MicroRNA-29Regulates The Proliferation Of Mouse Bone Marrow Mesenchymal Stem Cells By Targeting Robol

Part1:Silence Robo1charge proliferation of mesenchymal stem cells in mouse bone marrowObjective:To confirm if Robo1is the direct target gene of miR-29.Methods:Cultured in vitro mouse bone marrow the germplasm stem cells BMSCs, chemically synthesized siRNA targeting the silence Robo1liposomal transfection stained BMSCs, QPCR and Western blot verify silencing efficiency and Robol by MTT and BrdU assay was silent proliferation of BMSCs.Results:Liposomes Robo1-siRNA transfected BMSCs Robo1siRNA group Robo1expression amount compared to the Con-siRNA group, cut about3times, Robo1protein Robo1-siRNA group treated cells compared with at the Con-siRNA group has significantly lowered the con-siRNA and Robo1-siRNA were transfected BMSCs dye, MTT experiment and BrdU assay results BMSCs cell growth was significantly inhibited.Conclusion:The Robo1closely related to the growth of BMSCs in the Robo1silence, BMSCs growth was inhibited. Part2:Investigation of miR-29targeting the3’-UTR of RobolObjective:To confirm if Robol is the direct target gene of miR-29.Methods:Wild type and mutant Robo13’-UTR gene sequence, which were amplified by PCR, were connected to double endonuclease digestion PsiCHECKTM-2vector respectively and transformed into competent cells. Then examined positive clones by plasmid restriction enzyme digestion and sequenced. After miR-29and plasmid had been cotransfected into293T cell, collected the cell cracking cytosol and examined whether Robol was the direct target gene of miR-29through Dual-Luciferase Reporter Assay System.Results:MiR-29significantly inhibits the activity of luciferase compared with blank group (P<0.01) in the plasmid groups encoding Robo13’-UTR. The same result was dectected in miR-29group compared to the negative control group (P<0.01). This indicated that miR-29can combine with the3’-UTR of Robol, thereby inhibiting the activity of luciferase. In the experimental groups of plasmid encoding mutRobol3’-UTR, there was no statistical difference between miR-29group and blank group or negative control group, indicating that miR-29can not target the mutRobo13’-UTR to inhibit the activity of luciferase.Conclusion:Robo1is the target gene of miR-29. Part3Transfection of miR-29in mesenchymal stem cells and its expressionObject:Construct Lentivirus vector to express miR-29, produce pseudo viral particles and determine its titer.Methods:Lentiviral MicroRNAs expression vector was constructed with Gateway system. Mature miR-29, TRE promoter and eGFP sequences were inserted into plasmids to produce pUp-TRE, pDown-miR-29and pTail-IRES/eGFP; scramble sequence was set as negative control. pLV.EX3d.P/puro-TRE> miR-29>IRES/eGFP was obtained with incubation of donors and accepter vectors catalyzed by LR clonase. Plasmid was then sequenced and purified for lentivirus envelope.Envelope helper plasmids:pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5, with pLV.EX3d.P/puro-TRE-miR-29-IRES/eGFP or pLVrtTA/neo which contains the imperative elements for virus packaging, were co-transfected into293T cells with lipofectamine2000, according to the manufacture’s instructions for the generation of Lenti-miR-29-eGFP/puro or Lenti-rtTA/neo respectively.To perform lentiviral infections, the mice MSCs cells were plated at40%-50%confluence and incubated overnight. Cells were firstly treated by Lenti-rtTA/neo, Selection was terminated when control cells were completely dead and antibiotic free medium were used for propagation. Neomycin resistant cells were then infected by Lenti-miR-29-eGFP/puro and grown with2μg/ml Puromycin. Double resistance cells were ultimately obtained, and2μg/ml doxycycline was added to medium and intrigue expression of miR-29.Results:(1) MiR-29genomic sequence was amplified and lentiviral expression vector were constructed.(2) LV-miR-29pseudo virus was packaged and the titer was determined;(3)293cell infected by LV-miR-29can over-express miR-29.Conclusion:LV-miR-29infection can enhance the expression level of miR-29in MSCs.