[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]
点击复制

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()
分享到:

《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.

参考文献/References:

[1] 尹周一, 王梦圆, 游伟程, 等. 2022 美国癌症统计报 告解读及中美癌症流行情况对比[J]. 肿瘤综合治疗 电子杂志, 2022, 8(2): 54-63. YIN Zhouyi, WANG Mengyuan, YOU Weicheng, et al. Interpretation on the report of American Cancer Statistics, 2022 and comparison of cancer prevalence in China and America[J]. Journal of Multidisciplinary Cancer Management(Electronic Version), 2022, 8(2): 54-63.
[2] 邹小农, 贾漫漫, 王鑫, 等. 《2020 全球癌症报告》 要点解读[J]. 中国胸心血管外科临床杂志, 2021,28(1): 11-18. ZOU Xiaonong, JIA Manman, WANG Xin, et al. Interpretation of the World Cancer Report 2020[J]. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2021, 28(1): 11-18.
[3] CHEN Xingyu, CHEN Haotian, YAO Honghui,et al. Turning up the heat on non-immunoreactive tumors: pyroptosis influences the tumor immune microenvironment in bladder cancer[J]. Oncogene,2021, 40(45): 6381-6393.
[4] 张思佳, 徐彩娜, 陈杰, 等. 通过调控肿瘤代谢增强 T 细胞抗肿瘤活性[J]. 应用化学, 2020, 37(9): 977- 984. ZHANG Sijia, XU Caina, CHEN Jie, et al. Enhancing T cell antitumor activity by regulating tumor metabolism[J]. Chinese Journal of Applied Chemistry,2020, 37(9): 977-984.
[5] 黄柱华, 苏文, 韩正全, 等. 非小细胞肺癌患者的肿 瘤转移与肿瘤浸润的CD8+T 细胞数量负相关[J]. 细 胞与分子免疫学杂志, 2019, 35(3): 266-270. HUANG Zhuhua, SU Wen, HAN Zhengquan, et al. Tumor metastasis in patients with non-small cell lung cancer is inversely correlated with the number of tumor-infiltrating CD8+T cells[J]. Chinese Journal of Cellular and Molecular Immunology, 2019, 35(3): 266- 270.
[6] 李翠霞, 苏秀兰. 长链非编码RNA (LncRNAs) 参 与肿瘤细胞免疫逃逸的研究进展[J]. 免疫学杂志,2019, 35(9): 817-822. LI Cuixia, SU Xiulan. Research progress of long-chain non-coding RNAs (LncRNAs) involved in tumor cell immune escape[J]. Immunological Journal, 2019,35(9): 817-822.
[7] ZHANG Zhengjia, HUANG Qingcai, YU Liuchunyang,et al. The role of miRNA in tumor immune escape and miRNA-based therapeutic strategies[J]. Front Immunol,2021, 12: 807895.
[8] ZHANG Caixiang, WANG Wenying, LIN Jun, et al. LncRNA CCAT1 promotes bladder cancer cell proliferation, migration and invasion[J]. International Braz J Urol, 2019, 45(3): 549-559.
[9] 李聪聪, 赵金艳, 吴姣, 等. miR-155 研究进展[J]. 生物技术通报, 2018, 34(11): 70-82. LI Congcong, ZHAO Jinyan, WU Jiao, et al. Research progress on miR-155[J]. Biotechnology Bulletin, 2018,34(11): 70-82.
[10] 陈倩云, 范恒. miR-155 调控T 细胞分化与功能的研 究进展[J]. 中国免疫学杂志, 2016, 32(7): 1065-1069. CHEN Qianyun, FAN Heng. Research progress of miR- 155 regulation of T cell differentiation and function[J]. Chinese Journal of Immunology, 2016, 32(7): 1065- 1069.
[11] 白盈盈, 朱光旭, 潘兴华. 长链非编码RNA 对结直 肠癌潜在诊断价值的研究进展[J]. 现代检验医学杂 志, 2018, 33(1): 161-164. BAI Yingying, ZHU Guangxu, PAN Xinghua. Research progress on the potential diagnostic value of long noncoding RNA in colorectal cancer[J]. Journal of Modern Laboratory Medicine, 2018, 33(1): 161-164.
[12] LI J L, LI R, GAO Y, et al. LncRNA CCAT1 promotes the progression of preeclampsia by regulating CDK4[J]. European Review for Medical and Pharmacological Sciences, 2018, 22(5): 1216-1223.
[13] ZHANG Shouhua, XIAO Juhua, CHAI Yong, et al. LncRNA-CCAT1 promotes migration, invasion, and EMT in intrahepatic cholangiocarcinoma through suppressing miR-152[J]. Digestive Diseases and Sciences, 2017, 62(11): 3050-3058.
[14] SUN W, SHEN N M, FU S L. Involvement of LncRNAmediated signaling pathway in the development of cervical cancer[J]. European Review for Medical and Pharmacological Sciences, 2019, 23(9): 3672-3687.
[15] HU Min, ZHANG Qi, TIAN Xiaohui, et al. LncRNA CCAT1 is a biomarker for the proliferation and drug resistance of esophageal cancer via the miR-143/PLK1/ BUBR1 axis[J]. Molecular Carcinogenesis, 2019,58(12): 2207-2217.
[16] L?Li, JIA Jianqin, CHEN Jin. The LncRNA CCAT1 upregulates proliferation and invasion in melanoma cells via suppressing miR-33a[J]. Oncology Research,2018, 26(2): 201-208.
[17] 胡诗芸, 奕天飞, 王家立, 等. 长链非编码RNA 在肿瘤免疫微环境中的作用[J]. 生命科学, 2022,34(2): 212-219. HU Shiyun, YI Tianfei, WANG Jiali, et al. Research progress on long non coding RNA in tumor immune microenvironment[J]. Chinese Bulletin of Life Sciences, 2022, 34(2): 212-219.
[18] YU Jing, JIANG Lijuan, GAO Yutao, et al. LncRNA CCAT1 negatively regulates miR-181a-5p to promote endometrial carcinoma cell proliferation and migration[J]. Experimental and Therapeutic Medicine,2019, 17(5): 4259-4266.
[19] HAN Chunyong, LI Xuebiao, FAN Qian, et al. CCAT1 promotes triple-negative breast cancer progression by suppressing miR-218/ZFX signaling[J]. Aging, 2019,11(14): 4858-4875.
[20] YOU Zonghao, LIU Chunhui, WANG Can, et al. LncRNA CCAT1 promotes prostate cancer cell proliferation by interacting with DDX5 and miR-28- 5P[J]. Molecular Cancer Therapeutics, 2019, 18(12): 2469-2479.
[21] BAYRAKTAR R, VAN ROOSBROECK K. MiR-155 in cancer drug resistance and as target for miRNAbased therapeutics[J]. Cancer Metastasis Reviews,2018, 37(1): 33-44.
[22] SHAO Chuchu, YANG Fengming, QIN Zhiqiang, et al. The value of miR-155 as a biomarker for the diagnosis and prognosis of lung cancer: A systematic review with meta-analysis[J]. BMC Cancer, 2019, 19(1): 1103.
[23] XIN Xiaoru, LU Yanan, XIE Sijie, et al. MiR-155 accelerates the growth of human liver cancer cells by activating CDK2 via targeting H3F3A[J]. Molecular Therapy Oncolytics, 2020, 17: 471-483.
[24] JI Yun, WRZESINSKI C, YU Zhiya, et al. MiR- 155 augments CD8+ T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic γc cytokines[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(2): 476-481.

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