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

doi:10.3877/cma.j.issn.2095-1221.2021.01.002

Abstract

Abstract:

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

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.

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Figures/Tables 11

References 25

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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

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

分组	实验次数	ZNF667-AS1
Het-1A	3	1.00±0.08
Eca109	3	0.29±0.04^a
EC1	3	0.36±0.05^a
TE1	3	0.33±0.06^a
F值		291.489
P值		< 0.001

分组	实验次数	ZNF667-AS1
Het-1A	3	1.00±0.08
Eca109	3	0.29±0.04^a
EC1	3	0.36±0.05^a
TE1	3	0.33±0.06^a
F值		291.489
P值		< 0.001

分组	实验次数	细胞活力（﹪）	细胞克隆形成数（个）	增殖相关蛋白
分组	实验次数	细胞活力（﹪）	细胞克隆形成数（个）	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 ± 3.75^ab	34.26± 3.52^ab	0.26±0.05^ab	0.79±0.09^ab
F值		193.782	148.921	144.848	185.007
P值		< 0.001	< 0.001	< 0.001	< 0.001

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