Abstract:

BACKGROUND: The use of mesenchymal stem cells in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cells are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that miRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cells.
OBJECTIVE: To observe the chondrogenic effect of human bone marrow mesenchymal stem cells transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury.
METHODS: miRNA microarray technology was applied to detect miRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cells were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cell counting kit-8 was used to determine the cell proliferation, the differentiation of bone marrow mesenchymal stem cells towards chondrocytes was verified by type II collagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II collagen and aggrecan mRNA expression at 21 days of chondrogenic induction.
RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cells. miRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and Collagen type II were significantly increased in the miR-221-3p/222-3p inhibition group and cell proliferation was also inhibited significantly compared with non-transduced cells or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cells could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cells.