Abstract:

[Abstract] Objective   To establish method of constructing lentiviral vectors to express microRNA（miRNA） ''tough decoy''（TuD）and to detect the effects of the TuD on cellular endogenous miRNA level and cellular phenotypes. Methods Two-step cloning strategy was utilized to first generate a universal miRNA TuD frame vector，followed by constructing the TuD expression vector specially targeting miR- 203. The package of the recombinant lentivirus was performed in 293T cells. Then the rat bone marrow mesenchymal stem cells（BM-MSCs）were infected by the miR-203 TuD expression lentivirus. The pSIH1-H1-copGFP vector was also packaged and the BM-MSCs infected by this lentivirus were served as control. Endogenous miR-203 level in BM-MSCs was measured by quantitative RT-PCR，and cellular viability and apoptosis were detected by CCK-8 test and Annexin V-PI staining respectively. Results  The miR-203 TuD expression vector was successfully constructed and inserted sequence was validated. At the 3rd，6th and 9th days after in fected by the miR-203 TuD expression lentivirus，rat BM-MSCs exhibited a repressed endogenous miR-203 level. The miR-203 TuD also promoted viability and inhibited apoptosis of BM-MSCs. All these differences between miR-203 TuD group and control group were statistically significant. Conclusion  The two-step cloning method for the construction of miRNA TuD expression vector is simple and efficient. The miRNA TuD can effectively suppress the level of the target miRNA and affect cellular phenotypes.

Key words: mesenchymal stem cells, lentivirus, cell cycles, microRNAs, cellular viability, genetic vectors