The Role Of MiR-21 In P12^CDK2AP1 Regulation And That Proliferation Or Invasion In Cultured Cells

Head and neck cancers represent about 5% of total tumors. 80-90% of these cancers are constituted of squamous cell carcinomas. Despite a rapid progress in diagnostics and therapy, the overall 5-year survival of this type cancer is among the lowest of the major tumor types. Tumor-suppressor P12CDK2AP1 negatively regulates cyclin dependent kinase 2 (CDK2) activities and suppresses DNA replication, more important, it is downregulated in head and neck squamous cell carcinomas (HNSCCs). However, its role in HNSCCs has not been well studied. So, it is important significantly to clarify the role of P12CDK2AP1 in HNSCCs carcinogenesis. This study indicates that miR-21 expression plays a key role in regulating P12CDK2AP1 in HNSCCs and may serve as a target for effective therapies.Objectives:1. To investigate the role of P12CDK2AP1 played in HNSCCs carcinogenesis.2. To investigate the regulation of P12CDK2AP1, and proliferation/invasion by miRNAs.Methods:1. A third generation of self-inactivating lentivirus vector containing a CMV-driven EGFP reporter and a U6 promoter upstream of cloning restriction sites (AgeI and EcoRI) to allow the introduction of oligonucleotides encoding short hairpin RNAs (shRNAs) was purchased from Genechem Co., Ltd. We constructed three si-CDK2AP1 lentiviruses KD-1, KD-2, and KD-3 targeting human CDK2AP1 mRNA respectively. HEK293T cells were transfected with 5μg of the lentiviral vectors along with 9μg of ViraPower Packaging mix (Invitrogen) by calcium phosphate transfection method.2. CDK2AP1 mRNA and protein expression were measured by Quantitative Real Time PCR and Western blot analysis.3. According to the CDK2AP1 GenBank accession No. NM₀04642, we predicted the miRNA target of CDK2AP1 by means of bioinformatics searches. miR-21 was chose for the following research.4. We cloned a reporter plasmid containing potential miR-21 binding sites within 3'-UTR of CDK2AP1 for further validation by luciferase reporter assays.5. Effects of pre-miR-21 or anti-miR-21 transfected on cultured cells were detected, including proliferation activity analyzed by MTT assay and Flow cytometric analysis, and invasion-related processes analysis by soft-agar assay and Transwell invasion assay.Results:1. CDK2AP1 mRNA and P12CDK2AP1 expression were efficiently knockdown in lenti-shCDK2AP1 transduced HaCaT cells.2. Knockdown of P12CDK2AP1 significantly promotes HaCaT cells growth and cell cycle transition in vitro.3. The 3'-UTR of CDK2AP1 might being a target sequence for miR-21.4. P12CDK2AP1 and miR-21 expression correlates inversely in HaCaT and Tca8113 cell lines.5. miR-21 downregulates P12CDK2AP1 expression and promotes cultured cell proliferation and invasion.Conclusions:1. In this study we have constructed a lentivirus vector for delivery of siRNAs and demonstrated its ability to efficiently downregulate the expression of a target gene in the infected cells. It is also demonstrated that P12CDK2AP1 downregulation promotes proliferation in cultured cells.2. Our study shows that tumor suppressor P12CDK2AP1 is downregulated by miR-21 at the post-transcriptional level, through a specific target site (nt349–370) within the 3'-UTR. It also demonstrated that miR-21 promotes invasion and proliferation in cultured cells.