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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (01) : 8 -15. doi: 10.3877/cma.j.issn.2095-1221.2021.01.002

所属专题： 文献；

论著

LncRNA ZNF667-AS1靶向miR-31-5p对食管癌细胞增殖、迁移和凋亡的机制研究
邱卫明1, 王建荣1, 徐志敏1, 缪玖麟1, 陈保华1, 汪芝珍1,†()   
  1. 1. 335000　鹰潭，联勤保障部队第908医院普通外科
  • 收稿日期:2020-06-03 出版日期:2021-02-01
  • 通信作者: 汪芝珍

Mechanisms of LncRNA ZNF667-AS1 targeting miR-31-5p in proliferation, migration and apoptosis of esophageal carcinoma cells

Weiming Qiu1, Jianrong Wang1, Zhimin Xu1, Jiulin Miao1, Baohua Chen1, Zhizhen Wang1,†()   

  1. 1. General Surgery, 908th Hospital of Joint Logistics Support Force, Yingtan 335000, China
  • Received:2020-06-03 Published:2021-02-01
  • Corresponding author: Zhizhen Wang
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引用本文：

邱卫明, 王建荣, 徐志敏, 缪玖麟, 陈保华, 汪芝珍. LncRNA ZNF667-AS1靶向miR-31-5p对食管癌细胞增殖、迁移和凋亡的机制研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(01): 8-15.

Weiming Qiu, Jianrong Wang, Zhimin Xu, Jiulin Miao, Baohua Chen, Zhizhen Wang. Mechanisms of LncRNA ZNF667-AS1 targeting miR-31-5p in proliferation, migration and apoptosis of esophageal carcinoma cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(01): 8-15.

目的

探究长链非编码RNA （lncRNA）ZNF667-AS1通过靶向miR-31- 5p对食管癌细胞增殖和迁移的影响及潜在的机制。

方法

采用实时荧光定量PCR（qPCR）技术检测ZNF667-AS1在食管癌细胞Eca109、EC1、TE1和正常食管上皮细胞Het- 1A的表达水平，并选择表达差异最大的细胞株进行后续实验。采用脂质体转染技术将pcDNA3.1- ZNF667-AS1过表达重组载体质粒转染至人食管癌Eca109细胞，实验分为对照组（未行转染的Eca109细胞）、pcDNA3.1组（转染空载体质粒的Eca109细胞）和ZNF667- AS1组（转染pcDNA3.1- ZNF667- AS1质粒的Eca109细胞），qPCR技术检测pcDNA3.1- ZNF667-AS1的转染效果，噻唑蓝比色法（MTT）和平板克隆实验检测过表达ZNF667-AS1对Eca109细胞增殖能力的影响，Transwell实验检测Eca109细胞迁移能力，流式细胞术检测Eca109细胞凋亡率，Western blot检测Eca109细胞中G1/S-特异性周期蛋白-D1（Cyclin D1）、细胞周期依赖性激酶抑制因子（p21）、上皮标志物上皮型钙黏蛋白（E-cadherin）、间充质标志物神经型钙黏蛋白（N-cadherin）、波形蛋白（Vimentin）、B细胞淋巴瘤/白血病-2相关蛋白X（Bax）和B细胞淋巴瘤/白血病-2（Bcl-2）的表达水平，生物信息学预测ZNF667- AS1与miR- 31-5p的互补配对关系，荧光素酶报告实验和qPCR技术验证ZNF667-AS1和miR-31-5p的靶向调控关系。多组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。

结果

与Het- 1A细胞相比，食管癌Eca109、EC1和TE1细胞中ZNF667-AS1的表达水平均降低（1.00±0.08比0.29±0.04，0.36±0.05，0.33±0.06），差异有统计学意义（P < 0.001）。与ZNF667-AS1组相比，对照组及pcDNA3.1组中ZNF667-AS1的表达水平（3.14±0.32比1.00±0.10，0.94±0.09），细胞活力[（63.42±3.75）﹪比100.00±4.85、（97.69±4.57）﹪]，细胞克隆形成数[（34.26±3.52）个比（95.64±10.22）个、（92.80±10.04）个]，Cyclin D1表达水平（0.26±0.05比0.75±0.08、0.71±0.07），迁移细胞数[（20.24±2.35）个比（63.55±6.04）个、（60.02±6.12）个]，细胞中E-cadherin表达水平（0.19±0.02比0.48±0.05，0.49±0.05）和miR- 31-5p的表达水平（0.30±0.03比1.00±0.10、0.95±0.09）均升高，p21表达水平（0.79±0.09比0.24±0.05，0.26±0.06），细胞凋亡率[（29.17±1.26）﹪比（3.41±0.73）﹪，（3.72±0.78）﹪]，细胞中N-cadherin（0.87±0.09比0.42±0.04，0.40±0.04）、Vimentin表达水平（0.82±0.08比0.44±0.04，0.44±0.04）均降低，差异有统计学意义（P均< 0.001），与对照组比较，pcDNA3.1组ZNF667-AS1的表达水平，细胞活力，细胞克隆形成数，Cyclin D1和p21表达水平，迁移细胞数，细胞中E-cadherin、N-cadherin、Vimentin和miR-31-5p表达水平，细胞凋亡率差异无统计学意义（P均> 0.05）。生物信息学预测发现ZNF667-AS1和miR-31-5p存在靶向结合位点；荧光素酶报告实验结果显示，与空载转染组比较，miR-31-5p组ZNF667-AS1-Wt相对荧光素酶活性（1.00±0.10比0.19±0.02）降低（P < 0.05），ZNF667-AS1-Mut相对荧光素酶活性比较，差异无统计学意义（P > 0.05）。

