Changes And Correlation Analysis Of P62 Protein Expression After Denosumab Treatment Of Giant Cell Tumor Of Bone

Objectives:Denosumab is the only targeted drug for the treatment of giant cell tumor of bone(giant cell tumor of bone,GCTB),which detailed mechanism of action is not completely clear.Previous studies found that p62 protein was highly expressed in GCTB tissues,and down-regulation of it expression could inhibit the proliferation and invasion of GCTB neoplastic stromal(NS)cells,which may be related to the efficacy of denosumab.This study,we will detect the changes in the expression of p62 protein before and after the treatment of GCTB by denosumab and analyze the correlation between p62 protein and denosumab efficacy.Methods:1.Collect 6 pairs of paired GCTB fresh tissue specimens before and after treatment with denosumab.RT-qPCR and Western Blot were used to detect p62 mRNA and protein.ELISA was used to detect p62 protein in the serum of 9 pairs of paireds.To compare the changes of p62 expression before and after denosumab treatment of GCTB.2.40 paraffin specimens before and after the treatment of GCTB with denosumab were collected.p62 protein was detected by IHC.And to analyze the correlation between P62 protein expression level and clinical features such as GCTB clinical Campanacci grade,pathological fracture and recurrence.3.Fresh GCTB tissue(T)and transferred GCTB fresh tissue(M)were collected respectively for primary cell culture,and NS(T strain and M strain)were isolated and identified.Lentiviral plasmid transfection was used to obtain NS with low expression of p62 and stable passage,which were treated and grouped with denosumab.Including GCTB control group:(4th generation NS);shp62 group:silenced p62 gene NS;shp62-Denosumab group:silenced p62 gene NS treated by denosumab;NC group:shP62 negative control;NC-Denosumab group:shp62 negative control were treated with denosumab.4.Western Blot and immunofluorescence were used to detect the changes of p62 protein in NS of different groups treated with denosumab.We used CCK8 to detect cell proliferation,flow cytometry to detect cell apoptosis and cell cycle and transwell to detect changes in cell migration and invasion ability.Observe the inhibitory effect of denosumab on NS with different p62 protein expression levels.Results:1.p62 mRNA and protein expression were decreased after denosumab treatment compared with before treatment in fresh GCTB tissue samples,with statistical significance(all P<0.05).There was no difference in the expression of p62 protein in serum samples before and after denosumab treatment.2.The expression level of p62 protein after denosumab treatment was decreased compared with that before treatment in paraffin samples of GCTB,with statistical significance(P<0.05).There was no correlation between the decrease level and the clinical Cammpanacci stage and pathological fracture in GCTB(all P>0.05),which was negatively correlated with tumor recurrence(P<0.05).3.Primary cells were isolated from the sample of the lower femur(T strain)from GCTB and the scapular metastasis sample in the upper humerus(M strain)from GCTB.All tumor tissues were composed of m’ultinucleated giant(MNG)cells and NS,few MNG were found in metastatic tissues.After 48 hours of passage,the number of MNG decreased gradually and almost disappeared after 2-3 passages.The remaining cells were positive for H3.3G34W immunofluorescence staining,which indicated that they were NS.The fourth generation cells were used for subsequent experiments..Lentiviral plasmid transfection was used to obtain NS with low expression of p62 and stable passage.4.The p62 protein of NS in different groups of T and M strain was treated with denosumab 800μg/ml respectively,and the p62 protein decreased to varying degrees.The expression of p62 protein in shp62-denosumab group was decreased compared with the control group(NC-denosumab group),the difference was statistically significant(P<0.05).The proliferation level of NS in T and M strains shp62-denosumab group was lower than that in the control group,and the difference was statistically significant(P<0.0001,P<0.01).The NS of T and M strains shp62-denosumab group stagnated significantly in G0/G1 phase compared with the control group,the difference between the two was statistically significant(all P<0.05).The apoptosis rates of NS in the T strain and M strain shp62-denosumab group were 27.730%and 45.960%,which were significantly higher than those in the control group(15.399%and 39.750%),the difference between the two was statistically significant(P<0.01,P<0.05).The migration number of NS in T and M shp62-denosumab group was 30.330±0.881 and 61.000±7.550,which were lower than that in control group(111.000±3.055 and 148.000±4.619),the difference between the two was statistically significant(P<0.0001,P<0.001).The invasion number of NS in T and M shp62-denosumab group was 51.000±1.528 and 58.670±2.603,which were lower than those in control group(73.330±1.856 and 97.670±5.548),the difference between the two was statistically significant(P<0.001,P<0.05).Conclusions:LAfter denosumab treatment of GCTB,p62 protein expression was down-regulated,which may be related to the efficacy of denosumab.2.Denosumab inhibited the proliferation,migration and invasion of NS by down-regulating the expression of p62 protein.3.After down-regulating the expression of p62 protein,denosumab further inhibits cell proliferation and migration and invasion by inducing apoptosis of NS.