山东医药

目的 探讨中心体相关激酶2(NEK2)基因敲低对多发性骨髓瘤（MM）细胞介导骨质破坏及核因子κB(NF-κB)信号通路的影响。方法 取MM细胞系（MM. 1S细胞、RPMI 8226细胞），均设置NEK2敲低组、阴性对照组，分别用慢病毒载体-NEK2-RNA干扰、病毒CON077进行感染，用实时荧光定量反转录聚合酶链式反应（RT-qPCR）法检测NRK2 mRNA表达以确认转染效率，选择敲低效率低的细胞与MM细胞共培养。提取骨髓基质细胞（BMSC），将其分为阴性对照组、NEK2敲低组，分别加入RPMI 8226细胞、敲低NEK2的RPMI 8226细胞共培养，RT-qPCR法检测细胞内骨质破坏相关指标NF-κB受体活化因子配体（RANKL）、骨保护蛋白（OPG）mRNA表达。取NEK2敲低组、阴性对照组MM细胞，用CCK-8法检测细胞增殖（以吸光度值表示），流式细胞术检测细胞凋亡率，Western blotting法检测NF-κB信号通路相关蛋白NF-κB、乙酰肝素酶（HPSE）表达，RT-qPCR法检测NF-κB、HPSE mRNA表达。结果 MM. 1S细胞、RPMI-8226细胞NEK2敲低组NEK2 mRNA相对表达量均低于其阴性对照组（P均<0.05），基因敲低效率分别为92.2%、64.0%。与BMSC的阴性对照组比较，NEK2敲低组RANKL mRNA表达低（P<0.05），而OPG mRNA表达高，但差异无统计学意义（P>0.05）。与MM. 1S细胞、RPMI-8226细胞的阴性对照组比较，其NEK2敲低组吸光度值低，细胞凋亡率高，NF-κB、HPSE mRNA相对表达量低（P均<0.05）,NF-κB、HPSE蛋白表达低。结论 NEK2基因敲低可能通过调控NF-κB信号通路抑制MM细胞增殖、促进其凋亡，降低HPSE表达，从而抑制MM细胞介导的骨质破坏。

Objective To investigate the effects of centrosome-associated kinase 2(NEK2) gene knockdown on multiple myeloma(MM) cell-mediated bone destruction and the nuclear factor kappa B(NF-κB) signaling pathway.Methods MM cell lines(MM. 1S cells and RPMI 8226 cells) were used, with both NEK2 knockdown group and negative control group established for each. Lentiviral vectors carrying NEK2-RNA interference(shRNA) or control virus(CON077) were used for transfection. NEK2 mRNA expression was detected by real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR) to confirm transfection efficiency. Bone marrow stromal cells(BMSCs) were divided into the negative control group and NEK2 knockdown group, which were co-cultured with RPMI 8226 cells or NEK2-knockdown RPMI 8226 cells. RT-qPCR was used to measure the mRNA levels of bone destruction-related markers, including receptor activator of NF-κB ligand(RANKL) and osteoprotegerin(OPG). MM cells from the NEK2 knockdown group and negative control group were selected. Cell proliferation(absorbance value) was assessed using the CCK-8 assay, apoptosis rates were measured by flow cytometry, and NF-κB pathway-related proteins [NF-κB, heparanase(HPSE)] and their mRNA levels were detected by Western blotting and RT-qPCR, respectively. Results NEK2 mRNA expression was significantly lower in the NEK2 knockdown groups of MM. 1S and RPMI 8226 cells than in the negative control groups(both P<0. 05), with knockdown efficiencies of 92. 2% and 64. 0%, respectively. Compared with the negative control group of BMSCs, the NEK2 knockdown group had lower RANKL mRNA expression(P<0. 05), while OPG mRNA expression was higher; no statistically significant difference was found(P>0. 05). Compared with the negative control groups of MM. 1S cells and RPMI-8226 cells, the NEK2 knockdown group of MM. 1S and RPMI 8226 cells had lower absorbance values, higher apoptosis rates, lower NF-κB and HPSE mRNA levels, and higher NF-κB and HPSE protein expression levels(all P<0. 05). Conclusion NEK2 knockdown may inhibit MM cell proliferation, promote apoptosis, and up-regulate HPSE expression by modulating the NF-κB signaling pathway, thereby suppressing MM cell-mediated bone destruction.