Study on total flavonoids from Selaginella uncinata in inhibiting malignant behavior of colorectal cancer cells through circular RNA circ_0006528 pathway
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摘要:目的 探讨翠云草总黄酮对结直肠癌细胞恶性生物学行为的影响及可能机制。方法 体外培养结直肠癌细胞Caco-2, 用不同剂量(10、20、40 μg/mL)翠云草总黄酮干预24 h、或转染环状RNA circ_0006528小干扰RNA至Caco-2细胞后培养24 h、或用40 μg/mL的翠云草总黄酮干预转染circ_0006528过表达载体的Caco-2细胞24 h, 然后用检测细胞存活与生长比色法(MTT)、克隆形成实验、划痕实验和流式细胞术分别检测细胞活性、克隆形成数、迁移和凋亡。Western Blot检测Bax和Bcl-2蛋白表达，实时荧光定量聚合酶链反应(qRT-PCR)检测circ_0006528和miR-330-3p表达。双荧光素酶报告基因实验验证circ_0006528和miR-330-3p调控关系。结果 Caco-2细胞经翠云草总黄酮干预后，细胞活性、克隆形成数、迁移距离和细胞中Bcl-2蛋白、circ_0006528的表达均降低，而凋亡率和Bax蛋白、miR-330-3p的表达升高，差异有统计学意义(P < 0.05), 且呈剂量依赖性。干扰circ_0006528后, Caco-2细胞活性、克隆形成数、迁移距离和Bcl-2蛋白表达降低，细胞凋亡率和Bax蛋白表达升高，差异有统计学意义(P < 0.05)。circ_0006528靶向负调控miR-330-3p。过表达circ_0006528逆转了翠云草总黄酮对Caco-2细胞增殖、迁移和凋亡及miR-330-3p表达的影响。结论 翠云草总黄酮可能通过调控circ_0006528/miR-330-3p轴，抑制结直肠癌细胞增殖和迁移，并促进细胞凋亡。Abstract:Objective To investigate the effect of total flavonoids from Selaginella uncinata on malignant biological behavior of colorectal cancer cells and its possible mechanism.Methods Colorectal cancer cells Caco-2 were cultured in vitro. After Caco-2 cells were treated with different concentrations (10, 20 and 40 μg/mL) of total flavonoids from Selaginella uncinata for 24 hours, or Caco-2 cells were transfected with circ_0006528 small interfering RNA, or Caco-2 cells that transfected with circ_0006528 overexpression vector were treated with 40 μg/mL total flavonoids from Selaginella uncinata for 24 hours, methyl thiazolyl tetrazolium (MTT) assay, clone formation assay, scratch assay and flow cytometry were used to detect cell proliferation viability, the number of colony formation, migration and apoptosis, respectively. The expressions of Bax and Bcl-2 proteins were detected by western blot, and the expressions of circ_0006528 and miR-330-3p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The regulatory relationship between circ_0006528 and miR-330-3p was verified by dual luciferase reporter gene assay.Results After Caco-2 cells were intervened with total flavonoids from Selaginella uncinata, cell viability, the number of clone formation, migration distance and the expression of Bcl-2 protein as well as circ_0006528 in Caco-2 cells were significantly decreased, but apoptosis rate and the expression of Bax protein and miR-330-3p were significantly increased (P < 0.05), and they were dose-dependent. After circ_0006528 interference, Caco-2 cell activity, the number of clone formation, migration distance and Bcl-2 protein expression were significantly decreased, while apoptosis rate and Bax protein expression were significantly increased (P < 0.05). The circ_0006528 negatively regulated miR-330-3p by targeting. Overexpression of circ_0006528 reversed the effects of total flavonoids from Selaginella uncinata on proliferation, migration and apoptosis of Caco-2 cells and expression of miR-330-3p.Conclusion The total flavonoids from Selaginella uncinata may inhibit the proliferation and migration of colorectal cancer cells and promote cells apoptosis by regulating the circ_0006528/miR-330-3p axis.
