Department of Urinary Surgery (I), The Second Affiliated Hospital of Qiqihar Medical University, Qiqihar. P.R. China.
Eur Rev Med Pharmacol Sci. 2020 Aug;24(16):8349-8358. doi: 10.26355/eurrev_202008_22631.
To investigate the effect and mechanism of miRNA-34a overexpression on proliferation and migration of PC3 prostate cancer cells.
Benign prostatic hyperplasia tissue (30 cases), prostate cancer tissue (30 cases), and prostate paracancerous tissue (30 cases) were collected. Levels of miRNA-34a in these fresh tissues were measured by fluorescence quantitative PCR. PC3 cells were divided into non-loaded group and overexpression group. Cells in the non-loaded group were transfected with non-loaded plasmid. Cells in the overexpression group were transfected with miRNA-34a plasmid, and the miRNA-34a level was determined by fluorescence quantitative PCR to confirm the overexpression. Cell proliferation was analyzed by CCK-8 assay. Cell migration rate was measured by cell scratch assay. Flow cytometry was used to detect apoptosis and analyze cell cycle. Western blot was used to measure the expression levels of β-catenin, E-cadherin and Vimentin.
The expression level of miRNA-34a in prostate cancer tissue was significantly lower than that in prostate paracancerous tissue. Dual-Luciferase reporter gene assay was used to analyze the transcriptional activity of Wnt1 gene. The proliferation and migration of PC3 cells were significantly decreased after overexpression of miRNA-34a, and the differences were statistically significant compared with those in the non-loaded group (p<0.05). Flow cytometry analysis showed that in the overexpression group, the apoptotic rate, as well as the proportion of cells in the G2 phase, was significantly higher than that in the non-loaded group (p<0.05). The β-catenin level in the nucleus of PC3 cells was significantly reduced after overexpression of miRNA-34a. The total protein levels of β-catenin and Vimentin were significantly decreased, whereas the level of E-cadherin in the overexpression group was apparently increased, compared with that in the non-loaded group. The Dual-Luciferase reporter gene showed a decrease in the relative fluorescence intensity of Wnt1 after overexpression of miR-34a (p<0.05).
Overexpression of miRNA-34a inhibits Wnt/β-catenin pathway by regulating the transcriptional activity of Wnt1, thereby regulating the proliferation and migration of PC3 cells and promoting apoptosis.
研究 miRNA-34a 过表达对 PC3 前列腺癌细胞增殖和迁移的影响及其机制。
收集 30 例良性前列腺增生组织、30 例前列腺癌组织和 30 例前列腺癌旁组织，采用荧光定量 PCR 检测这些新鲜组织中 miRNA-34a 的水平。将 PC3 细胞分为空载组和过表达组，空载组转染空载质粒，过表达组转染 miRNA-34a 质粒，荧光定量 PCR 检测 miRNA-34a 水平以证实过表达，CCK-8 法分析细胞增殖，细胞划痕实验检测细胞迁移率，流式细胞术检测细胞凋亡并分析细胞周期，Western blot 检测β-catenin、E-cadherin 和 Vimentin 表达水平。
与前列腺癌旁组织相比，前列腺癌组织中 miRNA-34a 的表达水平明显降低。双荧光素酶报告基因检测分析 Wnt1 基因的转录活性。过表达 miRNA-34a 后，PC3 细胞的增殖和迁移明显下降，与空载组相比差异有统计学意义（p<0.05）。流式细胞术分析显示，过表达组细胞的凋亡率及 G2 期细胞比例明显高于空载组（p<0.05）。过表达 miRNA-34a 后，PC3 细胞核内β-catenin 水平明显降低，β-catenin 和 Vimentin 的总蛋白水平明显降低，E-cadherin 水平明显升高，与空载组相比差异有统计学意义。双荧光素酶报告基因显示过表达 miR-34a 后 Wnt1 的相对荧光强度降低（p<0.05）。
miRNA-34a 通过调控 Wnt1 的转录活性抑制 Wnt/β-catenin 通路，从而调控 PC3 细胞的增殖和迁移，促进细胞凋亡。
