食管癌肿瘤细胞中Per2、HDAC1、E-钙黏蛋白mRNA表达及其意义

林招贤1; 朱立桓1; 黄建源1; 陈志耀2; 黄阳昀1; 林兴1; 吴榕龙1; 欧德杉1; 叶明凡1

doi:10.3760/cma.j.issn.1673-9752.2020.03.019

食管癌肿瘤细胞中Per2、HDAC1、E-钙黏蛋白mRNA表达及其意义

Expression and significance of Per2 mRNA, HDAC1 mRNA and E-cadherin mRNA in esophageal cancer cells

摘要

摘要: 目的:探讨食管癌肿瘤细胞中Per2、HDAC1、E-钙黏蛋白mRNA表达及其意义。
方法:采用实验研究方法。体外培养人食管癌细胞系KYSE-150细胞至对数生长期设为实验组，体外培养人正常食管上皮细胞至对数生长期设为对照组。观察指标：(1)细胞增殖活力。(2)细胞迁移和侵袭能力情况。(3)初始生理状态下Per2、HDAC1、E-钙黏蛋白mRNA表达情况。(4)Per2激动剂和抑制剂处理后Per2、HDAC1、E-钙黏蛋白mRNA表达情况。(5)HDAC1抑制剂处理后Per2、E-钙黏蛋白mRNA表达情况。正态分布的计量资料以±s表示，组内比较采用t检验；组间比较采用t检验或协方差分析。
结果:(1)细胞增殖活力：实验组和对照组细胞增殖活力分别为0.90%±0.14%和0.52%±0.08%，两组比较，差异有统计学意义(t=5.166，P<0.05)。(2)细胞迁移和侵袭能力情况：实验组和对照组发生迁移细胞数分别为(173±41)个和(50±15)个，两组比较，差异有统计学意义(t=6.274，P<0.05)。实验组和对照组发生侵袭细胞数分别为(86±27)个和(21±9)个，两组比较，差异有统计学意义(t=5.153，P<0.05)。(3)初始生理状态下Per2、HDAC1、E-钙黏蛋白mRNA表达情况：初始生理状态下，实验组细胞Per2、HDAC1、E-钙黏蛋白mRNA表达水平分别为11.7±2.7、20.4±6.6、12.4±2.5；对照组细胞上述指标分别为2.4±0.5、8.5±2.2、27.3±4.5，两组比较，差异均有统计学意义(t=5.782，2.982，-5.034，P<0.05)。(4)Per2激动剂和抑制剂处理后Per2、HDAC1、E-钙黏蛋白mRNA表达情况：Per2激动剂处理后，实验组细胞Per2、HDAC1、E-钙黏蛋白mRNA表达水平分别为13.1±2.2、22.4±6.2、16.6±4.2；对照组细胞上述指标分别为9.9±3.1、18.4±5.6、15.3±2.3。实验组细胞Per2激动剂处理后上述指标与初始生理状态下比较，差异均无统计学意义(t=-4.300，10.087，-4.187，P>0.05)；对照组细胞Per2激动剂处理后上述指标与初始生理状态下比较，差异均有统计学意义(t=-4.846，3.501，9.294，P<0.05)。Per2激动剂处理后，两组细胞Per2和E-钙黏蛋白mRNA表达水平比较，差异均无统计学意义(F=1.000，7.582，P>0.05)；两组细胞HDAC1 mRNA表达水平比较，差异有统计学意义(F=1.724，P<0.05)。Per2抑制剂处理后，实验组细胞Per2、HDAC1、E-钙黏蛋白mRNA表达水平分别为4.1±1.7、7.5±2.2、22.8±4.2；对照组细胞上述指标分别为3.1±0.9、9.3±3.2、28.4±5.8。实验组细胞Per2抑制剂处理后上述指标与初始生理状态下比较，差异均有统计学意义(t=12.124，5.105，-10.245，P<0.05)；对照组细胞Per2抑制剂处理后上述指标与初始生理状态下比较，差异均无统计学意义(t=-2.815，1.568，-1.439，P>0.05)。Per2抑制剂处理后，两组细胞Per2和E-钙黏蛋白mRNA表达水平比较，差异均有统计学意义(F=22.965，82.134，P<0.05)；两组细胞HDAC1 mRNA表达水平比较，差异无统计学意义(F=6.416，P>0.05)。(5)HDAC1抑制剂处理后Per2、E-钙黏蛋白mRNA表达情况：HDAC1抑制剂处理后，实验组细胞Per2、E-钙黏蛋白mRNA表达水平分别为13.4±3.5、24.2±3.4，对照组细胞上述指标分别为3.1±1.2、26.8±5.2。实验组细胞HDAC1抑制剂处理后Per2 mRNA表达水平与初始生理状态下比较，差异无统计学意义(t=-3.959，P>0.05)；E-钙黏蛋白mRNA表达水平与初始生理状态下比较，差异有统计学意义(t=-21.977，P<0.05)。对照组细胞HDAC1抑制剂处理后Per2、E-钙黏蛋白mRNA表达水平与初始生理状态下比较，差异均无统计学意义(t=-1.440、1.058，P>0.05)。HDAC1抑制剂处理后，两组细胞Per2 mRNA表达水平比较，差异无统计学意义(F=2.004，P>0.05)，E-钙黏蛋白mRNA表达水平比较，差异有统计学意义(F=325.800，P<0.05)。
结论:食管肿瘤细胞中Per2和HDAC1 mRNA表达水平升高，E-钙黏蛋白mRNA表达水平降低。Per2 mRNA高表达可能会激活下游靶向蛋白HDAC1的表达升高，抑制细胞表面E-钙黏蛋白mRNA表达水平。

