Construction And Identification Of Eukaryotic Expression Vector Of Hsa-miR-21

Background MicroRNAs (miRNAs)are a class of endogenous small non-coding RNAs. They are approximately 20 to 25 nucleotide long in eucaryotic organisms that genetically regulate the expression of some potential target mRNAs at the post-transcriptional level. They can specially recognize and integrate the site of 3'-UTR. They lead to the degradation or translational suppression of target mRNAs at the post-transcriptional level. MiRNAs regulate a variety of important biological processes.MiRNAs are aberrantly expressed in many cancers. Many of them are linked to different cancer-related processes. They may play a role of oncogene or tumor suppression . Researches have discovered that miR-21 is one of the most commonly oncogenes in many cancers. MiR-21 gene not only regulates cells differentiation, proliferation and apoptosis but also the invasion and metastasis of tumors. Objective To construct an eukaryotic expression vector of miR-21 and to transfect it into osteosarcoma cells MG63. So that miR-21 can be highly expressed in MG63 cells. Select with G418 for 4 weeks to obtain the stable cells. So this formed the basis for further investigating the function of miR-21 in proliferation, apoptosis, invasion and the mechanism of gene regulation in osteosarcoma.Methods (1) To extract the DNA of osteosarcoma cells MG63 with DNA kit following the instruction. According to the maturity sequence of miRNA and nucleotides (about 200nt) in the vicinity which accounted to 433nt in all to design the PCR primer. We also design the consensus primer T7 and BGH .Perform PCR to amplify oligonucleotides. After being annealed,oligonucleotides were ligated with linearized pcDNA 3.1(+) by T4 DNA ligase. To verified the by restriction enzyme digestion, bacterial colony PCR, and sequencing analysis. (2) The correct recombinant plasmid pcDNA3.1(+)- miR-21 was transfected in osteosarcoma cells MG63,and selected with G418 to obtain the stable cells. The level of miR-21 expression was verified by Northern blot and Real time RT-PCR. (3)After transfection,We detect the biologic changes of MG63 by Immunocytochemistry,Western blot and Flow cytometer .Results (1) The miR-21eukaryotic expression vector pcDNA3.1(+)- miR-21 was successfully constructed. The results of restriction enzyme digestion, bacterial colony PCR, and sequencing analysis showed that an approximately 433bp DNA fragment was inserted into pcDNA3.1(+) plasmid. (2) The correct recombinant plasmid pcDNA3.1(+)-miR-21 was transfected in osteosarcoma cells MG63,and selected with G418 to obtain the stable cells. The level of miR-21 expression was verified by Northern blot and Real time RT-PCR. Results showed that the level of miR-21 was higher than normal MG63 cells (P<0.05). (3)After transfection of the recombinant plasmid, the osteosarcoma cells MG63 have had some biocharacteristics changes. Immunocytochemistry shows that the selected cells'membranes were brown. Western blot shows that the expression level of PDCD4 was reduced. Flow cytometer shows that the cell cycle was changed greatly.Conclusion (1) The miR-21 eukaryotic expression vector was successfully constructed and effectively expressed in osteosarcoma MG63 cells. (2) After transfection of the recombinant plasmid pcDNA3.1(+)- miR-21,the expression level of miR-21 was highly expressed in osteosarcoma cells MG63. This will facilitate the further investigations of function and mechanism of miR-21 in osteosarcoma. (3) The biocharacteristics of selected osteosarcoma cells MG63 have had a series of changes. This suggests that miR-21 plays an important role in osteosarcoma.