[1]王军堂a,齐普良a,张金刚a,等.hsa_circ_0006950 调控miR-124-3p/EZH2 轴促进胰腺癌细胞增殖与迁移的机制研究[J].现代检验医学杂志,2022,37(05):93-99.[doi:10.3969/j.issn.1671-7414.2022.05.019]
　WANG Jun-tanga,QI Pu-lianga,ZHANG Jin-ganga,et al.Mechanism of hsa_circ_0006950 Regulating miR-124-3p /EZH2 Axis to Promote the Proliferation and Migration of Pancreatic Cancer Cells[J].Journal of Modern Laboratory Medicine,2022,37(05):93-99.[doi:10.3969/j.issn.1671-7414.2022.05.019]
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hsa_circ_0006950 调控miR-124-3p/EZH2 轴促进胰腺癌细胞增殖与迁移的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年05期
页码:
93-99
栏目:
论著
出版日期:
2022-09-15

文章信息/Info

Title:
Mechanism of hsa_circ_0006950 Regulating miR-124-3p /EZH2 Axis to Promote the Proliferation and Migration of Pancreatic Cancer Cells
文章编号:
1671-7414（2022）05-093-07
作者:
王军堂1a; 齐普良1a; 张金刚1a; 阿吉德1b; 何 婧2
1. 青海省人民医院a. 急诊外科；b. 普外科, 西宁 810007；2. 武警青海省总队医院内二科, 西宁 810014
Author(s):
WANG Jun-tang1a;  QI Pu-liang1a;  ZHANG Jin-gang1a;  A Ji-de1b;  HE Jing2
1a.Department of Emergency Surgery；1b.Department of General Surgery, Qinghai Provincial People’s Hospital， Xining 810007,China；2.the Second Department of Internal Medicine，Qinghai Provincial Armed Police Corps Hospital，Xining 810014, China
关键词:
胰腺癌; hsa_circ_0006950; 微小核糖核酸-124-3p; EZH2; 增殖; 克隆形成; 迁移
分类号:
R736.7；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.05.019
文献标志码:
A
摘要:
目的　检测环状核糖核酸（circular RNA ，cicrRNA）hsa_circ_0006950 在胰腺癌（pancreatic cancer，PC）中的表达，探讨其对胰腺癌细胞增殖、迁移的影响及其潜在分子作用机制。方法　利用实时荧光定量PCR(qRT-PCR) 检测hsa_circ_0006950 在胰腺癌组织及细胞中的相对表达水平；转染hsa_circ_0006950 干扰载体构建低表达细胞系，通过CCK-8实验、克隆斑点形成实验和Transwell 实验检测hsa_circ_0006950 对胰腺癌细胞增殖、克隆形成及迁移的影响；利用生物信息学网站预测hsa_circ_0006950 的miRNA 分子靶标及其调控网络（hsa_circ_0006950-miR-124-3p-EZH2），双荧光素酶基因报告实验验证hsa_circ_0006950 和miR-124-3p，miR-124-3p 和EZH2 的靶向结合关系；转染过表达/ 干扰miR-124-3p 和过表达EZH2 细胞系，探究miR-124-3p 和EZH2 及hsa_circ_0006950 调控miR-124-3p/EZH2 轴对胰腺癌细胞克隆形成及迁移的影响。结果　胰腺癌组织中hsa_circ_0006950 表达明显高于癌旁正常组织（3.57±0.52 vs 1.01±0.03），差异有统计学意义（t=21.980，P ＜ 0.001）。胰腺癌细胞PANC-1（7.51±0.62），AsPC-1（5.26±0.45），Capan-2（3.69±0.38），SW1990（3.25±0.32）and BXPC-3（3.86±0.35）中hsa_circ_0006950 相对表达明显高于正常胰腺导管上皮细胞HPDE6-C7（1.00±0.01）中的表达，差异有统计学意义（F=88.585，P ＜ 0.001）。与对照组相比，干扰hsa_circ_0006950 表达组细胞增殖能力（0.79±0.17 vs 1.83±0.42），克隆形成率（51.42%±5.84% vs 78.76%±13.65%）和迁移数目（104.64±24.73 vs 218.21±31.57）明显降低，差异均有统计学意义（t=3.976，3.190，4.905，P=0.016，0.033，0.008）。miR-124-3p 是hsa_circ_0006950 的下游靶基因，EZH2 是miR-124-3p 的直接靶标。hsa_circ_0006950 靶向负调控miR-124-3p，miR-124-3p 靶向负调控EZH2。与对照组相比，过表达miR-124-3p 组PC 细胞增殖（0.21±0.16 vs1.75±0.47），克隆形成率（47.85%±4.13% vs 81.54%±2.33%）和细胞迁移数目（118.74±24.65 vs 202.36±31.45）明显降低，差异均有统计学意义（t=5.378, 14.317, 3.390, 均P ＜ 0.001）；共转染过表达EZH2 后，miR-124-3p 对细胞增殖、克隆形成率及迁移能力的抑制作用被逆转。在干扰hsa_circ_0006950 组细胞中同时下调miR-124-3p 或过表达EZH2 后，hsa_circ_0006950 对细胞克隆形成及迁移的抑制作用被逆转恢复。结论　胰腺癌中hsa_circ_0006950 显著高表达，抑制其表达可以抑制胰腺癌细胞的增殖及迁移；hsa_circ_0006950 可能通过靶向下调miR-124-3p 表达，进而上调EZH2 表达来发挥作用，参与胰腺癌的发生发展。
Abstract:
