Abstract:
Objective:To investigate the relationship between astrocyte elevated gene-1 (AEG-1) and microRNA-885-5p, observe the influence of microRNA-885-5p on MHCC-97H migration and invasion, identify the target gene of microRNA-885-5p and uncover the mechanisms of AEG-1 promoting cancer metastasis.
Methods: The experimental study was adopted. The AEG-1 overexpressed plasmid constructed and the siRNA of silent AEG-1 synthesized were transiently transfected into MHCC-97H cells, respectively. For plasmid transfection, MHCC-97H cells tranfected with empty Vector NC were divided into group A as the control, and MHCC-97H cells tranfected with AEG-1 overexpressed plasmid were divided into group B. For AEG-1 siRNA transfection, MHCC-97H cells tranfected with negative siRNA were divided into group C as the control, and MHCC-97H cells tranfected with AEG-1 siRNA were divided into group D. The relative expressions of microRNA-885-5p in MHCC-97H cells of group A, B, C, D were measured by fluorescent quantitative polymerase chain reaction(PCR). For microRNA-885-5p simulator transfection, MHCC-97H cells tranfected with negative microRNA simulator were divided into group E as the control, and tranfected with microRNA-885-5p simulator were divided into group F. Transwell assay was used to evaluate MHCC-97H migration and invasion. Targetscan software was used to predict the target gene of microRNA-885-5p. MHCC-97H cells cotransfected with reporter plasmid of target gene and negative control siRNA were divided into group G as the control, and MHCC-97H cells cotransfected with reporter plasmid of target gene and microRNA-885-5p simulator were divided into group H. MHCC-97H cells cotransfected with reporter plasmid of target gene of binding region mutation and negative control siRNA were divided into group I as the control, MHCC-97H cells cotransfected with reporter plasmid of target gene of binding region mutation and microRNA-885-5p simulator were divided into group J. The luciferase activities of MHCC-97H cells in group G, H, I, J were tested. MHCC-97H cells transfected with negative control siRNA were divided into group K, and tranfected with microRNA-885-5p simulator were divided into group L. Western blot test was used to detect the relative expression of target gene of microRNA-885-5p. MHCC-97H cells transfected with siRNA of target gene of microRNA-885-5p were divided into group M, and transfected with negative control siRNA were divided into group N. Transwell assay was used to evaluate MHCC-97H migration and invasion in group M and group N. Measurement data with normal distribution were presented as
±s and comparison between groups was done using the t test.
Results:The relative expressions of microRNA-885-5p in MHCC-97H cells of group A and group B were (10.68±1.32)×10 -4 and (5.02± 0.20)×10 -4, respectively, showing significant difference between the 2 groups (t=7.357, P<0.05). The relative expressions of microRNA-885-5p in MHCC-97H cells of group C and group D were (11.04±0.97)×10 -4 and (24.15±3.71)×10 -4, respectively, showing significant difference between the 2 groups (t=5.920, P<0.05). The results of MHCC-97H migration showed that the numbers of MHCC-97H cells in lower chambers of transwell plate of group E and group F were 1 452±212 and 778±95, showing significant difference between the 2 groups (t=5.018, P<0.05). The results of MHCC-97H invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate of group E and group F were 1 237±238 and 470±70, showing significant difference between the 2 groups (t=5.353, P<0.05). MMP9 was predicted as a candidate for target gene. The luciferase activity of MHCC-97H cells was 0.73±0.03 in group G and 0.46±0.04 in group H, showing significant difference between the 2 groups (t=8.623, P<0.05) . The reporter plasmid of binding region mutation mutMMP9 was detected. The luciferase activity of MHCC-97H cells was 0.69±0.03 in group I and 0.64±0.08 in group J, showing no significant difference between the 2 groups (t=0.934, P>0.05) . The relative expressions of MMP9 in MHCC-97H cells of group K and group L were 0.75±0.03 and 0.25±0.03, respectively, showing significant difference between the 2 groups (t=19.086, P<0.05). The results of MHCC-97H migration and invasion showed that the numbers of MHCC-97H cells in lower chambers of transwell plate were 1 210±163 and 1 192±170 in group M, 537±16 and 374±55 in group N, showing significant differences between the 2 groups (t=7.111, 7.916, P<0.05).
Conclusions:AEG-1 downregulates the expression of microRNA-885-5p, the target gene of which is MMP9. MicroRNA-885-5p inhabits the migration and invasion of hepatoma cells by negative regulation of MMP9.
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