[Construction of beta-catenin miRNA-expressing vectors and test of silencing effect in adipose-derived stem cells from Sprague-Dawley rats].

ABSTRACT

UNLABELLED OBEJECTIVE To construct miRNA-expressing plasmid vector for interfering the expression of beta-catenin in adipose-derived stem cells from Sprague-Dawley rats. METHODS: The double strands oligonucleotides of miR-expressing cassette were ligated into plasmid pSWH to generate pSWH-miR. Then the PCR product of enhanced green fluorescence protein (EGFP) open reading frame (ORF) fragment was inserted into BamH I and Xbo I digested pSWH-miR to generate pSWH-EGFP-miR. The adipose-derived stemcells from rats (rADSCs) were transfected with pSWH-EGFP-miR respectively. The expression of beta-catenin was determined by Western blot at 72 h post-transfection. RESULTS: Five miRNA-expressing plasmid vectors were constructed (miR-780, miR-796, miR-1467, miR-1948, miR-1960). miR-780 and miR-796 had the best silencing effect on the expression of beta-catenin (P < 0.05), less did the miR-1960 (P < 0.05), but not the miR-1467 and the miR-1948 (P < 0.05). CONCLUSION: miR-780 and miR-796 could interference the beta-catenin in ADSCs with high efficiency.