The Inhibition Of MiRNA-338-5p On Proliferation And Invasion Of Glioma And The Research On CTBP2 Protein Targeted Radionuclide Imaging

Objective:1.Evaluate the expression of miR-338-5p in glioma tissues and the correlations between expression of miR-338-5p and the clinical-pathological characteristics of glioma patients.2.Explore its role in glioma cell proliferation and identify its target genes in the hope of identifying a new target for further diagnostic and treatment research in glioma.3.The phage peptide library technique was introduced to screen out and verify the winning binding peptide towards new target protein.4.Tumor targeted ability was evaluated in a tumor-bearing mouse model.Methods:The expression of miR-338-5p in 44 glioma tissues and normal tissues were detected by real time PCR(RT-PCR).The relationship between miR-338-5p and clinical-pathological characteristics of glioma patients were analyzed.Based on the expression of miR-338-5p in glioma tissues,we chose two glioma cell lines with lower miR-338-5p expression,for rescue restoration of miR-338-5p in further functional study.We then verified the rescue restoration of miR-338-5p which was transfected with miRNA-338-5p mimics ow negative control by RT-PCR in T98 G and U87 cell lines.The cell proliferation was measured by CCK-8 and cell cycle analysis was executed by flow cytometry in the biological functional study respectively.By bio-informatics method,we predict the potential targets of miR-338-5p in glioma.Luciferase reporter assay was used to verify the candidate genes.Western blot was also performed to detect the expression levels of protein.By bio-informatics method,a conservative sequence was identified on the surface of CTBP2 protein.The peptide was synthesized by solid phase method.Targeting this peptide,we used the classical phage peptide library display technique to search potential target-binding peptide.After that,phage clones that bear preferable binding affinity to target CTBP2 conservative peptide were obtained through total four rounds of screening processes,among which,seven clones were randomly selected and then sequenced,based on the single strained DNA fragments.ELISA analysis was performed to check the binding affinity of the final selected phage clones.TCB(VSKYHLHSRARL)with the highest binding affinity was chosen as the winner peptide.In the radionuclide imaging study,TCB was labeled with 99 mTc.Using U87 tumor cell bearing mouse model,we performed tumor imaging study 30 min,60 min and 90 min after injection of 100 ?L 99mTc-HYNIC-TCB(11.1MBq/kg)through the mouses' tail vein,the specific recognizing and binding ability of 99mTc-HYNIC-labelled TCB to tumor was evaluated.The tumor bearing mouse was sacrificed at 0.5,1h,2h and 4h after injection of 100 u L of 99mTc-HYNIC-labeled TCB(with a radioactivity of 11.1 MBq per kilogram).Then,organs including lung,liver,kidney,intestine,spleen,heart,muscle,bone,tumor tissues and peripheral blood samples were obtained and then weighed.Samples from each organ or tissue were separately placed on a gamma-counter for radioactive count evaluation.The percentage of ID/g was calculated after calibration.The bio-distribution profile of 99mTc-HYNIC-labeled TCB in U87 tumor cell line loaded mouse models was investigated.Results:1.miR-338-5p expression was significantly down-regulated in the glioma tissue as compared with normal tissue samples(p<0.05).Moreover,miR-338-5p expression was found to be correlated with WHO glioma grade and Karnofsky performance status.2.Compared with negative control,the expression of miR-338-5p in glioma cell line T98 G and U87 increased significantly after transfected with miR-338-5p mimics(p<0.05).The cell functional assay showed that the proliferation ability of T98 G and U87 was significantly decreased(p<0.05).The cell cycle was arrested in G1 phase when miR-338-5p was up-regulated.3.Using bio-informatics,we found that miR-338-5p potentially target CTBP2.Luciferrase reporter assay confirmed that CTBP2 was a direct target of miR-338-5p.Western blot demonstrated miR-338-5p suppressed the expression of CTBP2 to inhibit proliferation and invasion of glioma.The CTBP2 was verified as a target for further imaging and treatment studies.4.The highly conservative sequence on the surface of CTBP2 protein was successfully synthesized: R158YAYEVPIREEKGHQ172.Taking this target peptide for binding assay,we finally identified a peptide with optimum binding affinity: TCB(VSKYHLHSRARL).The KD value between TCB and the conservative peptide was 3.15 n M.5.The labeling rate of 99mTc-HYNIC-TCB was 95%.The SPECT imaging study demonstrated that 90 min after injection,the tumor was with favorable imaging quality in mouse model.Conclusion:1.miR-338-5p executes its inhibition of proliferation and invasion of glioma by down-regulation of CTBP2 protein.2.We successfully obtained the optimum specific binding peptide TCB(VSKYHLHSRARL)to CTBP2 using phage peptide library technique.99 mTc was successfully labeled on this peptide.3.By targeting CTBP2 on glioma,we successfully imaged tumor of glioma using 99mTc-HYNIC-TCB SPECT,which will provide basis for further research on diagnosis,pathological classification,treatment and mechanism of glioma.