Abstract: Objective:To investigate the expression of Per2 mRNA, HDAC1 mRNA and E-cadherin mRNA in esophageal cancer cells and their significance.
Methods:The experimental study was conducted. Human normal esophageal epithelial cells as the control group and human esophageal cancer cell line KYSE-150 cells as the experimental group were cultured in vitro to logarithmic growth stage. Observation indicators: (1) the proliferation of cells; (2) the migration and invasion of cells; (3) the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state; (4) the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA after cells were treated with Per2-agonists or inhibitors; (5) the expression of Per2 mRNA and E-cadherin mRNA after cells were treated with HDAC1 inhibitors. Measurement data with normal distribution were represented as Mean±SD, the t test was used for comparison within groups and the t test or ANCOVA were used for comparison between groups.
Results:(1) The proliferation of cells: the cell proliferation of the experimental group and control group were 0.90%±0.14% and 0.52%±0.08%, with a significant difference between the two groups (t=5.166, P<0.05). (2) The migration and invasion of cells: the numbers of cell migration and invasion for the experimental group were 173±41 and 86±27, versus 50±15 and 21±9 for the control group, with significant differences between the two groups (t=6.274, 5.153, P<0.05). (3) The expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state: the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA in cells of initial physiological state for the experimental group was 11.7±2.7, 20.4±6.6, and 12.4±2.5, respectively, versus 2.4±0.5, 8.5±2.2, and 27.3±4.5 for the control group, with significant differences between the two groups (t=5.782, 2.982,-5.034, P<0.05). (4) The expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA after cells were treated with Per2-agonists or inhibitors: after cells were treated with Per2-agonists, the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA were 13.1±2.2, 22.4±6.2, 16.6±4.2 for the experimental group, and 9.9±3.1, 18.4±5.6, 15.3±2.3 for the control group, respectively. There was no significant difference in the expression of Per2 mRNA, HDAC1 mRNA, or E-cadherin mRNA of the experimental group between cells being treated with and without Per2-agonists (t=-4.300, 10.087,-4.187, P>0.05). There were significant differences in the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA of the control group between cells being treated with and without Per2-agonists (t=-4.846, 3.501, 9.294, P<0.05). There was no significant difference in the expression of Per2 mRNA or E-cadherin mRNA between the experimental group and control group after cells were treated with Per2-agonists (F=1.000, 7.582, P>0.05), while there was a significant difference in the expression of HDAC1 mRNA between the two groups (F=1.724, P<0.05). After cells were treated with Per2-inhibitors, the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA were 4.1±1.7, 7.5±2.2, 22.8±4.2 for the experimental group, and 3.1±0.9, 9.3±3.2, 28.4±5.8 for the control group, respectively. There were significant differences in the expression of Per2 mRNA, HDAC1 mRNA, and E-cadherin mRNA of the experimental group between cells being treated with and without Per2-inhibitors (t=12.124, 5.105,-10.245, P<0.05). There was no significant difference in the expression of Per2 mRNA, HDAC1 mRNA, or E-cadherin mRNA of the control group between cells being treated with and without Per2-inhibitors (t=-2.815, 1.568,-1.439, P>0.05). There were significant differences in the expression of Per2 mRNA and E-cadherin mRNA after cells were treated with Per2-inhibitors between the experimental group and control group (F=22.965, 82.134, P<0.05), while there was no significant difference in the expressions of HDAC1 mRNA between the two groups (F=6.416, P>0.05). (5) The expression of Per2 mRNA and E-cadherin mRNA after cells were treated with HDAC1 inhibitors: after cells were treated with HDAC1 inhibitors, the expression of Per2 mRNA and E-cadherin mRNA were 13.4±3.5, 24.2±3.4 for the experimental group, and 3.1±1.2, 26.8±5.2 for the control group, respectively. There was no significant difference in the expression of Per2 mRNA of the experimental group between cells being treated with and without HDAC1-inhibitors (t=-3.959, P>0.05). There was a significant difference in the expression of E-cadherin mRNA of the experimental group between cells being treated with and without HDAC1-inhibitors (t=-21.977, P<0.05). There was no significant difference in the expression of Per2 mRNA or E-cadherin mRNA of the control group between cells being treated with and without HDAC1-inhibitors (t=-1.440, 1.058, P>0.05). After cells were treated with HDAC1-inhibitors, there was no significant difference in the expressions of Per2 mRNA between the experimental group and control group (F=2.004, P>0.05), while there was a significant difference in the expression of E-cadherin mRNA between the two groups (F=325.800, P<0.05).
Conclusions:Human esophageal cancer cells have an elevated expression of Per2 mRNA and HDAC1 mRNA, and a reduced expression of E-cadherin mRNA. The overexpression of Per2 mRNA may activate the expression of downstream targeting protein HDAC1, and inhibit the expression of cell surface E-cadherin mRNA.

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