Expression And Significance Of MiR - 124 Target Gene Capn4 In Glioma And Its Related Molecular Mechanism

Aims:To detect the expression level of Capn4in glioma and normal brain tissues, and definite whether difference between glioma and normal brain tissues exist and analyse the the significance of difference. Establishing an Anti-capn4glioma cell line, and researching the role of Capn4in GBM cell biological functions. To analyse the expression level of Capn4in glioblastoma, and definite whether Capn4has prognostic significance for glioblastoma patients. The miRNA prediction website predict that the Capn4was one of miRNA-124targets, and we will verify this hypothesis.Materials and methods:Immunohistochemistry was used to detect the expression of the Capn4in300samples consisting of13normal brain tissues,8adjacent tissues and279glioma tissues. The difference between tissues was investigated and the signification of difference was analysed. Then, we selected U251glioblastoma cell line, and made a virus plasmid transfectthe hairpin RNA (shRNA-Capn4) of anti-Capn4into U251glioblastoma cell line to establish stable siRNA-Capn4-U251cell line as the Experimental Group; For the Control Group, we made a blank virus plasmid transfectinto U251glioblastoma cell tumor, and establish stable eGFP-U251cell line. Reverse transcription was used to make a real-time quantitative test of PCR and Western Blotting, which can reflect the expression level of mRNA and protein in the Experimental Group and Control Group, to confirm theCapn4showed low expression levelsin siRNA-Capn4-U251cells. To test the Cell migration, invasion and proliferation abilities of the eGFP-U251cell line as Control Group, U251cell line as blank Control Group, and shRNA-Capn4-U251cell line as Experimental Group through the cell function test (Scratch test, Transwell, MTT). Definite the impact that Capn4makes to the biological behavior of the glioma cells through comparing the differences of Cell biological characteristics between the three groups. In the third part, the expression of the Capn4in94cases of glioblastoma tumors samples was detected by immunohistochemistry. After completing clinicopathologic information of patients (including follow-up data), the SPSS19.0software was used to analysis the relationship between high/low expression of Capn4and OS, PFS of the94cases of GBM patients (Using Kaplan-Meier univariate analysis and Cox multivariate regression analysis). Finally, after miR-124transfection experience, testing the mRNA and protein level of Capn4to verify whether Capn4is the target gene of miR-124in vitro model.Results:In the first part, the Capn4positive rate of normal tissues and adjacent tissues was significant lower than glioma tissues (p<0.001). In addition, the Capn4positive rate of HGG tissues was significant higher than LGG tissue (p<0.001). In the second part, after the down-regulation of Capn4expression level in the U251cell, the migration and invasion abilities of GBM cells decreased significantly and proliferation ability not significantly affected.In the third part, among the94GBM cases,52cases (55%) are high expressed while42cases (45%) low expressed. The overall survival time (p=0.032for univariate analysis? p=0.048, HR=1.541,95%CI:1.004-2.055for multivariate regression analysis) and progression-free survival time (p=0.038for univariate analysis? p=0.019, HR=2.23495%CI:1.151-4.757for multivariate regression analysis) of GBM patients with high expressed Capn4are shorter than that of GBM patientswith low expressed Capn4, which is statistically significant。In the fourth part, the expression of Capn4in U251GBM cells was significantly increased after the transfection of miRNA-124, while the mRNA and protein expression level of Capn4significantly decreased, which is confirmed by RT-qPCR and Western blotting. Thus, the hypothesis that Capn4is target gene of miR-124was vetified.Conclusions:Capn4is the target gene of miR-124. Capn4high was common in glioma, special for HGG. Capn4high was considered to be a novel negative prognostic biomarker for GBM patients in our data. The role of Capn4was regulating cell migration and invasion. It may be used as a new target in therapeutics strategies of GBM...