[Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes].

OBJECTIVE

To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) ( CS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes .

METHODS

Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with CS to form CS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), CS/GV268 empty plasmid transfection group (group B), and CS/GV268-miR-140 transfection group (group C). CS/GV268 and CS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR.

RESULTS

RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B ( <0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated ( <0.05). There was no significant difference in above indexes between groups A and B ( >0.05).

CONCLUSION

Exogenous gene can be carried into the chondrocytes by CS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured , which provides important experimental basis for the treatment of cartilage damage diseases.