MiR-21Regulats The Expression Of MAP2K3and Its Effect On The Proliferation And Apoptosis Of HepG2

Objective To construct the recombined adenovirus vector, which can efficientlyinhibits maturation miR-21, and to explore its effects and mechanisms on hepatoma cell lineHepG2. The aim of this experiment is to explain tumor molecular mechanisms of miR-21gene, provide clinic with more information about liver cancer related control measures, andprovide reference data and new ideas for clinical treatment. The research results are expectedto provide a new theoretical basis for in-depth study of miR-21gene and its molecularregulatory mechanisms in the course of tumorigenesis, and laid a foundation of drugresearch To prevent or treat cancer.Methods （1）Oligonucleotides miR-21S were synthesized，which is designed bymiRNA sponge technology. This sequence is perfectly complementary to miR-21and repeatseight times. The resulting extracted amplicons were first cloned into the pAdTrack-CMVvector. To generate recombinant adenoviral plasmid expressing genes, each high quality PmeIlinearized transfer vector were transformed into electro competent E. coli BJ5183containingsupercoiled pAdEasy-1to prompt homologous recombination between them, followed by16-18hr incubation on LB medium containing kanamycin. Plasmid pAd/miR-21/inhibitorwas transfected into293A cells for packaging of the adenovirus using liposome，293A cellswere then transduced with an appropriately diluted adenovirus supernatant for the titration ofvirus titer．（2）Using miRanda, Target Scan and the PicTar target gene prediction software tofind the potential target genes of miR-21（microRNA-21）, which was the high degree ofconservatism, combined with lower free energy, and related to innate immune response. Weselected MAP2K3as a target gene of miR-21. Firefly Luciferase expression vector wasconstructed to verify the relationship between miR-21and MAP2K33’UTR of MAP2K3wasPCR amplified and inserted into pMIR-Report Luciferase vector. pMIR-Report/MAP2K3and pAd/pri-miR-21were co-transfected into293T cells with lipofectanine regent. After48hourincubation, the cells were harvested and subjected to dual Luciferase assay, and RenillaLuciferase used as internal control. Point mutation was used to mutate the binding site ofmiR-21in MAP2K3-3’UTR to detect the inhibition effect.（3）HepG2cells was infected byrecombinant adenovirus Ad/miR-21/inhibitor, Ad/pri-miR-21or Ad/con.Cells were harvested48hours after infection,and microRNAs and total protein was isolated,then the method ofReal-time PCR and Western blot were used to confirm the relationship between miR-21andMAP2K3.（4）HepG2cells was infected by recombinant adenovirus Ad/miR-21/inhibitor.Cells were harvested24h,48h,72h, and test cell proliferation and apoptosis level.（5）Fourtheen liver tumor samples and matched controls in the experimental panel were obtainedfrom Medical Pathology Department,General Hospital，Ningxia Medical University from2007to2009. Protein expression ofMAP2K3was evaluated by immunohistochemistry using rabbit anti-MAP2K3antibodyResults Bioinformatic analysis suggested several possible binding sites betweenmiR-21and MAP2K3. Through the PCR、restriction enzyme digestion、DNA sequencing andexpression of GFP, recombinant adenoviral vector miR-21S gene was constructedsuccessfully. The experiment verified that the miR-21could reduce the expression level ofMAP2K3protein, palt an important role of regulation in the occurrence of liver cancer. Therecombinant adenovirus can inhibite the proliferation and promote the apoptosis of HepG2cells.Conclusions We constructe human pAd/miR-21/inhibitor recombinant adenovirussuccessfully, and verify that the miR-21can negatively regulate expression of target geneMAP2K3and play an important role in the occurrence of liver cancer.This reserch providessafeguard for gene therapy by targeting microRNA-21for the future.