Tetrandrine Regulates the Proliferation, Apoptosis, Migration and Invasion of Esophageal Cancer Cells Via the miR-149-5p/HMGA2 Axis
CHANG Jian1*, LIU Qing’e1, YAN Lijuan1, TIAN Li1, LI Sen1, LI Zhiguo2, XU Jianping1
This study aims to investigate the effects of Tet (tetrandrine) on the proliferation, apoptosis, mi gration and invasion of esophageal cancer cells through the miR-149-5p/HMGA2 (high mobility group protein A2) axis. The expression levels of miR-149-5p and HMGA2 mRNA in esophageal cancer cell lines (EC9706, Eca109, TE-13, TE-1) and normal esophageal epithelial cells HEECs were detected by qRT-PCR, and the best cell lines were screened for subsequent experiments; the optimal cell line EC9706 obtained through screening was treated with different concentrations (5, 10, and 20 μmol/L) of Tet for 48 h respectively. CCK-8 assay was used to detect cell viability to obtain appropriate Tet concentration. EC9706 cells in logarithmic growth phase were randomly divided into the Control group, Tet group, Tet+inhibitor negative control (inhibitor NC) group, and Tet+miR-149 5p inhibitor (miR-149-5p inhibitor) group. qRT-PCR was used to detect the expression levels of miR-149-5p and HMGA2 mRNA in each cell; flow cytometry was used to detect apoptosis in each group; Western blot was used to detect the expression levels of cellular epithelial marker proteins E-cadherin, CyclinD1, mesenchymal marker N cadherin, Bax (B-lymphoma-2-associated protein), and HMGA2; Transwell was used to measure cell migration and invasion; CCK-8 assay was used to detect cell proliferation; and dual-luciferase experiment was used to examine the targeting relationship between miR-149-5p and HMGA2. 10 μmol/L Tet was the best concentration in this study. HMGA2 was a targeted binding site for miR-149-5p. Compared with HEEC cells, the expression of miR-149-5p in esophageal cancer cell lines was obviously decreased, and the expression of HMGA2 mRNA was increased (P<0.05), among which EC9706 cells had the most obvious changes and they were used as the best experimental cells for subsequent experiments. Compared with the Control group, the D450 value, the numbers of migrating and invasive EC9706 cells, the expression of CyclinD1, N-cadherin and HMGA2 in the Tet group at 24 h and 48 h were obvi ously decreased, and the apoptosis rate and the expression of Bax and E-cadherin were obviously increased (P<0.05); compared with Tet+inhibitor NC group, the D450 value, the numbers of migrating and invasive EC9706 cells, the ex pression of CyclinD1, N-cadherin and HMGA2 in the Tet+miR-149-5p inhibitor group at 24 h and 48 h were obvi ously increased, and the apoptosis rate and the expression of Bax and E-cadherin were obviously decreased (P<0.05). Tet may up-regulate miR-149-5p and inhibit the expression of HMGA2, thereby inducing apoptosis of esophageal cancer cells and inhibiting their proliferation, migration and invasion.
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