objective　 To detect the expression of cicrRNA hsa_circ_0006950 in pancreatic cancer (PC) and investigate the effect of cicrRNA hsa_circ_0006950 on the proliferation and migration of PC cells and its potential molecular mechanism. Methods　QRT-PCR was used to detect the relative expression level of hsa_circ_0006950 in pancreatic cancer tissues and cells. Low expression cell lines were constructed by transfection of hsa_circ_0006950 interference vector, and the effects of hsa_ circ_0006950 on PC cell proliferation, clonal formation and migration were detected by CCK-8, clone spot formation assay and Transwell assay. Predicted miRNA molecular target and regulatory network of hsa_circ_0006950 (hsa_circ_0006950-miR-124- 3p-EZH2) by bioinformatics website，targeted binding relationship between hsa_ circ_0006950 and miR-124-3p, miR-124-3p and EZH2 was verified by double luciferase gene report experiment. Over expression/interference of miR-124-3p and over expression of EZH2 cell lines were transfected to explore the effects of miR-124-3p, EZH2 and hsa_circ_0006950 regulating miR-124-3p/EZH2 axis on the formation and migration of PC cell clones.Results　The expression of Hsa_circ_0006950 in PC tissues (3.57±0.52 vs 1.01±0.03) was significantly higher than that in adjacent normal tissues，the difference was statistically significant (t=21.980, P ＜ 0.001).In PC cell PANC-1 (7.51±0.62), AsPC-1 (5.26±0.45), Capan-2 (3.69±0.38), SW1990 (3.25±0.32), the relative expression of hsa_circ_0006950 in BXPC-3 (3.86±0.35) was significantly higher than that in HPDE6-C7 (1.00±0.01) in normal pancreatic duct epithelial cells, the difference was statistically significant (F=88.585, P ＜ 0.001).Compared with the control group, the proliferation ability and clone formation rate of hsa_circ_0006950 expression group were 0.79±0.17 vs 1.83±0.42, and 51.42±5.84 vs 78.76±13.65 and the number of migrations (104.64±24.73 vs 218.21±31.57） were significantly decreased，the differences were statistically significant（t =3.976, 3.190, 4.905, P =0.016， 0.033，0.008).MiR-124-3p is the downstream target gene of hsa_circ_0006950, and EZH2 is the direct target of miR-124-3p. Hsa_circ_0006950 negatively regulates miR-124-3p, and miR-124-3p negatively regulates EZH2.Compared with the control group, PC cell proliferation in miR-124-3p overexpressed group (0.21±0.16 vs 1.75±0.47 ), the clone formation rate (47.85%±4.13% vs 81.54%±2.33%) and the number of cell migration (118.74±24.65 vs 202.36±31.45) were significantly decreased，the differences were statistically significant (t=5.378, 14.317, 3.390,P ＜ 0.001). After co-transfection and over expression of EZH2, the inhibition of miR-124-3p on cell clonal formation rate and migration ability was reversed.After downregulation of miR-124-3p or over expression of EZH2 in hsa_circ_0006950 group, the inhibition of hsa_circ_0006950 on cell cloning and migration was reversed and restored. Conclusion　Hsa_circ_0006950 was highly expressed in pancreatic cancer, and inhibition of its expression could inhibit the proliferation and migration of pancreatic cancer cells. hsa_circ_0006950 may play a role in the occurrence and development of pancreatic cancer by down-regulating the expression of miR-124-3p and then up-regulating the expression of EZH2.

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备注/Memo

备注/Memo:
基金项目：青海省卫生计生系统科研项目（2017-wjzdx-23）：青海省卫生健康系统指导性科研项目（2020-wjzdx-27）。
作者简介： 王军堂（1977-），男，本科，主治医师，研究方向：胰腺肿瘤的临床治疗与基础研究，E-mail: wang87632@163.com。
通讯作者：阿吉德（1979-），男，博士，主治医师，E-mail:ye19790309@126.com。
更新日期/Last Update: 2022-09-15