骨保护素/核因子κB受体活化因子配体影响肺癌细胞下颌骨与股骨转移差异的初步研究

Preliminary study on osteoprotegerin/receptor activator of nuclear factor-κB ligand expression in mandible and femur on site selectivity of bone metastasis of lung cancer cells

目的探究下颌骨和股骨中骨保护素(OPG)与核因子κB受体活化因子配体(RANKL)的比值是否参与调控肺癌细胞骨转移部位差异。方法采用小鼠Lewis肺癌细胞构建全身性肺癌细胞骨转移小鼠模型及股骨局部肺癌细胞骨转移模型,苏木素-伊红染色与显微CT(MicroCT)检测肿瘤细胞定植与骨组织破坏情况,组织蛋白酶K免疫组织化学染色检测骨组织中破骨细胞活化情况,转录组测序(RNA-seq)及实时定量聚合酶链反应(RT-qPCR)检测骨组织中OPG mRNA/RANKL mRNA比值。结果全身性肺癌细胞骨转移小鼠模型证实:相较于股骨,下颌骨不易发生肺癌细胞骨转移。分析生理情况下小鼠下颌骨与股骨的差异,发现在肺癌细胞骨转移低发的下颌骨中破骨细胞数目明显少于转移高发的股骨,且OPG mRNA/RANKL mRNA比值明显高于股骨(P<0.01)。进一步分析发生肿瘤细胞转移的骨组织中破骨细胞活化情况及OPG mRNA/RANKL mRNA比值发现,肺癌细胞骨转移引起的骨破坏主要发生在组织蛋白酶K阳性破骨细胞集中分布的区域,发生肿瘤细胞转移的骨组织中破骨细胞数目明显多于非骨转移组(P<0.01),且OPG mRNA/RANKL mRNA比值明显低于非骨转移组(P<0.000 1)。结论在小鼠肺癌细胞骨转移低发的下颌骨与高发的股骨中,破骨细胞活化情况及OPG mRNA/RANKL mRNA比值存在明显差异,提示骨组织可能通过OPG/RANKL影响破骨细胞活化进而参与调控肺癌细胞骨转移部位差异。

Objective To explore the involvement of osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand(RANKL)ratio in mandible and femur on the site selectivity of bone metastasis of lung cancer cells.Methods Mouse Lewis lung cancer cells were used to construct mouse models of systemic/local lung cancer bone metastasis.Hematoxylin-eosin staining and micro-computed tomography(MicroCT)analysis were used to determine the selectivity of tumor metastasis amongst different bone tissues.Immunohistochemical staining of cathepsin K(CTSK)was performed to detect osteoclast activation in bone tissues.RNA sequencing(RNA-seq)and real time-quantitative polymerase chain reaction(RT-qPCR)were conducted to measure OPG mRNA/RANKL mRNA in bone.Results Using the mouse model of systemic lung-cancer cell bone metastasis,we established that the bone metastasis was less likely to occur in the mandible than the femur.Under physiological conditions,taking the mandible and femur as representative bone tissues with low/high incidence of lung cancer-cell metastasis,we found that the mandible exhibited a significantly fewer osteoclast number and higher OPG mRNA/RANKL mRNA ratio than the femur(P<0.01).Further investigations suggested that lung cancer cell-induced bone destruction mainly distributed in areas concentrated with CTSK-positive osteoclasts,with a significantly greater osteoclast number(P<0.01)and lower OPG mRNA/RANKL mRNA ratio in the metastasized bone tissue compared with the non-metastasized bone tissue(P<0.0001).Conclusion Differences in osteoclast activation and OPG mRNA/RANKL mRNA ratio levels exist in bone tissues with low/high incidence of lung cancer-cell bone metastasis,suggesting that OPG/RANKL may affect osteoclast activation,and is therefore involved in the site selectivity of lung cancer-cell bone metastasis.