group was higher than control group (P=0.010), and 786-O-shCapn4 group’s FAK and NF-κB phosphorylation level in down group was significantly lower than control group (P=0.010); FAK specific inhibitor (PF573228) or NF-κB inhibitor QNZ could inhibit cancer cell growth rate which induced by overexpression of Capn4. Conclusion Capn4 induces human renal cancer cell proliferation by activating NF-κB signaling pathway through FAK phosphorylation. Key words: Renal cell carcinoma; Calpain small subunit 1; Focal adhesion kinase; Nuclear factor kappa-light-chain-enhancer of activated B cells