Effects Of LncRNA-PVT1 On Advanced Glycation End Products Induced Autophagy In Human Chondrocytes

OBJECTIVE : To study the effect of LncRNA-PVT1 on advanced glycation end products(AGEs)induced autophagy's activity of human chondrocytes and ECM degeneration.MATERIALS AND METHODS:(1)The expressions of COL2A1,MMP13,and LncRNA-PVT1 were detected by qPCR after the treatment of human chondrocytes at 12 h,24h,and 48 h,respectively.(2)Treatment human cartilage cells with AGEs 12 h,24h,48 h respectively,then detect the expression of Beclin1,LC3 ? protein with Western Blot.Before AGEs treatment,chondrocytes were transfected with autophagy double-labeled adenovirus(mRFP-GFP-LC3)to produce fusion marker proteins to observe the number of mRFP-GFP-LC3.(3)Chondrocytes were transfected with si-NC and three kinds of si-PVT1 respectively,and their interference efficiency was detected by fluorescence quantitative PCR.(4)CCK-8 assay was used to determine the proliferation activity of si-NC and si-PVT1 transfected chondrocytes at 0h,12 h,24h and 48 h.(5)Treatmented with si-PVT1 transfected chondrocytes after 48 h,fluorescence quantitative PCR were used to detect COL2A1,MMP13 expression,detecting cell Beclin1,LC3 ? protein expression with Western Blot,electron transmission microscope(TEM)was for couting autophagosome number inside the cell,Before si-RNA transfaction,chondrocytes were transfected with autophagy double-labeled adenovirus(mRFP-GFP-LC3)to produce fusion marker proteins to observe the number of mRFP-GFP-LC3.RESULTS:(1)MMP13 gene expression increased gradually within 48 h,COL2A1 decreased gradually after AGEs(100mg/L)treatment.After cells were treated with AGEs(100mg/L),LC3?,Beclin1 protein expression in 24 h reached maximum.After 24 h,LC3? began to decline.LC3?,Beclin1 expression of 12 h and 24 h respectively compared with control group,the difference was statistically significant;the difference of LC3?,Beclin1 expression of 24 h and 48 h was statistically significant.After 12 h and 24 h treatment of cell,the number of mRFP-GFP-LC3 was significantly higher than that of the control group.At 48 h,the number of mRFP-GFPLC3 in cells was significantly lower than that at 24 h.LncRNA-PVT1 expression increased gradually within 48 h after AGEs(100mg/L)treatment.(2)The interference ratio of the three types of si-PVT1 were all significant,which was statistically different from that of the si-NC group,but the interference ratio of si-PVT1-2 were the most obvious.(3)The difference in cell proliferation activity between siRNA-PVT1 group and siNC group at 12 h,24h and 48 h was statistically significant after treatment.(4)After AGEs treatment of chondrocytes for 48 h,the mRNA expression of MMP13 decreased in si-PVT1 transfected cells,which was significantly different from that in siNC group.The mRNA expression of COL2A1 was increased in si-PVT1 cells,which was significantly different from that of siNC group.Which deal with chondrocytes after 48 h,LC3 ?,Beclin1 protein expression in si-PVT1-2 group increased compared with siNC group,the difference was statistically significant.At high power field,the mRFP-GFP-LC3 volume in the si-PVT1 group was higher than that in the siNC group,and the difference is statistically significant.Observed with TEM,the number of autophagosomes in the si-PVT1 group was higher than that in the siNC group.CONCLUSIONS:The autophagy activity of chondrocytes increase in the early stage and decrease after AGEs treated.LncRNA-PVT1 inhibite autophagy activity of chondrocytes induced by AGEs in the late stage,promote cartilage matrix degeneration,and decrease the cell proliferation activity.