HOXB3促进骨肉瘤细胞增殖、克隆形成、迁移和抑制细胞凋亡的机制研究

Mechanism of HOXB3 Promoting Proliferation,Cloning Formation,Migration and Inhibiting Apoptosis of Osteosarcoma Cells

目的探究同源异型盒基因B3(homeobox genes B3,HOXB3)对骨肉瘤细胞增殖、克隆形成、迁移和凋亡的影响及其潜在分子机制。方法检测2020年1月~12月在雅安市第二人民医院骨科行手术治疗的30例骨肉瘤患者病理组织及邻近正常组织中HOXB3表达;通过转染干扰siRNA敲低HOXB3表达,验证其转染效率;采用细胞增殖实验、克隆形成实验、细胞划痕实验及细胞凋亡实验分别探究敲低HOXB3对骨肉瘤细胞增殖、克隆形成、迁移和凋亡的影响;分析CDCA3在骨肉瘤中的表达特征及与HOXB3的相关性,通过染色质免疫共沉淀技术(chromatin immunoprecipitation,ChIP)分析和荧光素酶报告基因实验验证HOXB3和细胞分裂周期相关蛋白3(cell division cycle associatedfprotein 3,CDCA3)结合关系;验证HOXB3和CDCA3表达调控对骨肉瘤细胞生物学行为的作用机制。结果30例骨肉瘤临床组织中HOXB3表达(41.154±8.626)较邻近正常组织(22.857±7.512)显著升高,差异有统计学意义(t=8.975,P<0.001)。敲低HOXB3表达后,骨肉瘤细胞增殖率、克隆形成率和迁移率明显降低(F=37.477~399.842,均P<0.001),细胞凋亡率显著升高(F=10.556,P=0.011)。骨肉瘤组织中CDCA3表达(38.624±7.736)明显高于邻近正常组织(21.875±6.482),差异有统计学意义(t=9.090,P<0.001);HOXB3与CDCA3表达呈正相关(r=0.736,P<0.01)。CDCA3是HOXB3的靶基因;敲低HOXB3表达显著降低了骨肉瘤细胞中CDCA3表达(F=694.283,P<0.001)。HOXB3可结合CDCA3的启动子区域正调控CDCA3的表达;在敲低HOXB3表达的细胞中回补CDCA3,逆转了HOXB3对骨肉瘤细胞增殖、克隆形成、迁移、凋亡的抑制/促进作用。结论HOXB3在骨肉瘤中表达上调,其可能通过与CDCA3启动子区域结合,正向调控CDCA3表达促进了骨肉瘤细胞的增殖、克隆形成及迁移,抑制了细胞凋亡,可作为临床骨肉瘤治疗的潜在药物靶点。

Objective To investigate the effects of HOXB3 on proliferation,clone formation,migration and apoptosis of osteosarcoma cells and its potential molecular mechanism.Methods HOXB3 expression in pathological tissues and adjacent normal tissues of 30 patients with osteosarcoma who underwent surgical treatment in the Department of Orthopedics of the Second People’s Hospital of Ya’an from January 2020 to December 2020 was detected.The expression of HOXB3 was knocked down by interfering siRNA transfection,and the transfection efficiency was verified.The effects of knockdown HOXB3 on proliferation,clone formation,migration and apoptosis of osteosarcoma cells were investigated by cell proliferation assay,clone formation assay,cell scratch assay and apoptosis assay.The expression characteristics of CDCA3 in osteosarcoma and its correlation with HOXB3 were analyzed.The binding relationship between HOXB3 and CDCA3 was verified by Chchip analysis and luciferase reporter assay.To verify the mechanism of HOXB3 and CDCA3 expression regulation on the biological behavior of osteosarcoma cells.Results HOXB3 expression in 30 cases of osteosarcoma(41.154±8.626)was significantly higher than that in adjacent normal tissues(22.857±7.512),the difference was statistically significant(t=8.975,P<0.001).After knockdown of HOXB3 expression,the proliferation rate,clone formation rate and migration rate of osteosarcoma cells were significantly decreased(F=37.477~399.842,P<0.001),and the apoptosis rate was significantly increased(F=10.556,P=0.011).CDCA3 expression in osteosarcoma tissues(38.624±7.736)was significantly higher than that in adjacent normal tissues(21.875±6.482),the difference was statistically significant(t=9.090,P<0.001).HOXB3 was positively correlated with CDCA3 expression(r=0.736,P<0.01).CDCA3 was the target gene of HOXB3.Knocking down HOXB3 expression significantly decreased CDCA3 expression in osteosarcoma cells(F=694.283,P<0.001).HOXB3 could bind to the promoter region of CDCA3 to positively regulate the expression of CDCA3.CDCA3 supplementation in the cells that knocked down HOXB3 expression reversed the inhibition/promotion of HOXB3 on proliferation,clone formation,migration and apoptosis of osteosarcoma cells.Conclusion HOXB3 expression is up-regulated in osteosarcoma.HOXB3 may positively regulate the expression of CDCA3 by binding to the CDCA3 promoter region to promote the proliferation,clonal formation and migration of osteosarcoma cells,and inhibit cell apoptosis.HOXB3 can be used as a potential drug target for clinical treatment of osteosarcoma.