Role and mechanism of Annexin A9 in the proliferation of human colon carcinoma cell line SW620

Abstract

Objective To investigate the role of Annexin A9 (ANXA9) in the proliferation of human colon cancer cell line SW620 and the underlying mechanism. Methods The expression of ANXA9 in human colon cancer cell lines HT29, SW620, SW480, LoVo and SW1116 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. SW620 cell lines with the strongest expression of ANXA9 was transfected with 40 nmol/L ANXA9-small interfering RNA (siRNA) to inhibit the expression of ANXA9. Cell counting kit-8 (CCK-8) assay and flow cytometry (FCM) were correspondingly used to examine the effect of transfection on the activity and cell cycle of SW620 cells. The expression of proliferating cell nuclear antigen (PCNA), Cyclin D, Cyclin E, p21 and p16 was detected by Real-time PCR and Western blotting. Results The mRNA and protein expression levels of ANXA9 in SW620 cells were the highest in human colon cancer cell lines HT29, SW620, SW480, LoVo and SW1116 and mRNA expressions were as following: 0.027±0.004, 0.041±0.005, 0.028±0.003, 0.033±0.003, 0.025±0.005; protein expressions were as following: 0.287±0.076, 1.246±0.103, 0.326±0.040, 0.387±0.097, 0.295±0.064 (F=8.212, P=0.003; F=81.728, P=0.000). Expressions of ANXA9 mRNA and protein in transfection group, control group, blank group were (0.209±0.016, 0.317±0.104), (0.957±0.070, 0.647±0.155), (1.031±0.171, 0.727±0.145). The ANXA9 mRNA and protein expression in the transfection group was significantly lower than in the control and blank group (F=54.072, P=0.000; F=7.601, P=0.023). Results of CCK-8 showed that cell activity in transfection group, control group, blank group was as following: 24 h (0.605±0.024, 0.612±0.022, 0.628±0.027), 48 h (0.810±0.049, 1.196±0.118, 1.308±0.070), 72 h (1.145±0.070, 1.531±0.062, 1.454±0.081). Cell activity in transfection group were significantly lower than the control and blank group (F=38.742, P=0.000; F=32.56, P=0.000). The percentage of cells in G0/G1 phase, G2/M phase, S phase was (66.870±3.850)%, (15.060±2.480)%, (18.080±1.420)% in transfection group; 50.070±3.370)%, (32.520±2.500)%, (17.410±0.920)% in control group; (48.740±2.170)%, (32.390±1.820)%, (19.210±0.390)% in blank group. The percentage of G0/G1 phase cells in the transfection group increased (F=29.752, P=0.001), and the proportion of G2/M phase decreased (F=57.860, P=0.000), while the proportion of S phase cells had no significant change (F=1.568, P=0.284). The expression of Cyclin D1 and Cyclin E1 was significantly lower after ANXA9-siRNA transfection (mRNA: F=462.821, 5.512, P=0.000, 0.044; protein: F=15.148, 8.300, P=0.005, 0.019), and that of p21 was significantly increased (mRNA: F=6.742, P=0.032; protein: (F=21.230, P=0.002). Conclusion ANXA9 may participate in the proliferation of colon cancer cell line SW620 possibly through the expression of Cyclin D1, Cyclin E1, p21. Key words: Colon cancer; Annexin A9; Proliferation