miR-124靶向调控CM...胶质瘤细胞杀伤效应机制研究_秦君翔.pdf

94现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023miR-124 靶向调控 CMTM6 增强 CIK 对胶质瘤细胞 杀伤效应机制研究秦君翔，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）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 domain-containing family 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(0.3250.031 vs 1.0040.086)显著降低，CMTM6 mRNA 表达(3.1670.324 vs 0.9810.089)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%vs 98.26%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(0.4350.042 vs 0.8970.061，0.3540.029 vs 0.7250.068)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(1.1420.121 vs 0.8970.061，1.3260.125 vs 0.7250.068)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。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中图分类号：R739.41；R730.43文献标识码：A文章编号:1671-7414（2023）01-094-06doi:10.3969/j.issn.1671-7414.2023.01.018Study on the Mechanism of miR-124 Targeted Regulating of CMTM6 to Enhance the Cytotoxic Effect of CIK on Glioma CellsQIN Jun-xiang，HUANG Jin-feng，YUAN Xue-gang，HU Zhen-kun，XIAO Sheng（Department of Neurosurgery，Ezhou Central Hospital，Hubei Ezhou 436000,China）Abstract:ObjectiveTo 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.MethodsFrom 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(0.3250.031 vs 1.0040.086)was significantly lower,the expression of CMTM6 mRNA(3.1670.324 vs 0.9810.089)was significantly increased,the differences were statistically significant(t=39.051,26.337,all P0.001)，and there was a negative correlation between them(r2=0.262).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：。95现代检验医学杂志第 38 卷第 1 期2023 年 1 月J Mod Lab Med,Vol.38,No.1,Jan.2023Compared 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(0.4350.042 vs 0.8970.061,0.3540.029 vs 0.7250.068)in C6 and U87 cells co-cultured with miR-124 mimic and CIK cells was significantly decreased,while the expression of CMTM6 protein(1.1420.121 vs 0.8970.061,1.3260.125 vs 0.7250.068)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).ConclusionCIK cells could effectively kill glioma cells.MiR-124 can enhance the killing effect of CIK cells on glioma cells by targetingly inhibiting CMTM6.Keywords:glioma;microRNA-124;chemokine-like factor-like MARVEL transmembrane domain-containing family member 6;cytokine induced killer cell虽然近来胶质瘤(glioma)治疗取得长足进展，但接受手术或联合放化疗的胶质母细胞瘤患者 2 年和 5 年生存率仅 25%和 10%，且患者中位生存期并没有显著延长，依然严重威胁人类健康1。细胞因子诱导杀伤细胞（cytokine induced killer cells，CIK）治疗不仅对肾癌、胰腺癌细胞2等具有杀伤作用，还可有效治疗肝癌、胶质瘤等3-4，但其作用机制尚不完全明确。成熟微小核糖核酸（miR-NA）在细胞增殖、凋亡、分化及肿瘤等疾病发展中起重要作用5-6。miR-124 参与调控神经细胞分化过程和神经元特异性基因的表达，影响脑胶质瘤发展7-8。含 MARVEL 跨膜结构域的趋化素样因子家族基因 6（chemoki