| Objective:To study the role and mechanism of SREBP-2 in osteoclast differentiation and bone metastasis of breast cancer,and to clarify the regulatory mechanism of SREBP-2 in bone damage caused by bone metastasis of breast cancer,and to provide a novel therapeutic target for osteolysis caused by bone metastasis of breast cancer.Methods:BMMs derived from bone marrow of mice were extracted to induce osteoclast differentiation in vitro.Osteoclasts were determined by TRAP staining,and the expression of osteoclast related genes and SREBPs genes were detected by qPCR.Small interference and plasmid transfection were used to silence and over-express SREBP-2 gene,respectively,to verify the effect of srebp-2 silencing and over-expression on osteoclast differentiation.WB and ChIP studied the mechanism of SREBP-2 in regulating osteoclast differentiation.Immunohistochemistry was used to analyze the expression of SREBP-2 in tumor tissues and paracancer tissues of breast cancer patients.CCK-8,clone formation experiment,flow cytometry and Transwell experiment were used to determine the influence of SREBP-2 on the proliferation,apoptosis,invasion and migration of breast cancer cells.Finally,the effect of SREBP-2 inhibition on osteolysis induced by bone metastasis of breast cancer was evaluated by Micro-CT scanning,bone histomorphometry and immunohistochemical analysis.Results:During osteoclastogenesis,the expression of SREBP-2 was significantly increased,while silencing the expression of SREBP-2 significantly inhibited the differentiation and bone resorption of osteoclasts,SREBP-2 overexpression saved the state of osteoclast differentiation damage after inhibiting SREBP-2 or inhibiting CREB.ChIP analysis showed that SREBP-2 directly regulated the transcription of NFATcl to influence osteoclast differentiation.On the other hand,we found that the expression of SREBP-2 in breast cancer was higher than that in para-carcinoma tissues,and database analysis suggested that the expression of SREBP-2 was correlated with metastatic free survival rate of breast cancer patients.Moreover,we found that inhibition of SREBP-2 or CREB could inhibit the migration and invasion of breast cancer cells as well as the protein expression of MMP-2 and MMP-9,and the inhibition effect was saved after the overexpression of SREBP-2.Finally,we found that the use of SREBP-2 inhibitor Fatostatin alleviated the osteolysis caused by bone metastasis of breast cancer cells.Conclusion:In this study,we showed that SREBP-2 is induced in a RANKL-CREB dependent pathway and plays a positive role in the regulation of osteoclast formation.SREBP-2 was found to transcriptionally control the expression of NFATcl by binding to SRE in the promoter of NFATc1.In addition,we found elevated expression of SREBP-2 in breast cancer tissue samples,and the high SREBP-2 expression was positively correlated with invasive breast carcinomas and predictor of poor prognosis.Targeted inhibition of SREBP-2 exert anti-migration and anti-invasion effects on breast cancer cells in vitro and protected against breast cancer-induced osteolysis in vivo.Thus,SREBP-2 represents a novel molecular target for the development of specific therapeutic agents for the treatment of osteolytic bone lesions induced by breast cancer metastases. |
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