[1]秦君翔,黄锦峰,袁学刚,等.miR-124 靶向调控CMTM6 增强CIK 对胶质瘤细胞杀伤效应机制研究[J].现代检验医学杂志,2023,38(01):94-99.[doi:10.3969/j.issn.1671-7414.2023.01.018]
　QIN Jun-xiang,HUANG Jin-feng,YUAN Xue-gang,et al.Study on the Mechanism of miR-124 Targeted Regulating of CMTM6 to Enhance the Cytotoxic Effect of CIK on Glioma Cells[J].Journal of Modern Laboratory Medicine,2023,38(01):94-99.[doi:10.3969/j.issn.1671-7414.2023.01.018]
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miR-124 靶向调控CMTM6 增强CIK 对胶质瘤细胞杀伤效应机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年01期
页码:
94-99
栏目:
论著
出版日期:
2023-01-15

文章信息/Info

Title:
Study on the Mechanism of miR-124 Targeted Regulating of CMTM6 to Enhance the Cytotoxic Effect of CIK on Glioma Cells
文章编号:
1671-7414（2023）01-094-06
作者:
秦君翔; 黄锦峰; 袁学刚; 胡振坤; 肖 胜
（鄂州市中心医院神经外科，湖北鄂州 436000）
Author(s):
QIN Jun-xiang; HUANG Jin-feng; YUAN Xue-gang; HU Zhen-kun; XIAO Sheng
（Department of Neurosurgery，Ezhou Central Hospital，Hubei Ezhou 436000, China）
关键词:
胶质瘤; 微小核糖核酸-124; 含MARVEL 跨膜结构域的趋化素样因子家族基因6; 细胞因子诱导杀伤细胞
分类号:
R739.41；R730.43
DOI:
10.3969/j.issn.1671-7414.2023.01.018
文献标志码:
A
摘要:
目的 研究细胞因子诱导杀伤（cytokine induced killer ，CIK）细胞对胶质瘤细胞杀伤效应的影响以及微小核糖核酸（miRNA，miR）-124 在此过程中的调节作用及可能机制。方法 收集2017 年3 月～ 2019 年10 月鄂州市中心医院神经外科收治的30 例脑胶质瘤患者的癌组织及癌旁组织标本，通过qRT-PCR 法检测脑胶质瘤及癌旁组织中miR-124，含MARVEL 跨膜结构域的趋化素样因子家族基因6 (chemokine-like factor-like MARVEL transmembrane domaincontainingfamily member 6，CMTM6) mRNA 表达量及相关性。诱导培养CIK 细胞后，分别与胶质瘤细胞C6 和U87共培养，另转染miR-124 mimic，inhibitor 及阴性对照序列，通过CCK-8 实验检测CIK 细胞及转染对胶质瘤细胞的杀伤效应；双荧光素酶报告实验分析miR-124 与CMTM6 基因的靶向关系，qRT-PCR 和Western blot 检测胶质瘤细胞中miR-124，CMTM6 mRNA 及蛋白表达。结果 与癌旁组织比较，脑胶质瘤组织中miR-124 表达(0.325±0.031 vs1.004±0.086) 显著降低，CMTM6 mRNA 表达(3.167±0.324 vs 0.981±0.089) 显著升高，差异有统计学意义（t=39.051，26.337，均P ＜ 0.001），二者呈负相关（r2=0.262）。与转染阴性对照比较，miR-124 mimic 转染及其与CIK 细胞共培养对C6(72.84%±3.81% vs 99.67%±3.45%，51.46%±3.18% vs 74.59%±3.47%)，U87 细胞(71.93%±3.94% vs98.26%±3.59%，52.67%±3.24% vs 76.28%±3.64%) 增殖均有显著抑制作用，差异有统计学意义（q=16.340，15.486,14.087, 13.886，均P ＜ 0.001）。双荧光素酶报告实验显示，miR-124 和CMTM6 具有明显的靶向关系。Western blot 结果显示，与转染阴性对照+CIK 组比较miR-124 mimic 与CIK 细胞共培养的C6，U87 细胞CMTM6蛋白表达(0.435±0.042vs 0.897±0.061，0.354±0.029 vs 0.725±0.068) 显著降低，转染miR-124 inhibitor 与CIK 细胞共培养的C6，U87 细胞CMTM6 蛋白表达(1.142±0.121 vs 0.897±0.061，1.326±0.125 vs 0.725±0.068) 显著增加，差异均有统计学意义（q=13.817，10.839, 7.327，17.558，均P ＜ 0.001）。结论 CIK 细胞可有效杀伤胶质瘤细胞，miR-124 可通过靶向抑制CMTM6 增强CIK 细胞对胶质瘤细胞的杀伤效应。
Abstract:
