Lnc-AK077216基于OPG/RANKL/RANK通路对破骨细胞分化成熟的调控作用

Regulatory effect of Lnc-AK077216 on osteoclast differentiation and maturation through OPG/RANKL/RANK pathway

摘要:

目的 基于骨保护蛋白（OPG）/细胞核因子κB受体活化因子配体（RANKL）/细胞核因子κB受体活化因子（RANK）通路探究Lnc-AK077216对破骨细胞（OC）分化成熟的调控作用。方法 取对数生长期RAW264.7小鼠单核巨噬细胞，采用脂质体转染法将Lnc-AK077216-shRNA、Control-shRNA转染至RAW264.7细胞，分别设为转染组、空载组。另取未作处理细胞设为对照组。采用荧光显微镜观察转染率，并采用实时荧光定量聚合酶链反应（qRT-PCR）测定转染后Lnc-AK077216 mRNA相对表达量；抗酒石酸酸性磷酸酶（TRAP）染色检测诱导分化7 d后的OC数及阳性染色面积；采用qRT-PCR检测诱导分化培养3 d后各组OPG、RANKL、RANK mRNA相对表达量；采用Western blotting检测诱导分化培养3 d后各组OPG、RANKL、RANK蛋白相对表达量。结果 转染48 h后，荧光显微镜显示转染效率> 70％；转染48 h后转染组Lnc-AK077216 mRNA相对表达量低于对照组和空载组（P <0.05）；诱导分化前RAW264.7细胞多呈圆形且形态规则，诱导分化7 d后经TRAP染色，可观察到呈阳性的多核巨细胞，细胞有伪足、突起，且体积较大，表明生成OC；转染组OC数少于对照组和空载组（P <0.05），阳性染色面积小于对照组和空载组（P <0.05）；转染组OPG mRNA和蛋白相对表达量高于对照组和空载组（P <0.05），RANKL、RANK mRNA和蛋白相对表达量均低于对照组和空载组（P <0.05）。结论 敲低Lnc-AK077216可抑制RAW264.7细胞向OC分化，其调控机制可能与上调OPG mRNA和蛋白表达，下调RANKL、RANK mRNA和蛋白表达有关。

Abstract:

Objective To explore the regulatory effect of Lnc-AK077216 on osteoclast differentiation and maturation based on the OPG/RANKL/RANK pathway.Methods Mononuclear macrophages of RAW264.7 mice in logarithmic phase were taken, Lnc-AK077216-shRNA and Control-shRNA were transfected into RAW264.7 cells by liposome transfection method, which were set as transfection group and empty group. The other untreated cells were set as the control group. The transfection efficiency was observed with a fluorescence microscope, and the relative expression of Lnc-AK077216 mRNA after transfection was measured by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect OC number and positive staining area after 7 days of induced differentiation. qRT-PCR was used to determine the relative expression of OPG, RANKL, and RANK mRNA in each group after 3 days of induced differentiation. Western blot was used to determine the relative expression of OPG, RANKL, and RANK proteins in each group after 3 days of induced differentiation.Results After 48 hours of transfection, fluorescence microscopy showed that the transfection efficiency was > 70%. After 48 hours of transfection, the relative expression levels of Lnc-AK077216 mRNA in the transfection group were lower than those in the control group and the empty group (P <0.05). Before induction of differentiation, RAW264.7 cells were mostly round and regular in shape. After 7 days of induction, they were stained with TRAP. The formation of multinucleated giant cells with TRAP staining was observed. The cells had pseudopods and protrusions, and the volume was large, indicating that OC was generated. The number of OC in the stained group was less than that in the control group and the empty group (P < 0.05). The area of positive staining in the stained group was less than that in the control group and the empty group (P < 0.05). The relative expression of OPG mRNA and protein in the transfection group were higher than those in the control group and the empty group (P < 0.05). The relative expression levels of RANKL and RANK mRNA and the protein in the transfection group were lower than those in the control group and the empty group (P < 0.05).Conclusion Knockdown of Lnc-AK077216 can inhibit the differentiation of RAW264.7 cells to OC. The regulatory mechanism may be related to up-regulation of OPG, and down-regulation of RANKL and RANK.