hsa-miR-140 as a Potent Cell Cycle Regulator in Glioblastoma by Inhibiting CDKs

Background and Aim: Glioblastoma multiforme (GBM) is the mostcommon malignant primary brain tumor among adults and one of themost lethal cancers. The median survival of patients is 12–15 months afterinitial diagnosis due to radiation therapy and chemotherapy resistance.Hence new therapeutic approaches are required. GBM is first cancer,which studied by The Cancer Genome Atlas Research (TCGA). Accordingto this study, 78.9% of tumors had one or more alteration affecting Rbfunction: 7.6% by direct RB1 mutation/deletion, 15.5% by amplificationof CDK4/6, and the remainder via CDKN2A deletion.Methods: miRNAs play a role of gene’s regulators by affecting the 3 UTRregion of its mRNA. We have chosen CDK4 and CDK6, upregulatedgenes in GBM and used TargetScan and miRWalk databases to reach alist of miRNAs, which can exert cell cycle arrest via inhibiting CDK4/6transcription/translation. The hsa-miR-140-5p was the most probablemiRNA having wanted function. hsa-miR-140 was cloned in the pLenti-III-eGFP vector. HEK 293T cells were used to package virus particlesby using lentiviral packaging plasmids, psPAX, pMD2.G and desirablevector. U-251 cell line is our model for this study. Cells were transducedby lentiviruses carrying hsa-miRNA-140 and after 72 hours cell wastrypsinized for RNA extraction and cDNA synthesis. Real-Time PCRmethod was used to find out the alteration of gene expression’s levelin treated cells versus the control group. Internal control is beta-2-microglobulin (B2M) gene.Results: Over 80% of U-251 cells have been transduced successfullyafter 48 and emitting green fluorescent light under a fluorescencemicroscope. REST software is used for data analysis and results areshown below. Expression of CDK6 is reduced significantly (P<0.001) to0.158-fold. Although the expression of CDK4 is reduced to 0.201-fold, itis not significant. (N=2)Conclusion: Needless cell proliferation caused by uncontrolled cellcycle and eventually cancer appeared. CDK4 and CDK6 are genesthat overexpressed in glioma tissue, pair with Cyclin D and let cellspass G1 checkpoint and enter to S phase. In this study, we have shownhas-miR-140 can be a good suppressor of CDK6, which is a promisingapproach to stop cancer proliferation. This study should examine in otherglioma cell lines such as U87 and A172. In addition, more assays like cellmigration and luciferase should be done to approve our claim.

کلیدواژه ها:

Glioblastoma ، Cyclin-dependent kinase 4 ، Cyclin-dependent kinase 6 ، MicroRNAs ، miR-140