结论

过表达ZNF667-AS1能抑制食管癌Eca109细胞的增殖、迁移并诱导其凋亡，其机制可能与ZNF667-AS1靶向调控miR-31-5p有关。

关键词: 长链非编码RNA, miR-31-5p, 食管癌细胞, 增殖, 迁移, 凋亡
Objective

To investigate the effects and mechanisms of long-chain non-coding RNA (lncRNA) ZNF667-AS1 on the proliferation and migration of esophageal cancer cells by targeting miR-31-5p.

Methods

Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression levels of ZNF667-AS1 in esophageal cancer cells Eca109, EC1, TE1 and normal esophageal epithelial cells Het-1A, and the cell lines with the greatest difference in expression of ZNF667-AS1 were selected for subsequent experiments. PcDNA3.1-ZNF667-AS1 overexpression recombinant vector plasmid was transfected into human esophageal cancer Eca109 cells by liposome transfection technology. Three groups involved in this experiment, including control group (Eca109 cells untransfected) , pcDNA3.1 group (Eca109 cells transfected with empty vector plasmid) , and ZNF667-AS1 group (Eca109 cells transfected with pcDNA3.1-ZNF667-AS1 plasmid) . qPCR detected pcDNA3.1-ZNF667- transfection effect of AS1. Thiazolyl blue colorimetric assay (MTT) and plate cloning formation assay were used to detect the proliferation rate of Eca109 cells. Transwell assay to detect the migration ability of Eca109 cells. Flow cytometry was used to detect the apoptosis rate of Eca109 cells. The expression levels of G1 / S-specific cyclin-D1 (Cyclin D1) , cell cycle-dependent kinase inhibitor (p21) , and epithelial marker E-cadherin, mesenchymal markers N-cadherin, Vimentin, B-cell lymphoma/leukemia-2 related protein X (Bax) , and B-cell lymphoma/leukemia-2 (Bcl-2) proteins in Eca109 cells were detected by Western blot. Bioinformatics was used to predicte the complementary pairing relationship between ZNF667-AS1 and miR-31-5p. The luciferase reporter assay and qPCR technique was used to verify the targeted regulation relationship between ZNF667-AS1 and miR-31-5p. One-way analysis of variance was used to compare the difference among multiple groups, and SNK-q test was used to compare the difference between groups.

Results

The expression levels of ZNF667-AS1 in normal esophageal epithelial Het-1A cells was higher than that in esophageal cancer Eca109, EC1 and TE1 cells (1.00±0.08 vs 0.29±0.04, 0.36±0.05, 0.33±0.06) , and the difference was statistically significant (P < 0.001) . Compared with the ZNF667-AS1 group, the expression level of ZNF667-AS1 in the control group and pcDNA 3.1 group (3.14±0.32 vs 1.00±0.10, 0.94±0.09) , cell viability [ (63.42±3.75) ﹪vs 100.00 ± 4.85, (97.69±4.57) ﹪], cell clonogenic number (34.26±3.52 vs 95.64±10.22, 92.80±10.04) . Cyclin D1 expression level (0.26±0.05 vs 0.75±0.08, 0.71± 0.07) , number of migrating cells (20.24±2.35 vs 63.55±6.04, 60.02±6.12) , E-cadherinherin expression level in cells (0.19 ±0.02 vs 0.48 ±0.05, 0.49 ±0.05) and the expression levels of miR- 31- 5p (0.30±0.03 vs 1.00 ± 0.10, 0.95±0.09) were all elevated, p21 expression level (0.79±0.09 vs 0.24±0.05, 0.26±0.06) , apoptosis rates [ (29.17±1.26) ﹪vs (3.41±0.73) ﹪, (3.72± 0.78) ﹪) ].The expression of N-cadherin (0.87±0.09 vs 0.42±0.04, 0.40±0.04) and vimentin expression (0.82±0.08 vs 0.44±0.04, 0.44±0.04) were significantly decreased (P < 0.001) . Compared with the control group, the expression levels of ZNF667-AS1, cell viability, number of cell clone formation, expression levels of Cyclin D1 and p21, number of migrating cells, expression levels of E-cadherin, N-cadherin, Vimentin and miR-31-5p in the pcDNA3.1 group, and the apoptotic rate were not significantly different (all P > 0.05) . Bioinformatics predictions revealed that ZNF667- AS1 and miR-31-5p had targeted binding sites; the results of luciferase reporter assay showed that the relative luciferase activity of ZNF667-AS1-Wt in the miR-31-5p group was decreased (1.00±0.10 vs 0.19±0.02) (P < 0.05) . Compared with the empty transfection group, the relative luciferase activity of ZNF667-AS1-Mut was not significantly different (P > 0.05) .

