Study On Effects Of MiR-21-5p On The Biological Character In Hep2 Cells Via Regulating Its Target Gene Laryngeal Carcinoma Related Gene 1 Expression

[Objective] To determine the functional site between mi R-21-5p and laryngeal carcinoma related gene 1, to study the mechanism of mi R-21-5p on the biological character in Hep2 cells via regulating its target gene laryngeal carcinoma related gene 1 expression. [Methods] After predicting the two binding sites between mi R-21-5p and LCRG1 in the Preliminary experiments, wild type and mutant were contructed. The mutant sequence made the seed area of corresponding mi RNA can not complementarily combine with LCRG1 3’ UTR. Then testing the luciferase activity of LCRG1 3’UTR and using mutant experiment to determine the binding sites. Using mi R-21-5p mimic and mi R-21-5p inhibitor to transfect Hep2 cells instantaneously, Real-time PCR was used to assess the mi RNA expression in Hep2 cells. Then Western blot assay was used to validate the protein level of LCRG1. MTT assay, Wound scratch assay, migration experiment, invasion experiment and flow cytometry were used to detect the influence of the biological character in Hep2 cells. [Results] ①The first binding site between mi R-21-5p and LCRG1 3’UTR in wild type can make the luciferase activity significantly lower, and the luciferase activity in wild type or mutant carrier of the second binding sites were commonly reduced. The results showed that the first binding site between mi R-21-5p and LCRG1 3’UTR was the functional site. ② Instantaneously transfected mi R-21-5p mimic, the expression of mi R-21-5p was significantly upregulated in Hep2 cells(P<0.05). And instantaneously transfected mi R-21-5p inhibitor, the expression of mi R-21-5p was significantly downregulated in Hep2 cells( P<0.05). The results showed that the transfection experiment was successful. ③The upregulation of mi R-21-5p can suppress the protein level of LCRG1, and the downregulation of mi R-21-5p can promote the protein level of LCRG1. The Western blot assay showed, compared with the mimic NC transfected group and the untransfected group, the protein level of LCRG1 was significantly decreased in the mi R-21-5p mimic transfected group(P<0.05). And compared with the inhibitor NC transfected group and the untransfected group, the protein level of LCRG1 was significantly increased in the mi R-21-5p inhibitor transfected group(P<0.05). ④The upregulation of mi R-21-5p can promote the proliferation, migration and invasion in Hep2 cells by suppressing LCRG1, and the downregulation of mi R-21-5p can suppress the proliferation, migration and invasion in Hep2 cells by promoting LCRG1. The MTT assay showed, compared with the mimic NC transfected group and the untransfected group, the growth rate of cells was significantly increased in the mi R-21-5p mimic transfected group(P<0.05). The results showed that the upregulation of mi R-21-5p can promote the proliferation in Hep2 cells by suppressing LCRG1. And compared with the inhibitor NC transfected group and the untransfected group, the growth rate of cells was significantly decreased in the mi R-21-5p inhibitor transfected group(P<0.05). The results showed that the downregulation of mi R-21-5p can suppress the proliferation in Hep2 cells by promoting LCRG1. The wound scratch assay, migration and invasion experiment showed, compared with the mimic NC transfected group and the untransfected group, the migration and invasion was significantly increased in the mi R-21-5p mimic transfected group(P<0.05). The results showed that the upregulation of mi R-21-5p can promote the migration and invasion in Hep2 cells by suppressing LCRG1. And compared with the inhibitor NC transfected group and the untransfected group, the migration and invasion was significantly decreased in the mi R-21-5p inhibitor transfected group(P<0.05). The results showed that the downregulation of mi R-21-5p can suppress the migration and invasion in Hep2 cells by promoting LCRG1. In addition, the flow cytometry showed, compared with the inhibitor NC transfected group and the untransfected group, the cell percentage of G1 phase and the rate of cell apoptosis were significantly increased. The results showed that the downregulation of mi R-21-5p can enhance cell apoptosis in Hep2 cells by promoting LCRG1 and the cell cycle was mainly arrested in G1 phase. [Conclusions] The upregulation of mi R-21-5p can enhance the cell proliferation, migration and invasion via regulating its target gene laryngeal carcinoma related gene 1 expression in Hep2 cells.