[1]周立远,叶玉祥,林 琳,等.LncRNA CCAT1 调节miR-155 表达增强CD8+T 细胞对食管癌抗肿瘤活性的机制研究[J].现代检验医学杂志,2023,38(03):79-85.[doi:10.3969/j.issn.1671-7414.2023.03.014]
　ZHOU Li-yuan,YE Yu-xiang,LIN Lin,et al.Mechanism of LncRNA CCAT1 Regulating miR-155 Expression and Enhancing Anti-tumor Activity of CD8+T Cells Against Esophageal Cancer[J].Journal of Modern Laboratory Medicine,2023,38(03):79-85.[doi:10.3969/j.issn.1671-7414.2023.03.014]
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LncRNA CCAT1 调节miR-155 表达增强CD8+T 细胞对食管癌抗肿瘤活性的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年03期
页码:
79-85
栏目:
论著
出版日期:
2023-05-15

文章信息/Info

Title:
Mechanism of LncRNA CCAT1 Regulating miR-155 Expression and Enhancing Anti-tumor Activity of CD8+T Cells Against Esophageal Cancer
文章编号:
1671-7414（2023）03-079-07
作者:
周立远; 叶玉祥; 林 琳; 王东阳
（中国人民解放军联勤保障部队第九〇〇医院仓山院区普通胸泌外科，福州 350002）
Author(s):
ZHOU Li-yuan;  YE Yu-xiang;  LIN Lin;  WANG Dong-yang
（Department of General Thoracic and Urological Surgery, Cangshan Hospital of the Joint Logistics Support Force of the People’s Liberation Army of China, Fuzhou 350002, China）
关键词:
长链非编码RNA 结肠癌相关转录本1(LncRNA CCAT1);  微小核糖核酸-155（miR-155）; CD8+T; 食管癌; 抗肿瘤活性
分类号:
R735.1；R730.43
DOI:
10.3969/j.issn.1671-7414.2023.03.014
文献标志码:
A
摘要:
目的 研究长链非编码RNA 结肠癌相关转录本1(long non-coding RNA colon cancer-associated transcript-1，LncRNA CCAT1) 对CD8+T 细胞抗食管癌肿瘤活性的影响及其作用机制。方法 分离食管癌患者和健康受试者外周血单个核细胞(peripheral blood mononuclear cell，PBMC)，用qRT-PCR 法分别检测PBMC 中CCAT1 和miR-155 的表达。分别下调CCAT1 和miR-155 表达，采用流式细胞术检测CD8+T 细胞凋亡率，Western blot 检测Cleaved Caspase 3 蛋白表达，qRT-PCR 检测IL-2 mRNA 和IFN-γ mRNA 基因表达，分析CD8+T 细胞对食管癌细胞的杀伤作用。采用miRanda 和双荧光素酶实验研究CCAT1 与miR-155 之间的相互作用。分析上调LncRNA CCAT1 靶向miR-155 对CD8+T 细胞凋亡率、杀伤作用及其Eca-109 细胞增殖和迁移的影响。结果 食管癌患者PBMCs 中CCAT1 表达（2.58±0.28）高于健康受试者（1.35±0.34），miR-155 表达（0.53±0.09）低于健康受试者（1.54±0.29），差异具有统计学意义（t=12.04，21.05，均P <0.05）。下调CCAT1 后，CD8+T 细胞凋亡率（12.05%±0.28%）明显低于si-control 组（19.86±0.45）%，CD8+T细胞的细胞杀伤活性（0.49±0.05）% 明显高于si-control 组（0.34%±0.02%），差异具有统计学意义（t=9.82，-17.54，均P<0.05）。si-CCAT1 组细胞内Cleaved Caspase 3 蛋白表达（0.32±0.09）低于si-control 组（1.37±0.72），IL-2 mRNA（2.38±0.47）和IFN-γ mRNA（3.28±0.26 ）表达高于si-control 组（1.12±0.23，1.28±0.37），差异具有统计学意义（t=-20.05，-18.29，-19.86，均P<0.05）。CCAT1 和miR-155 之间存在互补碱基对，双荧光素酶实验结果显示CCAT1野生型和miR-155 模拟物共转染后miR-155 mimics 组细胞荧光素酶活性（0.41±0.08）显著低于对照组（1.24±0.18），差异具有统计学意义（t=17.92，P<0.01）。下调miR-155 后CD8+T 细胞凋亡率（24.87%±0.95%）明显高于inhibitorcontrol 组（18.24%±1.25%），CD8+T 细胞的细胞杀伤活性（0.26%±0.06%）明显低于inhibitor control 组（0.43%±0.05%），差异具有统计学意义（t=10.03，20.06，均P<0.05）。miR-155 inhibitor 组细胞内Cleaved Caspase 3 蛋白表达（1.07±0.23）高于inhibitor control 组（0.42±0.02），IL-2 mRNA（0.73±0.26） 和IFN-γ mRNA（0.54±0.18） 表达低于inhibitorcontrol 组（1.39±0.08，1.16±0.24），差异具有统计学意义（t=18.75，19.27，18.35，均P<0.01）。上调CCAT1 通过miR-155 促进CD8+T 细胞凋亡并降低细胞杀伤活性，且促进了Eca-109 细胞增殖和迁移。结论 LncRNA CCAT1 在食管癌患者中表达显著上调，敲低CCAT1 通过调节miR-155 表达可抑制CD8+T 细胞凋亡，增强细胞的抗肿瘤活性。
Abstract:
