Objective: To investigate the role and regulatory mechanism of ring finger protein 11 (RNF11) on Akt signaling pathway in the process of osteogenesis of bone marrow mesenchymal stem cells (BMSCs) to provide ideas for further clarifying its osteogenesis mechanism and its use in clinical treatment in the future.
Methods: BMSCs were isolated and cultured from fresh bone marrow of healthy donors and subcultured. The 4th generation cells were used in experiments after identification by flow cytometry, and osteogenic, chondrogenic, and adipogenic induction. BMSCs were cultured in osteogenic differentiation medium for 0-14 days. The degree of osteogenic differentiation was detected by Alizarin red staining and alkaline phosphatase (ALP) staining, and the protein expression of RNF11 was detected by Western blot. The 4th generation BMSCs were divided into blank control group (group A), empty lentivirus (Lv-NC) group (group B), and knockdown RNF11 (Lv-ShRNF11) group (group C). Osteogenesis was induced and cultured for 0-14 days. The expression of RNF11 protein was detected by Western blot, the degree of osteogenic differentiation was detected by Alizarin red staining and ALP staining, and the relative mRNA expressions of Runx2, osteocalcin (OCN), and osteopontin (OPN) were detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein relative expressions of Akt, Smad1/5/8, and β-catenin signaling pathway were detected by Western blot, expressed as the ratio before and after phosphorylation. In order to study the effect mechanism of RNF11 on Akt signaling pathway, the 4th generation BMSCs were divided into Lv-NC transfection group (group A1), Lv-ShRNF11 transfection group (group B1), and Lv-ShRNF11 transfection supplemented with Akt signaling pathway activator SC79 group (group C1). The protein relative expressions of RNF11 and Akt signaling pathway were detected by Western blot, the related osteogenesis indexes were detected by Alizarin red staining, ALP staining, and qRT-PCR.
Results: The flow cytometry, and osteogenic, chondrogenic, adipogenic induction culture identification showed that the isolated and cultured cells were BMSCs. The protein relative expression of RNF11 increased gradually with the extension of osteogenic differentiation time ( P<0.05); after knockdown RNF11, Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C were significantly lower than those in groups A and B, and qRT-PCR detection showed that the relative expression of Runx2, OCN, and OPN mRNA significantly decreased ( P<0.05). The protein relative expressions of RNF11 and Akt signaling pathway significantly increased with the extensions of osteogenic differentiation time ( P<0.05). After knockdown RNF11, the protein relative expression of Akt signaling pathway in group C was significantly lower than that in groups A and B ( P<0.05), while Smad1/5/8 and β-catenin signaling pathway had no significant effect ( P>0.05). Compared with group A1, the protein relative expression of RNF11 in groups B1 and C1 significantly decreased ( P<0.05). Compared with groups A1 and C1, the protein relative expression of Akt signaling pathway in group B1 was significantly lower ( P<0.05); Alizarin red and ALP stainings showed that the degree of osteogenic differentiation of BMSCs in group C1 were slightly lower than that of group A1 ( P>0.05), but significantly higher than that of group B1 ( P<0.05); qRT-PCR detection showed that the relative expressions of Runx2, OCN, and OPN mRNA in group C1 were slightly lower than those of group A1 ( P>0.05), but were significantly higher than those of group B1 ( P<0.05).
Conclusion: RNF11 promotes the differentiation of BMSCs into osteoblasts by positively regulating the activation level of Akt signaling pathway. RNF11 can be used as a potential target to improve the bone repair efficacy of BMSCs and treat bone metabolic diseases.
目的: 研究BMSCs成骨分化过程中环指蛋白11（ring finger protein 11，RNF11）对Akt信号通路的调节作用，为进一步阐明BMSCs成骨分化机制和用于临床治疗提供思路。.
方法: 从健康人体新鲜骨髓中分离培养BMSCs并传代，取第4代细胞经流式细胞术，成骨、成软骨和成脂诱导培养鉴定后用于实验。BMSCs成骨诱导分化培养0～14 d，通过茜素红染色和ALP染色检测其成骨分化程度，并用Western blot法检测RNF11蛋白表达。取第4代BMSCs，分为空白对照组（A组）、空载慢病毒（Lv-NC）组（B组）和敲低RNF11（Lv-ShRNF11）组（C组），成骨诱导分化培养0～14 d，采用Western blot检测RNF11蛋白表达，茜素红染色和ALP染色检测其成骨分化程度，14 d实时荧光定量PCR（real-time fluorescence quantitative PCR，qRT-PCR）检测BMSCs成骨标志物Runx2、骨钙素（osteocalcin，OCN）及骨桥蛋白（osteopontin，OPN）的mRNA相对表达量；采用Western blot检测Akt、Smad1/5/8及β-catenin信号通路蛋白相对表达量，以磷酸化前后比值表示。为研究RNF11对Akt信号通路的影响机制，取第4代BMSCs分为Lv-NC转染组（A1组）、Lv-ShRNF11转染组（B1组）和添加Akt信号通路激活剂SC79的Lv-ShRNF11转染组（C1组），14 d时采用Western blot检测RNF11和Akt信号通路蛋白相对表达量，茜素红染色、ALP染色及qRT-PCR检测成骨相关指标。.
结果: 流式细胞术及成骨、成软骨和成脂诱导培养鉴定显示分离培养细胞为BMSCs。RNF11蛋白相对表达量随成骨分化时间延长而逐渐增加（ P<0.05）；下调RNF11后，茜素红和ALP染色示C组相较于A、B组BMSCs成骨分化程度降低，qRT-PCR检测示Runx2、OCN、OPN mRNA相对表达量减少（ P<0.05）。随成骨分化时间延长，RNF11与Akt信号通路蛋白相对表达量均上升（ P<0.05）。下调RNF11后，C组相较于A、B组，其Akt信号通路蛋白相对表达量显著降低（ P<0.05），而对Smad1/5/8以及β-catenin信号通路蛋白相对表达量无明显影响（ P>0.05）。B1、C1组相较于A1组，其RNF11蛋白相对表达量减少（ P<0.05），B1组相较于A1、C1组，其Akt信号通路蛋白相对表达量降低（ P<0.05）；茜素红与ALP染色示C1组BMSCs成骨分化程度稍低于A1组（ P>0.05），而明显高于B1组（ P<0.05）；qRT-PCR检测示C1组Runx2、OCN、OPN mRNA相对表达量稍低于A1组（ P>0.05），而明显高于B1组（ P<0.05）。.
结论: RNF11通过正向调控Akt信号通路激活水平促进BMSCs向成骨细胞分化。RNF11可作为提高BMSCs修复骨缺损疗效以及治疗骨代谢病的潜在靶点。.
Keywords: Akt signaling pathway; Bone marrow mesenchymal stem cells; bone defect repair; osteogenesis; ring finger protein 11; tissue engineered bone.