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图 1 翠云草总黄酮对Caco-2细胞活性的影响
与对照组比较, *P < 0.05;
与翠云草总黄酮-L组比较, #P < 0.05;
与翠云草总黄酮-M组比较, △P < 0.05。![]()
图 2 翠云草总黄酮对Caco-2细胞集落形成和迁移距离的影响
A: 克隆形成实验检测翠云草总黄酮对Caco-2细胞集落形成的影响; B: 划痕实验检测翠云草总黄酮对Caco-2细胞迁移的影响。
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图 3 翠云草总黄酮对Caco-2细胞凋亡及Bax、Bcl-2蛋白表达的影响
A: 流式细胞术检测翠云草总黄酮对Caco-2细胞凋亡的影响; B: Western Blot检测翠云草总黄酮对Caco-2细胞中Bax和Bcl-2蛋白表达的影响。与对照组比较, *P < 0.05; 与翠云草总黄酮-L组比较, #P < 0.05; 与翠云草总黄酮-M组比较, △P < 0.05。
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图 4 翠云草总黄酮对Caco-2细胞中circ_0006528和miR-330-3p表达的影响
A: qRT-PCR检测翠云草总黄酮对Caco-2细胞中circ_0006528表达的影响;
B: qRT-PCR检测翠云草总黄酮对Caco-2细胞中miR-330-3p表达的影响。
与对照组比较, *P < 0.05; 与翠云草总黄酮-L组比较, #P < 0.05; 与翠云草总黄酮-M组比较, △P < 0.05。![]()
图 5 干扰circ_0006528对Caco-2细胞活性、凋亡和Bax、Bcl-2蛋白表达的影响
A: qRT-PCR实验检测转染si-circ_0006528的Caco-2细胞中circ_0006528表达; B: qRT-PCR实验检测干扰circ_0006528对Caco-2细胞中miR-330-3p表达的影响; C: 流式细胞术检测干扰circ_0006528对Caco-2细胞凋亡的影响; D: MTT实验检测干扰circ_0006528对Caco-2细胞活性的影响; E: Western Blot检测干扰circ_0006528对Caco-2细胞中Bax和Bcl-2蛋白表达的影响。与si-NC组比较, *P < 0.05。
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图 6 circ_0006528和miR-330-3p互补序列及荧光素酶活性检测结果
A: Circular RNA Interactome靶基因软件预测显示的circ_0006528和miR-330-3p的互补核苷酸序列;
B: 荧光素酶活性检测结果。
与miR-NC比较, *P < 0.05。![]()
图 7 过表达circ_0006528逆转翠云草总黄酮对Caco-2细胞增殖、凋亡和及Bax、Bcl-2蛋白表达的影响
A: qRT-PCR验检测翠云草总黄酮对转染pcDNA-circ_0006528的Caco-2细胞中circ_0006528表达的影响;
B: qRT-PCR检测翠云草总黄酮对转染pcDNA-circ_0006528的Caco-2细胞中miR-330-3p表达的影响;
C: 流式细胞术检测翠云草总黄酮对转染pcDNA-circ_0006528的Caco-2细胞凋亡的影响;
D: MTT实验检测翠云草总黄酮对转染pcDNA-circ_0006528的Caco-2细胞活性的影响;
E: Western Blot检测翠云草总黄酮对转染pcDNA-circ_0006528的Caco-2细胞中Bax和Bcl-2蛋白表达的影响。
与翠云草总黄酮组比较, *P < 0.05。表 1 翠云草总黄酮对Caco-2细胞集落形成和迁移距离的影响(n=3)(x±s)
组别 集落形成数/个 迁移距离/μm 对照组 115.00±5.35 166.42±8.94 翠云草总黄酮-L组 91.67±4.99* 132.25±6.44* 翠云草总黄酮-M组 71.67±2.62*# 97.89±3.24*# 翠云草总黄酮-H组 48.00±1.63*#△ 73.39±1.60*#△ 与对照组比较, *P < 0.05;
与翠云草总黄酮-L组比较, #P < 0.05;
与翠云草总黄酮-M组比较, △P < 0.05。
下载: 导出CSV
表 2 翠云草总黄酮诱导Caco-2细胞凋亡(n=3)(x±s)
组别 凋亡率/% Bax Bcl-2 对照组 7.40±0.31 0.15±0.01 0.73±0.05 翠云草总黄酮-L组 13.01±0.54* 0.36±0.02* 0.51±0.04* 翠云草总黄酮-M组 17.73±0.76*# 0.55±0.03*# 0.34±0.02*# 翠云草总黄酮-H组 22.42±1.04*#△ 0.79±0.05*#△ 0.13±0.01*#△ 与对照组比较, *P < 0.05; 与翠云草总黄酮-L组比较, #P < 0.05; 与翠云草总黄酮-M组比较, △P < 0.05。
下载: 导出CSV
表 3 干扰circ_0006528对Caco-2细胞集落形成、迁移和凋亡的影响(n=3)(x±s)
组别 集落形成数/个 迁移距离/μm 凋亡率/% Bax Bcl-2 si-NC组 115.00±7.48 167.04±9.48 7.84±0.67 0.15±0.01 0.72±0.05 si-circ_0006528组 41.00±0.82* 62.30±2.85* 24.46±0.85* 0.86±0.05* 0.09±0.01* 与si-NC组比较, *P < 0.05。
下载: 导出CSV
表 4 过表达circ_0006528逆转翠云草总黄酮对Caco-2细胞增殖、迁移和凋亡的影响(n=3)(x±s)
组别 circ_0006528 miR-330-3p 集落形成数/个 迁移距离/μm 凋亡率/% Bax Bcl-2 翠云草总黄酮 0.20±0.01 4.31±0.10 47.33±2.62 73.56±1.90 22.54±1.09 0.78±0.06 0.14±0.01 翠云草总黄酮+pcDNA-circ_0006528 0.83±0.06* 1.22±0.06* 103.33±3.86* 144.84±5.69* 8.63±0.54* 0.19±0.01* 0.64±0.05* 与翠云草总黄酮组比较, *P < 0.05。
下载: 导出CSV
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