P62 Protein Regulates The Proliferation And Invasion Of Giant Cell Tumor Of Bone Mononuclear Stromal Cells Through RANKL-RANK-NF-Kb Signaling Pathway

Objectives:Previous studies have found that p62 protein is highly expressed in giant cell tumor of bone,and down-regulating p62 protein expression can inhibit the proliferation and invasion of giant cell tumor of bone mononuclear stromal cells.This study will explore the regulatory mechanism of the protein,whether it is through the RANKL-RANK-NF-κb pathway,whether the p62 protein activates NF-κb signaling through the classic or non-classical pathway,and whether it depends on autophagyMethods:1.Remove fresh tissue of giant cell tumor of bone for cell isolation and primary culture.Immunofluorescence was used to detect RANKL protein and histone H3.3 G34W.The isolated cells were identified as mononuclear stromal cells.In the aging experiment,cells with active proliferation are selected for subsequent experiments.2.Treatment of mononuclear stromal cells with denosumab,blocking RNAKL signal,detection of changes in cell proliferation ability by CCK8 kit,flow cytometry,and observation of changes in the invasion ability of mononuclear stromal cells by cell invasion and migration experiments;meanwhile,Western Blot and Immunofluorescence was used to detect changes in downstream signal p62 protein expression and NF-κb activation.3.Lentiviral plasmid transfection was used to up-regulate or down-regulate P62 gene expression.Western Blot and Immunofluorescence were used to observe the effect of p62 protein expression level on the activity of NF-κb signal key kinase IκBα.Observe the changes of autophagy LC3B II/I protein expression.Results:1.Primary cells of giant cell tumor of bone were isolated,mainly composed of mononuclear stromal cells and osteoclast-like multinucleated giant cells.Osteoclast-like cells began to decrease after 48 hours of passage,and basically disappeared by passage 2-3.Cell detection found that RANKL protein was localized in the cytoplasm and histone H3.3 G34W was located in the nucleus.Both marker proteins were specifically expressed in the remaining cells,confirming that the isolated cells were mononuclear stromal cells;The stromal cells were stained withβ-galactosidase and found that the fourth-generation cells were active and the proportion of senescent cells was the lowest.The fourth-generation mononuclear stromal cells were used for subsequent experiments.2.The optimal concentration of Denosumab blocking RNAKL is 800μg/ml,and the average OD value of cell proliferation at 24h,48h,72h and 96h when treated with this concentration is 0.568±0.023,0.595 ± 0.012,0.706 ± 0.022,0.921 ± 0.049,the mean values of relative OD values of cell proliferation in the control group at 24h,48h,72h,and 96h were:1.404 ± 0.040,1.758± 0.064,1.889 ± 0.613,2.584± 0.098,and the difference between the two groups is statistically significant(P<0.05);The percentage of apoptosis of mononuclear stromal cells in the group treated with denosu,ab was 23.615%,and the percentage of apoptosis of the NC control group was 2.836%.The difference between the two groups is statistically significant(P<0.05);after treatment with denosumab,the invasion and migration ability of mononuclear stromal cells decreased significantly compared with the control group,and the difference between the two groups was statistically significant(P<0.001).The expression levels of RANKL protein,p62 protein and histone H3.3 G34W in Western Blot and immunofluorescence detection were reduced to varying degrees in Denosumab treatment group compared with the control group,and The difference between the two groups is statistically significant(P<0.05);Western Blot detection found that NF-κb related subunits P65 and P52 protein expression decreased,and the difference between the two groups was statistically significant(P<0.05).3.Western Blot results showed that shP62 down-regulated group had a lower phosphorylation level of NF-κb key kinase IκBα than NC control group,and the difference between the two groups was statistically significant(P<0.05).Immunofluorescence results showed that the exprssi on levels of the main subunits P65 and P52 in shP62 down-regulation group were significantly lower than those in NC control group,and the difference between the two groups was statistically significant(P<0.05).Western Blot test found no significant change in the LC3BⅡ/Ⅰof shP62 down-regulation group compared with the NC control group,No statistical significance between the two groups(ns).Immunofluorescence results showed that there was no significant correlation between p62 protein expression and autophagy LC3BⅡ/Ⅰ protein expression,No statistical significance between the two groups(ns).Conclusions:1.p62 protein can regulate the proliferation and invasion of monocytes through RANKL-RANK-NF-κb signaling;2.p62 protein can activate NF-κb signaling through classical and non-classical pathways,and is independent of autophagy response regulation.