Repairing Articular Cartilage Defects In Rabbits With Bifunctional Short Peptide-conjugated-chitosan-mediated Microrna-140 Gene Transfer

Objective:MicroRNA-140（miR-140）plays an important role in the differentiation and metabolism of chondrocytes.However,the effect and mechanism of miR-140 in the repair process of cartilage defects need to be further studied.Here,we investigated the effect and mechanism of nucleus localization signal linked nucleic kinase substrate short peptide-conjugated chitosan(^NNSCS)mediated the miR-140gene transfer on articular cartilage defects in rabbits.Methods:MiR-140 was amplified from human genomic DNA isolated from whole blood and was inserted into GV268 vector to generate recombinant plasmid GV268-miR-140.The nucleus localization signal linked nucleic kinase substrate short peptide（NNS）was conjugated to chitosan to form ^NNSCS,then ^NNSCS was combined with pEGFP-C1 vector to form ^NNSCS/pEGFP complexes.Chondrocytes were isolated from articular cartilage of newborn rabbits and were identified by toluidine blue staining.^NNSCS/pEGFP complexes were transiently transfected into the second generation chondrocytes.The transfer efficiency of ^NNSCS was measured by GFP positive cells.In vitro:recombinant plasmid GV268-miR-140 and negative control plasmid GV268 were respectively combined with ^NNSCS to form ^NNSCS/GV268-miR-140 and ^NNSCS/GV268 complexes.The second generation chondrocytes were inoculated in cell culture plates,and the experiment was divided into 3 groups:transgenic group（group A）,negative control group（group B）and normal cell control group（group C）.^NNSCS/GV268-miR-140 and ^NNSCS/GV268 complexes were respectively transiently transfected into cells of group A and B.The same mass volume of ^NNSCS was added in group C.After transfection,real-time fluorescent quantitative PCR（RT-qPCR）was used to detect the expression of miR-140;MTT assay and Annexin V-FITC/PI double labeling assay were respectively used to detect the effects of miR-140 on proliferation and apoptosis of chondrocytes;RT-qPCR and western blot were respectively used to detect the expressions of chondrogenic related gene Sox9,Aggrecan and histone deacetylase 4（Hdac4）at mRNA and protein level.In vivo:eighteen healthy male New Zealand white rabbits were randomly divided into 3 groups:transgenic group（group A）,negative control group（group B）and sham-operated group（group C）,n=6.Full-thickness cartilage defects were made in the articular surface of the right lateral femoral trochlea of the knee in group A and B;the right lateral femoral trochlea articular surface was only exposed in group C.After 1 week of the surgery,intra-articular injection of ^NNSCS/GV268-miR-140 and ^NNSCS/GV268 complexes were respectively administrated 2 times a week for 7weeks in group A and group B,the same volume isotonic saline in group C.At 8weeks after the surgery,the rabbits were sacrificed and analyzed.The expressions of miR-140,Sox9,Aggrecan and Hdac4 were detected by RT-qPCR;the repair of cartilage in the defect zone was evaluated by HE staining,safranine O/fast green staining and Aggrecan immunohistochemistry.Results:The GV268-miR-140 eukaryotic expression plasmid was successfully constructed,and combined with ^NNSCS to form ^NNSCS/GV268-miR-140 complexes.^NNSCS could carry pEGFP-C1 into the chondrocytes,since about 40%of the cells were GFP-positive.In vitro:MTT showed that the proliferation activity in group A（150.3±10.3%）was significantly improved compared with those in group B（107.3±5.3%）and C（100.0±5.6%）（P<0.05）.Flow cytometry showed that the apoptosis rate in group A（7.05±0.263%）was significantly decreased compared with those in group B（10.37±0.163%）and C（9.87±0.093%）（P<0.05）.RT-qPCR showed that the expression of miR-140 in group A（5.87±0.543）was significantly higher than those in group B（1.27±0.143）and C（1.00±0.110）（P<0.05）;miR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan gene mRNA（Sox9:4.97±0.558,Aggrecan:3.00±0.140）and obviously down-regulated the expression of Hdac4 gene mRNA（0.63±0.060）compared with those in group B（Sox9:1.30±0.340,Aggrecan:1.23±0.080,Hdac4:1.08±0.063）and C（Sox9:1.00±0.130,Aggrecan:1.00±0.070,Hdac4:1.00±0.030）（P<0.05）.The results of western blot were consistent with RT-qPCR.MiR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan protein（Sox9:0.592±0.010,Aggrecan:2.287±0.117）and obviously down-regulated the expression of Hdac4 protein（0.451±0.008）compared with those in group B（Sox9:0.460±0.012,Aggrecan:1.593±0.009,Hdac4:0.752±0.025）and C（Sox9:0.464±0.008,Aggrecan:1.507±0.057,Hdac4:0.754±0.007）（P<0.05）.In vivo:RT-qPCR showed that the expression of miR-140 in group A（2.603±0.334）was significantly higher than those in group B（0.882±0.105）and C（1.022±0.225）（P<0.05）;miR-140 overexpression in group A obviously up-regulated the expressions of Sox9 and Aggrecan gene mRNA（Sox9:3.697±1.042,Aggrecan:2.726±0.430）compared with those in group B（Sox9:0.919±0.078,Aggrecan:0.880±0.104）and C（Sox9:1.008±0.143,Aggrecan:1.008±0.132）（P<0.05）.The mRNA level of Hdac4 gene in group B（4.434±1.187）was highest,and the others were in group A（2.552±0.182）and C（1.055±0.362）;miR-140 overexpression in group A obviously down-regulated the expression of Hdac4 gene mRNA compared with that in group B（P<0.05）.Cartilage repair appeared in group A,showing positive staining of HE,safranine O/fast green and Aggrecan;fibrous tissue proliferation and inflammatory cell infiltration were seen in the defect zone in group B without significant cartilage repairing.Conclusions:（1）Exogenous gene could be carried into the chondrocytes by ^NNSCS and expressed efficiently.（2）MiR-140 could improve the biological activity of chondrocytes cultured in vitro.（3）Intra-articular injection of ^NNSCS/GV268-miR-140complexes could improve the repairing of articular cartilage defect,which may be related to the up-regulation of Sox9 and Aggrecan gene expressions and the down-regulation of Hdac4 gene expression.