胶质瘤miR-218与细胞周期蛋白依赖性激酶6表达的相互关系及其对肿瘤细胞增殖和凋亡的影响

目的 探讨胶质瘤细胞中miR-218及细胞周期蛋白依赖性激酶6(CDK6)表达变化的相互关系及其对肿瘤细胞增殖及凋亡的影响.方法 采用组织微阵列、锁定寡核苷酸探针原位杂交及免疫组织化学(ABC法)方法,检测60例不同级别胶质瘤组织标本及10例对照脑组织中miR-218、CDK6及Ki-67抗原的表达状况,并分析三者之间的相互关系;胶质母细胞瘤细胞系(U87MG)分别被转染阴性对照序列(对照组)及miR-218 mimics(mimics组),采用即时定量聚合酶链反应(qRT-PCR)及免疫细胞化学方法 分别检测两组细胞中的miR-218水平及CDK6和Ki-67表达变化,用单细胞凝胶电泳检测凋亡细胞.结果 对照组、Ⅰ～Ⅱ级、Ⅲ级和Ⅳ级胶质瘤组miR-218阳性标记指数(LI,%)分别为22.45±0.59、4.00±1.07、1.87±1.06、0.94±0.78,四组间差异均有统计学意义(P<0.05);各组CDK6 LI分别为7.25±1.20、16.71±0.80、24.43±0.62、32.05±0.43,对照组与各胶质瘤组间及Ⅳ级组与Ⅰ～Ⅱ级组间差异有统计学意义(P<0.01);各组Ki-67阳性细胞密度分别为0.00±0.00、9.30±3.48、31.15±9.44、60.15±13.60[(±s)/0.05 mm2],各组间差异均有统计学意义(P<0.01);miR-218 LI与CDK6 LI及Ki-67阳性细胞密度间均呈显著性负相关(r=-0.480,-0.534,P<0.01),后两者间呈显著性正相关(r=0.530,P<0.01).mimics组的miR-218水平明显高于对照组,其CDK6及Ki-67 LI(14.74±1.19、30.88±3.31)均明显低于对照组(79.06±2.07、64.94±3.96,P<0.01),而凋亡指数(68.44±7.05)明显高于对照组(13.04±0.97),以上各指标两组间差异均有统计学意义(P<0.01).结论 miR-218表达水平是评价胶质瘤良恶性程度的重要参考指标;其表达异常减少可导致胶质瘤细胞CDK6表达及增殖活性增强;补充外源性miR-218可有效下调恶性胶质瘤细胞CDK6表达、抑制其增殖和促进其凋亡,故在恶性胶质瘤基因治疗中具有重要的潜在应用价值.

