Mechanism of microRNA-182 targeting and regulating forkhead box transcription factor 2 to promote the invasion, metastasis and angiogenesis of hepatocellular carcinoma

Abstract

Objective To investigate the expression of miRNA-182 in hepatocellular carcinoma (HCC) cell lines and tissues, to verify the targeted regulation relationship between miRNA-182 and forkhead box transcription factor 2 (FOXF2) and to explore whether miRNA-182 can promote the invasion, metastasis and angiogenesis of HCC by targeting FOXF2. Methods From January to October 2017, a total of 41 patients with primary HCC admitting to Cancer Hospital Affiliated to Zhengzhou University and receiving operation were enrolled. The expression of miRNA-182 in tumor tissues and normal paracancerous tissues was detected by quantitative real-time fluorescence polymerase chain reaction (qRT-PCR). The effects of overexpression of miRNR-182 and FOXF2 on invasion and metastasis of HCC were detected by Transwell transfer experiment and the Boyden invasion experiment. The effects of miRNA-182 and FOXF2 on angiogenesis of HCC were determinded by in vitro angiogenesis experiments. The targeted regulation relationship between miRNA-182 and FOXF2 was investigated by dual luciferase reporter gene. The expression of FOXF2 at mRNA and protein level after miRNA-182 and miRNA-182 scramble transfection was detected by qRT-PCR and Western blotting method. T test was performed for statistical analysis. Results The expression in miRNA-182 in HCC tissue was higher than that in normal tissues adjacent to cancer (5.41±1.72 vs. 1.80±0.76), and the difference was statistically significant (t=-5.764, P<0.01). The results of Transwell transfer experiment and the Boyden invasion experiment showed that the number of cells passing through the membrane of the small chamber of miRNA-182 over-expression group was higher than that of miRNA-182 scramble group (85.65±4.86 vs. 31.43±2.41, 55.34±5.66 vs. 19.13±2.12), the number of cells passing through the membrane of the small chamber of miRNA-182+ FOXF2 over expression group was less than that of simple miRNA-182 over expression group (11.21±3.16 vs. 31.43±2.41, 8.67±2.83 vs. 19.13±2.12), and the differences were statistically significant (t=15.110, 9.220, 5.443 and 4.410; all P<0.05). The activity of luciferase of wild FOXF2 and miRNA-182 cotransfection group was lower than that of miRNA-182 scramble group (0.43±0.10 vs. 0.97±0.05), and the difference was statistically significant (t=8.042, P=0.012). However the activity of luciferase of mutated FOXF2 and miRNA-182 cotransfection group was higher than that of miRNA-182 scramble group (0.97±0.47 vs. 1.06±0.52), and the difference was not statistically significant (t=0.934, P=0.402). The results of in vitro angiogenesis experiments demonstrated that the number of constituent vessels in miRNA-182 over expression group was higher than that in miRNA-182 scramble group (14.27±1.77 vs. 5.91±1.42), and after FOXF2 over-expressing plasmids cotransfected, the angiogenesis ability restored (1.78±0.71 vs. 14.27±1.77), and the differences were statistically significant (t=6.530 and 4.570, both P<0.05). The results of Western blotting indicated that the expression level of FOXF2 in miRNA-182 transfection group was lower than that of miRNA-182 scramble group (1.01±0.13 vs. 4.32±0.46), and the difference was statistically significant (t=7.420, P=0.012). The results of qRT-PCR showed that the expression level of FOXF2 mRNA of miRNA-182 transfection group was lower than that in miRNA-182 scramble group (0.591±0.284 vs. 1.534±0.345), and the difference was statistically significant (t=3.465, P=0.014). Conclusion miRNA-182 can promote the invasion and metastasis of HCC cell line by targeting regulation of FOXF2. Key words: Carcinoma, hepatocellular; miRNA-182; FOXF2; Invasion and metastasis; Angiogenesis