Objective To study the effect of cytokine induced killer (CIK) cellson the killing effect of glioma cells and the regulatory role and possible mechanism of microRNA-124 (miR-124) in this process. Methods From March 2017 to October 2019, 30 cases of glioma tissues and adjacent tissues in Department of Neurosurgery, Ezhou Central Hospital were collected. The expressions and correlation of miR-124 and chemokine-like factor-like MARVEL transmembrane domain-containing family member 6 (CMTM6) mRNA in glioma and adjacent tissues were detected by qRT-PCR. After inducing and cultruing CIK cells, they were co cultured with glioma cells C6 and U87 respectively, and were transfected with miR-124 mimic, inhibitor and negative control sequences. The killing effects of CIK cells and transfection on glioma cells was detected by CCK-8 experiment. The targeting relationship between miR-124 and CMTM6 gene was analyzed by double Luciferase Report, and the mRNA and protein expressions of miR-124 and CMTM6 in glioma cells were detected by qRT-PCR and Western blot respectively. Results Compared with that in adjacent tissues, the expression of miR-124 in gliomas (0.325±0.031 vs 1.004±0.086) was significantly lower, the expression of CMTM6 mRNA (3.167±0.324 vs 0.981±0.089) was significantly increased ,the differences were statistically significant(t=39.051, 26.337, all P<0.001)，and there was a negative correlation between them (r2=0.262).Compared with negative transfection control, the transfection of miR-124 mimic and its co-culture with CIK cells significantly inhibited the proliferation of C6 (72.84%±3.81% vs 99.67%±3.45 %, 51.46%±3.18% vs 74.59%±3.47%) and U87 (71.93%±3.94% vs 98.26%±3.59 %, 52.67%±3.24% vs 76.28%±3.64%) cells，the differences were statistically significant (q=16.340, 15.486, 14.087, 13.886, all P ＜ 0.001). Double Luciferase Report showed that miR-124 and CMTM6 had an obvious targeting relationship. Western blot results showed, compared with the negative transfection control +CIK group, that the expression of CMTM6 protein (0.435±0.042 vs 0.897±0.061, 0.354±0.029 vs 0.725±0.068) in C6 and U87 cells co-cultured with miR-124 mimic and CIK cells was significantly decreased, while the expression of CMTM6 protein (1.142±0.121 vs 0.897±0.061, 1.326±0.125 vs 0.725±0.068) in C6 and U87 cells co-cultured with miR-124 inhibitor and CIK cells was significantly increased ，the differences were statistically significant (q=13.817, 10.839, 7.327, 17.558, all P ＜ 0.001). Conclusion CIK cells could effectively kill glioma cells. MiR-124 can enhance the killing effect of CIK cells on glioma cells by targetingly inhibiting CMTM6.

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备注/Memo

备注/Memo:
作者简介： 秦君翔（1981-），男，硕士研究生，主治医师，研究方向：脑血管病诊断治疗，脑血管病介入诊断治疗，E-mail：aidd20220407@163.com。
更新日期/Last Update: 2023-01-15