Abstract：

Objectives To investigate the relationship between the expression of miR-218 and CDK6 in glioma cells, and their biological impacts on the tumor cell proliferation and apoptosis. MethodsExpression levels of miR-218 as well as CDK6 and Ki-67 proteins were analyzed in 60 cases of gliomas with various grades and 10 control brain tissue samples by tissue microarray, locked oligonucleotide probe in situ hybridization and immunohistochemistry. Glioblastoma multiform cell line (U87MG) was transfected with miR-218 mimics (mimics group) and a control sequence (control group), followed by qRT-PCR detection of miR-218 and immunocytochemical stain of CDK6 and Ki-67, respectively. Single cell gel electrophoresis was used to detect the presence of apoptotic cell. Results The miR-218 labeling indexes (LI) were statistically different (P<0.05) among all groups including control (22.45±0.59) and various glioma groups (grades Ⅰ-Ⅱ 4.00±1.07, grade Ⅲ 1.87±1.06 and grade Ⅳ 0.94±0.78, respectively). The CDK6 LI of the four groups was 7.25±1.20, 16.71±0.80, 24.43±0.62 and 32.05±0.43, respectively. Significant differences existed between the control group and the glioma groups, and between grade Ⅳ and grades Ⅰ-Ⅱ glioma groups (P<0.01). Ki-67 positive cell densities of the above four groups (0.00±0.00, 9.30±3.48, 31.15±9.44 and 60.15±13.60) were significantly different from one and another (P<0.01). The expression of miR-218 negatively correlated with CDK-6 LI (r=-0.480, P<0.01) and Ki-67 positive cell density (r=-0.534, P<0.01), while the latter two positively correlated with each other (r=0.530, P<0.01). U87MG transfection experiment showed that the miR-218 level of the mimics group was significantly higher than that of the control group (P<0.01). CDK6 and Ki-67 LI of the mimics group (14.74±1.19 and 30.88±3.31) were significantly lower than those of the control group (79.06±2.07 and 64.94±3.96, P<0.01), whilst its apoptotic index (AI) (68.44±7.05) was significantly higher than that of the control group (13.04±0.97, P<0.01). Conclusions The expression level of miR-218 is an important reference indicator for the assessment of the grade of gliomas. An aberrant decrease of its expression may lead to an increase of the CDK6 expression and proliferative activity of giloma cells. Introducing exogenous miR-218 may effectively down-regulate the CDK6 expression, inhibit cell proliferation and induce apoptosis of malignant giloma cells. These findings imply that miR-218 may serve as a therapeutic agent against malignant glioma.

Relationship between miR-218 and CDK6 expression and their biological impact on glioma cell proliferation and apoptosis

Objectives To investigate the relationship between the expression of miR-218 and CDK6 in glioma cells, and their biological impacts on the tumor cell proliferation and apoptosis. MethodsExpression levels of miR-218 as well as CDK6 and Ki-67 proteins were analyzed in 60 cases of gliomas with various grades and 10 control brain tissue samples by tissue microarray, locked oligonucleotide probe in situ hybridization and immunohistochemistry. Glioblastoma multiform cell line (U87MG) was transfected with miR-218 mimics (mimics group) and a control sequence (control group), followed by qRT-PCR detection of miR-218 and immunocytochemical stain of CDK6 and Ki-67, respectively. Single cell gel electrophoresis was used to detect the presence of apoptotic cell. Results The miR-218 labeling indexes (LI) were statistically different (P<0.05) among all groups including control (22.45±0.59) and various glioma groups (grades Ⅰ-Ⅱ 4.00±1.07, grade Ⅲ 1.87±1.06 and grade Ⅳ 0.94±0.78, respectively). The CDK6 LI of the four groups was 7.25±1.20, 16.71±0.80, 24.43±0.62 and 32.05±0.43, respectively. Significant differences existed between the control group and the glioma groups, and between grade Ⅳ and grades Ⅰ-Ⅱ glioma groups (P<0.01). Ki-67 positive cell densities of the above four groups (0.00±0.00, 9.30±3.48, 31.15±9.44 and 60.15±13.60) were significantly different from one and another (P<0.01). The expression of miR-218 negatively correlated with CDK-6 LI (r=-0.480, P<0.01) and Ki-67 positive cell density (r=-0.534, P<0.01), while the latter two positively correlated with each other (r=0.530, P<0.01). U87MG transfection experiment showed that the miR-218 level of the mimics group was significantly higher than that of the control group (P<0.01). CDK6 and Ki-67 LI of the mimics group (14.74±1.19 and 30.88±3.31) were significantly lower than those of the control group (79.06±2.07 and 64.94±3.96, P<0.01), whilst its apoptotic index (AI) (68.44±7.05) was significantly higher than that of the control group (13.04±0.97, P<0.01). Conclusions The expression level of miR-218 is an important reference indicator for the assessment of the grade of gliomas. An aberrant decrease of its expression may lead to an increase of the CDK6 expression and proliferative activity of giloma cells. Introducing exogenous miR-218 may effectively down-regulate the CDK6 expression, inhibit cell proliferation and induce apoptosis of malignant giloma cells. These findings imply that miR-218 may serve as a therapeutic agent against malignant glioma.