长链非编码RNA-TCONS00041960对大鼠激素性股骨头坏死模型中成脂和成骨基因表达的影响

Effect of long non-coding RNA-TCONS_00041960 on adipogenic and osteogenic gene expression in rat model of steroid-induced osteonecrosis of the femoral head

目的观察长链非编码RNA(lncRNA)-TCONS00041960在大鼠激素性股骨头坏死模型中对过氧化物酶体增殖物激活受体γ(PPARγ)和成骨基因runt相关转录因子2(Runx2)表达的调控作用。方法健康SD大鼠64只,随机分为4组:(1)正常对照组(Blank组)。每次臀肌注射2 ml生理盐水,连续3 d。(2)激素组(Dex组)。每次臀肌注射地塞米松20 mg/kg,连续3 d,制作大鼠激素性股骨头坏死模型。(3)无关序列组(Ex-NC+Dex组)。同法给予地塞米松,一侧股骨头内注入转染无关序列慢病毒载体的骨髓间充质干细胞(BMSCs)。(4)激素+重组慢病毒组(Ex-Lnc+Dex组)。同法给予地塞米松,一侧股骨头内注入转染重组lncRNA TCONS00041960 lncRNA载体的BMSCS。于实验第4、8周提取各组大鼠股骨头组织中总RNA及总蛋白,应用TaqMan实时定量聚合酶链反应(Real-time PCR)与蛋白质印迹法(Western blot)检测成脂基因PPARγ、成骨特异性转录因子Runx2 mRNA及其蛋白表达。两样本两组间比较采用t检验;数值变量资料统计采用方差分析,组间比较采用最小显著性差异法(LSD)。结果实验第4周,Ex-Lnc+Dex组股骨头细胞内PPARγ mRNA与蛋白呈低表达(1.129±0.024与0.366±0.007),与Blank组(1.001±0.055与0.352±0.003)接近,差异均无统计学意义(t4w-mRNA=3.052,P>0.05;t4w-蛋白=3.227,P>0.05);明显低于Dex组(2.351±0.086与0.836±0.032)、Ex-NC+Dex组(2.226±0.484与0.888±0.067),差异均有统计学意义(F4w-mRNA=296.841,P<0.01;F4w-蛋白=196.684,P<0.01)。Ex-Lnc+Dex组Runx2 mRNA与蛋白呈高表达(1.176±0.036与0.963±0.014),与Blank组(1.001±0.077与0.934±0.019)接近,差异均无统计学意义(t4w-mRNA=2.893,P>0.05;t4w-蛋白=2.154,P>0.05);明显高于Dex组(0.522±0.007与0.501±0.005)、Ex-NC+Dex组(0.614±0.003与0.521±0.008),差异均有统计学意义(F4w-mRNA=376.700,P<0.01;F4w-蛋白=2 239.834,P<0.01)。实验第8周各组表达量与第4周一致。结论重组lncRNA-TCONS00041960载体能够有效下调大鼠激素性股骨头坏死(ONFH)模型股骨头细胞内成脂基因表达,同时显著上调细胞内成骨基因表达,可高效预防激素性ONFH的发生。

Objective To explore the effect of long non-coding RNA(lncRNA)-TCONS_00041960 on adipogenic and osteogenic genes expression in rat model of steroid-induced osteonecrosis of the femoral head.Methods Totally,64 healthy SD rats were randomly divided into 4 groups:(1)normal control group(blank group)(2 ml physiological saline was injected into gluteal muscle for 3 days).(2)hormone group(Dex group)[Dexamethasone(20 mg/kg)was injected into gluteal muscle for 3 days to establish steroid-induced femoral head necrosis model].(3)unrelated sequence group(Ex-NC+Dex group)[Dexamethasone was given in the same way,and bone marrow mesenchymal stem cells(BMSCs)transfected with lentivirus of unrelated sequence were injected into one side of femoral head].(4)Dexamethasone+recombinant lentivirus group(Ex-Lnc+Dex group)[Dexamethasone was given in the same way,and BMSCs transfected with recombinant lentivirus vector lncRNA-TCONS_00041960 were injected into one side of femoral head].At 4th and 8th week,total RNA and total protein were extracted from the femoral head tissue of each group.The expression of the peroxisome proliferator-activated receptor-γ(PPARγ),runt-related transcription factor 2(Runx2)mRNA and protein was detected by TaqMan real-time quantitative polymerase chain reaction(Real-time PCR)and Western blotting at 4th week and 8th week of the experiment.The comparison between the two groups was performed by t test;the numerical variable data statistics were performed by analysis of variance,and the comparison between groups was performed by the least significant difference method(LSD).Results At 4 weeks,the expression levels of PPARγmRNA and protein in the femoral head cells in the Ex-Lnc+Dex group(1.129±0.024 and 0.366±0.007)were not significantly different from those in the blank group(1.001±0.055 and 0.352±0.003,t4w-mRNA=3.052,P>0.05;t4w-protein=3.227,P>0.05),but significantly lower than those in the Dex group(2.351±0.086 and 0.836±0.032)and Ex-NC+Dex group(2.226±0.484 and 0.888±0.067,F4w-mRNA=296.841,P<0.01;F4w-protein=196.684,P<0.01).The expression levels of Runx2 mRNA and protein in Ex-Lnc+Dex group(1.176±0.036 and 0.963±0.014)were not significantly different from those in the blank group(1.001±0.077 and 0.934±0.019,t4w-mRNA=2.893,P>0.05;t4w-protein=2.154,P>0.05),but significantly higher than those in the Dex group(0.522±0.007 and 0.501±0.005)and Ex-NC+Dex group(0.614±0.003 and 0.521±0.008,F4w-mRNA=376.700,P<0.01;F4w-protein=2239.834,P<0.01).At 8 weeks,the expression levels in all groups were consistent with those at the 4 weeks.Conclusion The recombinant lncRNA-TCONS_00041960 lentivirus vector can effectively down-regulate the expression of adipogenic gene in the femoral head cells of steroid-induced osteonecrosis of the femoral head(ONFH)model of rats in vivo,and significantly promote the expression of osteogenic gene in the cells at the same time,which can effectively prevent the occurrence of ONFH.