Related Research Of P62 Protein And Tumor Proliferation Of Giant Cell Tumor Of Bone

Objectives:To detect and analyze the expression and the correlation of P62、RANK、RANKL genes and protein in giant cell tumor of bone.To clarify the role of P62 protein expression in the tumor cell proliferation of giant cell tumor of bone.To find new molecular target for the clinical treatment of giant cell tumor of bone.Methods:1.P62 gene was detected the point mutation between Exons 7 and Exons 8 in 13 fresh tissues of giant cell tumor of bone by gene sequencing.The expressions of P62、RANK and RANKL mRNA and protein in 8 cases of giant cell tumor of bone and adjacent tissues were detected by qRT-PCR and Western blot,and the correlations of P62、RANK and RANKL mRNA and protein was analyzed.2.We retained fresh tissue of giant cell tumor of bone for cell separation and culture,and cultured mononuclear stromal cells.The expression of P62 gene was up-regulated or down-regulated by lentiviral plasmid transfection.The effects of P62 gene expression on cell proliferation、cell cycle and apoptosis of monocyte stromal cells were observed by CCK8 kit and flow cytometry.Results:1.Gene sequencing results showed that G1340A mutation occurred in the 1340 bp position of two samples between Exons 7 and Exons 8 of P62 gene in giant cell tumor bone.However,the amino acid R(arginine)at position 415 did not change.The results of qRT-PCR showed that the expression levels of P62 mRNA(P=0.0459)、RANK mRN A(P=0.0007)and RANKL mRNA(P=0.0001)in tissues of giant cell tumor of bone were significantly higher than those in paraneoplastic tissues.And there was positively correlated between RANKL and RANK(R2=0.5435),P62 was positively correlated with RANKL(R2=0.3505),P62 was positively correlated with RANK(R2=0.3325)(P<0.001).Western blot results showed that the expression levels of P62 protein(P=0.0023),RANK protein(P=0.0002)and RANKL protein(P=0.003)in the giant cell tumor of bone were significantly higher than those in the paraneoplastic tissues.And there was positively correlated between RANKL and RANK(R2=0.5207,P=0.0016),P62 was positively correlated with RANKL(R2=0.3476,P=0.0162),P62 was positively correlated with RANK(R2=0.4571,P=0.004).2.We separated the primary cells of giant cell tumor of bone and observed that the primary cells were mainly composed of osteoclast-like multinucleated giant cells and spindle-shaped mononuclear stromal cells.After 48 hours of passage,the osteoid-like multinucleated giant cells gradually decreased.After 2-3 generations of passage,it was found that osteoclast-like multinucleated giant cells disappeared,leaving only mononuclear stromal cells.We used 4th generation cells for experiments.P62 protein expression was down-regulated by plasmid transfection.The mean values of cell proliferation relative OD values in the 1-5 days of shP62 down-regulation group were found to be:1.081±0.076、1.192±0.015、1.226±0.012、1.249±0.019、1.386±0.038;The mean values of cell proliferation relative OD values in the NC control group for 1-5 days were:1.116±0.076、1.235±0.101、1.312±0.076,1.417±0.050、1.583±0.050;The difference of expression between the two groups was statistically significant on day 4(t=3.157,P=0.0343)and day 5(t=3.279,P=0.0305).The percentage of apoptosis in mononuclear stromal cells of shP62 down-regulated group was 37.8%,and the percentage of apoptosis in NC control group was 4.98%.The statistical difference was significant.The percentage of cell cycle distribution of mononuclear stromal cells in shP62 down-regulated group was 75.8%in G0/G1 phase,12.6%in S phase,and 11.6%in M/G2 phase.The percentage of cell cycle distribution of mononuclear stromal cells in the NC control group was 73.6%in G0/G1 phase,15.2%in S phase,and 11.1%in M/G2 phase,respectively.There was a statistically significant difference between the two groups.Conclusions:1.It does not found that the P62 gene is mutated in the Exons 7 and Exons 8 regions in giant cell tumor of bone.The expressions of P62、RANK and RANKL mRNA and protein were higher than those in the paraneoplastic tissues,and there was a positive correlation between P62、RANK and RANKL.2.P62 protein expression can promote the proliferation of mononuclear stromal cells of giant cell tumor of bone by regulating cell cycle and apoptosis.