Conclusion

Overexpression of ZNF667-AS1 can inhibit the proliferation, migration and induce apoptosis of Eca109 cells, which may be related to the targeted regulation of miR-31-5p by ZNF667-AS1.

Key words: Long non-coding RNA, miR-31-5p, Esophageal cancer cells, Proliferation, Migration, Apoptosis
表1 引物序列信息
引物名称 序列（5'-3'） 预期扩增产物长度（bp）
ZNF667-AS1 上游CATCACTACCATCCATCACTA 117
  下游CCAGGCAGAGAAGGATAA  
GAPDH 上游AGGTGAAGGTCGGAGTCAACG 165
  下游AGGGGTCATTGATGGCAACA  
表2 ZNF667-AS1在不同细胞系中的表达水平比较（ ± s）
分组 实验次数 ZNF667-AS1
Het-1A 3 1.00±0.08
Eca109 3 0.29±0.04a
EC1 3 0.36±0.05a
TE1 3 0.33±0.06a
F值   291.489
P值   < 0.001
图1 Western blot检测Eca109细胞中过表达ZNF667-AS1 CyclinD1和p21的影响
表3 过表达ZNF667-AS1对Eca109细胞活力、克隆形成数及增殖相关蛋白表达量的影响（ ± s）
分组 实验次数 细胞活力（﹪） 细胞克隆形成数（个） 增殖相关蛋白
Cyclin D1 p21
对照组 3 100.00 ± 4.85 95.64±10.22 0.75±0.08 0.24±0.05
pcDNA3.1组 3 97.69 ± 4.57 92.80±10.04 0.71±0.07 0.26±0.06
ZNF667-AS1组 3 63.42 ± 3.75ab 34.26± 3.52ab 0.26±0.05ab 0.79±0.09ab
F值   193.782 148.921 144.848 185.007
P值   < 0.001 < 0.001 < 0.001 < 0.001
图2 倒置显微镜下观察过表达ZNF667-AS1对Eca109细胞迁移影响（结晶紫染色，×400）
图3 Western blot检测Eca109细胞中过表达ZNF667-AS1对E-cadherin、N-cadherin和Vimentin的影响
表4 过表达ZNF667-AS1对Eca109细胞中E-cadherin、N-cadherin和Vimentin表达水平的影响（ ± s）
分组 实验次数 E-cadherin N-cadherin Vimentin
对照组 3 0.48±0.05 0.42±0.04 0.44±0.04
pcDNA3.1组 3 0.49±0.05 0.40±0.04 0.44±0.04
ZNF667-AS1组 3 0.19±0.02ab 0.87±0.09 ab 0.82±0.08 ab
F值   145.167 168.770 135.375
P值   < 0.001 < 0.001 < 0.001
图4 过表达ZNF667-AS1对Eca109细胞凋亡的影响
表5 过表达ZNF667-AS1对Eca109细胞中凋亡率及凋亡相关蛋白表达的影响（ ± s）
分组 实验次数 凋亡率（﹪） 凋亡相关蛋白
Bax Bcl-2
对照组 3 3.41±0.73 0.29±0.05 0.81±0.07
pcDNA3.1组 3 3.72±0.78 0.32±0.06 0.78±0.06
ZNF667-AS1组 3 29.17±1.26ab 0.85±0.08ab 0.32±0.05ab
F值   2162.480 214.344 185.155
P值   < 0.001 < 0.001 < 0.001
图5 生物信息学软件预测ZNF667-AS1和miR-31-5p靶向结合位点示意图
表6 过表达ZNF667-AS1对Eca109细胞相对荧光素酶活性的影响（ ± s）
分组 实验次数 ZNF667-AS1-Wt ZNF667-AS1-Mut
空载转染组 3 1.00±0.10 1.00±0.09
miR-31-5p组 3 0.19±0.02 0.99±0.09
t值   23.828 0.236
P值   < 0.001 0.817
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