Jing-min Zhang1, Cui-yun Sun, Shi-zhu Yu, Qian Wang, Tong-ling An, Yan-yan Li, Yan-ling Kong, Yan-jun Wen. 1. Department of Neuropathology, Tianjin Medical University General Hospital, Tianjin Neurologic Institute, Tianjin Key Laboratory of Injuries, Variations and Regeneration of Nervous System, Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin 300052, China.
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. METHODS: Expression 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.
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. METHODS: Expression 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.