Hao Jin, Jia Y U, Zi-Yu Wang, Ou Qiao, Jun Wang, Jiang Han, Zhao-Lian L I
the First People's Hospital of Yunnan Province Kunming 650032, China Department of General Surgery, Affiliated Hospital of Kunming University of Science and Technology Kunming 650032, China.
Department of Hepatobiliary Surgery, People's Hospital of Wuhan University Wuhan 430060, China.
Zhongguo Zhong Yao Za Zhi. 2020 Sep;45(18):4440-4447. doi: 10.19540/j.cnki.cjcmm.20200302.401.
The aim of this paper was to investigate the effect of flavonoids of Sophorae Fructus on the proliferation, migration and invasion of hepatocellular carcinoma cells and analyze the regulatory mechanism of LncRNA FBXL19-AS1/miR-342-3 p pathway. MTT assay and plate cloning assay were used to detect the effect of flavonoids of Sophorae Fructus at different concentrations(1, 5, 10 mg·mL~(-1)) on the proliferation of liver cancer Huh7 cells. The effect of flavonoids of Sophorae Fructus on the migration and invasion of Huh7 cells was examined by Transwell chamber assay. qRT-PCR was used to detect the effect of flavonoids of Sophorae Fructus on the expression levels of FBXL19-AS1 and miR-342-3 p in Huh7 cells. The dual luciferase reporter assay was used to detect whether FBXL19-AS1 targeted at miR-342-3 p. The effect on the inhibition of FBXL19-AS1 expression or FBXL19-AS1 overexpression and then the proliferation, migration and invasion of Huh7 cells were examined by the above methods. Gelatin zymography was used to detect the activities of MMP-2 and MMP-9. The expression levels of cyclinD1, p21, MMP-2 and MMP-9 proteins were detected by Western blot. Flavonoids of Sophorae Fructus significantly inhibited the proliferation, migration and invasion of Huh7 cells(P<0.05), promoted the expression of p21 protein(P<0.05), and inhibited the expressions of cyclinD1, MMP-2 and MMP-9(P<0.05) in a dose-dependent manner, and could reduce the activity of MMP-2 and MMP-9(P<0.05). The expression level of FBXL19-AS1 was significantly decreased in Huh7 cells treated with flavonoids of Sophorae Fructus(P<0.05), whereas the expression level of miR-342-3 p was significantly increased(P<0.05). The dual luciferase reporter assay confirmed that FBXL19-AS1 targeted at the inhibition of miR-342-3 p expression. After inhibiting the expression of FBXL19-AS1, the inhibition rate of cell proliferation was significantly increased(P<0.05), the number of cell clone formation was significantly reduced(P<0.05), the number of migrated cells and the number of invasive cells were significantly decreased(P<0.05), and the expression levels of cyclinD1, MMP-2 and MMP-9 were significantly decreased(P<0.05), the activities of MMP-2 and MMP-9 were significantly reduced(P<0.05), while the expression level of p21 protein was significantly increased(P<0.05). The overexpression of FBXL19-AS1 reversed the inhibitory effect of flavonoids of Sophorae Fructus on the proliferation, migration and invasion of Huh7 cells. Flavonoids of Sophorae Fructus could inhibite the proliferation, migration and invasion of hepatoma cells by regulating LncRNA FBXL19-AS1/miR-342-3 p pathway.
本文旨在研究槐角黄酮对肝癌细胞增殖、迁移和侵袭的影响，并分析长链非编码RNA FBXL19-AS1/微小RNA-342-3p通路的调控机制。采用MTT法和平板克隆法检测不同浓度（1、5、10mg·mL⁻¹）槐角黄酮对肝癌Huh7细胞增殖的影响。采用Transwell小室法检测槐角黄酮对Huh7细胞迁移和侵袭的影响。采用qRT-PCR法检测槐角黄酮对Huh7细胞中FBXL19-AS1和微小RNA-342-3p表达水平的影响。采用双荧光素酶报告基因检测法检测FBXL19-AS1是否靶向微小RNA-342-3p。采用上述方法检测抑制FBXL19-AS1表达或过表达FBXL19-AS1后对Huh7细胞增殖、迁移和侵袭的影响。采用明胶酶谱法检测基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）的活性。采用蛋白质免疫印迹法检测细胞周期蛋白D1、p21、MMP-2和MMP-9蛋白的表达水平。槐角黄酮显著抑制Huh7细胞的增殖、迁移和侵袭（P<0.05），促进p21蛋白表达（P<0.05），并呈剂量依赖性抑制细胞周期蛋白D1、MMP-2和MMP-9的表达（P<0.05），且能降低MMP-2和MMP-9的活性（P<0.05）。经槐角黄酮处理的Huh7细胞中FBXL19-AS1表达水平显著降低（P<0.05），而微小RNA-342-3p表达水平显著升高（P<0.05）。双荧光素酶报告基因检测证实FBXL19-AS1靶向抑制微小RNA-342-3p的表达。抑制FBXL19-AS1表达后，细胞增殖抑制率显著升高（P<0.05），细胞克隆形成数显著减少（P<0.05），迁移细胞数和侵袭细胞数显著减少（P<0.05），细胞周期蛋白D1、MMP-2和MMP-9的表达水平显著降低（P<0.05），MMP-2和MMP-9的活性显著降低（P<0.05），而p21蛋白表达水平显著升高（P<0.05）。FBXL19-AS1过表达逆转了槐角黄酮对Huh7细胞增殖、迁移和侵袭的抑制作用。槐角黄酮可通过调控长链非编码RNA FBXL19-AS1/微小RNA-342-3p通路抑制肝癌细胞的增殖、迁移和侵袭。
