| Purpose To examine the role of CMIP(C-Maf inducing protein)on cell proliferation and metastasis in human glioma cells,to study the downstream mechanism of CMIP in regulation of MDM2,and to analyze the expression levels and the clinical significance of CMIP in glioma patients.Methods In this study,we mainly carried out in vitro cell experiments and clinical pathological experiments.1.In vitro cell experiments:(1)Examine the m RNA levels of CMIP in human glioma cells A172,U251,and H4 by using real time PCR(RT-q PCR).(2)We determined the oncogene role of CMIP in promoting cell proliferation and metastasis in A172 cells through knockdown of CMIP(by si RNA method)and in U251 cells through over-expression of CMIP(by CMIP plasmids)by using MTT assay,cell colony formation assay,cell migration assay and cell invasion assay.Through the flow cytometry experiment,it showed that the proportion of cell apoptosis was changed obviously after A172 CMIP-si RNA.(3)Through the Western Blot experiment,we detected the change of MDM2 gene expression level in glioma cells A172 and U251 when CMIP was knocked down or over expressed,and further explored it as CMIP downstream regulation gene.2.Clinical pathological experiments: For study in patients,99 paraffin-embedded surgical glioma tissue specimens and 59 normal tissue specimens were collected at the First Affiliated Hospital of Anhui Medical University(Hefei,Anhui,China)between 2009 and 2015.No other diseases were diagnosed and no special therapies were used in these patients before surgery.The clinicopathological parameters were determined in the patients with glioma based on the World Health Organization grading systems.The patients with glioma were followed up for no less than 48 months.Inform consent was obtained from each patient before we performed this study.This study plan got the approval of the institutional review board.(1)The expression levels of CMIP in glioma tissues was detected by immunohistochemistry,and compared with normal glial tissues.(2)In the same case,the expression level of MDM2 was detected,and the relationship between CMIP gene was clarified.(3)The correlation between the expression of CMIP in glioma tissues and the pathological parameters of patients was analyzed.The parameters included patients' age,gender,histological grade and KPS score.(4)Kaplan-Meier curves were analyzed ccording to the relationship between CMIP expression levels in glioma tissues and the recurrence free survival rate(RFS)and overall survival rate(OS)of patients,and the correlation analysis was performed.Results 1.In vitro cell experiments:(1)To evaluate the base level of CMIP in different glioma cells,RT-q PCR was carried out in human glioma cells H4,A172 and U251.Among the three cell lines,m RNA level of CMIP was highest in A172 cells,was moderate in H4 cells and was lowest in U251 cells(H4,1.00±0.11;A172,2.21±0.18;U251,0.38±0.04).(2)In A172 cells,cell viability decreased significantly over a period of 96 hours after transfected with CMIP-si RNA compared with negative control si RNA(hour96 OD490,1.01±0.06 vs.0.71±0.09,P<0.05);meanwhile,cell viability increased significantly over a period of 96 hours in U251 cells after transfected withp IRESneo3-CMIP compared with p IRESneo3-Negtive control(hour96 OD490,1.11±0.12 vs.0.78±0.08,P<0.05).(3)Concordantly,CMIP-si RNA significantly decreased cell colony formation of A172 cells(45.2±9.8 vs.23.2±5.2,P<0.05)and p IRESneo3-CMIP significantly promoted cell colony formation of U251 cells respectively(24.7±4.5 vs.47.8±8.3,P<0.05).(4)Flow cytometry analysis of A172 cells showed that the apoptotic cell population was 3.101% in the negative control group,and was 4.423% in the CMIP-si RNA transfected group(P<0.05).(5)For further study,migration assay and invasion assay in human glioma cells were performed to examine the role of CMIP in cell metastasis.In A172 cells,both migration(171.6±3.2 vs.78.2±12.5,P<0.05)and invasion(149.3±9.6 vs.76.9±7.4,P<0.05)decreased significantly after transfected with CMIP-si RNA compared with control.Concordantly,both migration(42.2±4.8 vs.145.6±6.6,P<0.05)and invasion(48.3±4.2 vs.176.4±3.9,P<0.05)increased significantly in U251 cells with over expression of CMIP compared with control.(6)We selected several candidate genes to find the downstream mechanism of CMIP in human glioma cells.Among them,MDM2 decreased obviously after transfected with CMIP-si RNA in A172 cells,and increased significantly after transfected with p IRESneo3-CMIP in U251 cells.2.Study on clinical tissue samples(1)Protein levels of tissues from patients were detected using immunohistochemistry.CMIP expressed much higher in glioma tissues compared with normal tissues.As showed,38 out of 99 cases(38.4%)in glioma tissues were CMIP positive and 61 out of 99 cases(61.6%)were CMIP negative;12 out of 59 cases(20.3%)in adjacent normal tissues were CMIP positive and 47 out of 59 cases(79.7%)were CMIP negative.The difference of CMIP expression between glioma tissues and adjacent normal tissues was significant(P<0.05).(2)The percentage of CMIP high expressed tissues in high-grade gliomas(grade III-IV)(32/59,54.9%)was much higher than that in low-grade glioma(grade I-II)(8/40,20.8%)(P<0.01).(3)Immunohistochemical technique was taken to detect MDM2 expression in 99 patients with glioma.41 cases showed positive expression(41.4%),MDM2 protein was mainly located in tumor cell cytoplasm,and PEARSON correlation analysis showed that MDM2 and CMIP were significantly correlated in glioma expression(rs=0.433,p0.01).(4)The differences between CMIP expression with patients' age,gender or KPS were not significant(all P>0.1).(5)We made Kaplan-Meier curves in these 99 patients with glioma to analyze the association of CMIP expression with their RFS and OS rates.These patients were all followed up for more than 48 months.Compared with the CMIP low group,patients in the CMIP high group exhibited both lower RFS rate(P<0.05)and lower OS rate(P<0.01).Conclusion In this project,we have demonstrated that CMIP promoted proliferation,metastasis and invasion of human glioma cells,and the role of CMIP may be achieved by regulating MDM2.The expression levels of CMIP were significantly related to the prognosis and pathological parameters of patients,and CMIP could be used as a potential molecular target for clinical treatment of glioma. |
PDF Full Text Request