Objective To study the effect of long non-coding RNA colon cancer-associated transcript-1(LncRNA CCAT1) on the activity of CD8+T cells against esophageal cancer and its mechanism. Methods Peripheral blood mononuclear cell (PBMC) determination of esophageal cancer patients and healthy subjects were isolated. The expression of CCAT1 and miR-155 in PBMC was detected by qRT-PCR CCAT1 and miR-155 were down-regulated, respectively. The apoptosis rate of CD8+T cells was detected by flow cytometry. Expression of Cleaved Caspase 3 protein was detected by Western blot. The expression of IL-2 mRNA and IFN-γ mRNA gene was detected by qRT-PCR.The killing effect of CD8+T cells on esophageal carcinoma cells was analyzed. MiRanda and dual luciferase assay were used to study the interaction between CCAT1 and miR-155.The effect of upregulation of LncRNA CCAT1 targeting miR-155 on apoptosis rate of CD8+T cells, killing effect and proliferation and migration of Eca-109 cells was analyzed. Results The expression of CCAT1 (2.58±0.28) in PBMCs cells of patients with esophageal cancer was higher than that of healthy subjects (1.35±0.34), and the expression of miR-155 (0.53±0.09) was lower than that of healthy subjects (1.54±0.29), the difference was statistically significant (t=12.04, 21.05, all P<0.05). The apoptosis rate of CD8+T cells was significantly lower than that of si-control group (12.05%±0.28 % vs 19.86%±0.45%) after CCAT1 downregulation, and the cytokilling activity of CD8+T cells was significantly higher than that of si-control group (0.49%±0.05% vs 0.34%±0.02%), the differences were statistically significant (t=9.82, -17.54, all P<0.05). Cleaved Caspase 3 (0.32±0.09） protein expression in si-CCAT1 group was lower than that in si-control group（1.37±0.72), and IL-2 mRNA (2.38±0.47）and IFN-γ mRNA（3.28±0.26 ）expression were higher than that in si-control group（1.12±0.23, 1.28±0.37), the differences were statistically significant (t=-20.02, -18.29, -19.86, all P<0.05). There were complementary base pairs between CCAT1 and miR-155. The results of double luciferase experiment showed that the luciferase activity of miR-155 mimics group (0.41±0.08) was significantly lower than that of control group (1.24±0.18) after co-transfection of CCAT1 wild type and miR-155 mimics, the differences were statistically significant (t=17.92, P<0.01). The apoptosis rate of CD8+T cells after downregulation of miR- 155(24.87%±0.95%）was significantly higher than that of inhibitor control group（18.24%±1.25%), the cytokilling activity of CD8+T cells (0.26%±0.06%）was significantly lower than that of inhibitor control group （0.43%±0.05%), and the differences were statistically significant (t=10.03, 20.06, all P<0.05). Intracellular Cleaved Caspase 3 protein(1.07±0.23） expression was higher in miR-155 inhibitor group than in inhibitor control group （0.42±0.02), the expressions of IL-2 mRNA(0.73±0.26） and IFN-γ mRNA（0.54±0.18）were lower than those of inhibitor control group（1.39±0.08, 1.16±0.24), and the differences were statistically significant (t=18.75, 19.27, 18.35, all P<0.01). Up-regulation of CCAT1 promoted apoptosis of CD8+T cells and decreased cytokilling activity through miR-155, and promoted proliferation and migration of Eca-109 cells. Conclusion LncRNA CCAT1 expression was significantly up-regulated in patients with esophageal cancer. Knockdown of CCAT1 inhibited apoptosis of CD8+T cells and enhanced the antitumor activity of the cells by regulating miR-155 pathway.

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备注/Memo

备注/Memo:
作者简介：周立远(1993-)，男，硕士研究生，住院医师，研究方向：肺癌靶向治疗，恶性肿瘤生物学机制，E-mail：wal121537@126.com。
更新日期/Last Update: 2023-05-15