长链非编码RNA Crnde通过调节Wnt/β-连环蛋白信号通路影响小鼠成骨细胞增殖的研究  

Effect of long non-coding RNA Crnde on proliferation of mouse osteoblasts by regulating the Wnt/β-catenin signaling pathway

作　　者：孙新志[1] 廖鹰[2] 郭俊杰[1] 姬延辉 刘宏建[1] Sun Xinzhi;Liao Ying;Guo Junjie;Ji Yanhui;Liu Hongjian(Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Public Security Technology,Railway Police College,Zhengzhou 450053,China)

机构地区：[1]郑州大学第一附属医院骨科,450052 [2]郑州铁道警察学院刑事科学技术系,450053

出　　处：《中华实验外科杂志》2021年第4期708-711,共4页Chinese Journal of Experimental Surgery

摘　　要：目的观察长链非编码RNA Crnde通过调节Wnt/β-连环蛋白(β-catenin)信号通路对小鼠成骨细胞增殖的影响。方法培养MC3T3-E1成骨细胞,随后诱导分化3周,实时定量反转录聚合酶链反应(RT-qPCR)检测Crnde表达,应用CRISPR-Cas9方法构建Crnde基因敲除小鼠模型,同时以野生型小鼠作为对照,分为Crnde基因敲除小鼠组(n=25)和野生型小鼠组(n=25),小鼠处死前12 h腹腔注射BrdU进行染色观察成骨细胞增殖,检测血清1型前胶原N末端(P1NP)和Ⅰ型胶原羧基端交联肽(CTX-Ⅰ)表达,蛋白质印迹法(Western blot)检测骨组织Wnt和β-catenin蛋白表达,原位缺口末端标记法(TUNEL)染色检测细胞凋亡情况,分别运用Osteomeasure系统和MicroCT分析进行骨组织形态计量分析,双荧光素酶报告基因验证Crnde与Wnt蛋白的靶向调节关系。组间比较采用t检验。结果成骨细胞分化过程Crnde表达情况为在诱导的3周时间内,随着时间延长,Crnde表达明显增加,在第3周表达最高(P>0.05);Wnt和β-catenin在Crnde^(-/-)小鼠和正常小鼠中表达情况中,蛋白质印迹法(Western blot)结果显示,Crnde^(-/-)组中Wnt和β-catenin蛋白表达低于WT小鼠组(P<0.05);BrdU免疫荧光染色结果显示,正常对照组小鼠胫骨存在大量BrdU阳性细胞,而Crnde^(-/-)小鼠细胞显著减少;TUNEL染色显示正常对照组小鼠与Crnde^(-/-)小鼠小鼠凋亡成骨细胞数量差异无统计学意义(P>0.05);μCT和Osteomeasure系统结果显示,椎骨的组织形态分析BV/TV,WT组[(19.43±1.53)%],Crnde^(-/-)组[(20.14±1.56)%],Crnde^(-/-)鼠的小梁和皮质骨均显示出低骨量表型,Crnde^(-/-)小鼠的骨形成参数均与野生型小鼠差异有统计学意义(P<0.05);Crnde^(-/-)小鼠血清P1NP和CTX-I分别为(15.62±1.23)μg/L和(12.36±1.12)μg/L,WT小鼠血清P1NP和CTX-Ⅰ分别为(22.81±1.56)μg/L和(16.21±1.21)μg/L,Crnde^(-/-)小鼠组均比WT小鼠组明显下降(P<0.05);双荧光素酶报告基因检测结果显示Crnde模拟物显著降�Objective To investigate the effect of long non-coding RNA Crnde on the proliferation of mouse osteoblasts by regulating the Wnt/β-catenin signaling pathway.Methods MC3T3-E1 osteoblasts were cultured and then induced to differentiate for 3 weeks.The expression of Crnde was detected by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR).The Crnde gene knockout mouse model was constructed using CRISPR-Cas9 method.At the same time,wild-type mice were used as controls.They were divided into Crnde knockout mice group(n=25)and wild-type mice group(n=25).At 12 h after he mice were killed,BrdU was injected into the intraperitoneal cavity for staining to observe the proliferation of osteoblasts,and the N-terminal type 1 procollagen amino terminal peptide(P1NP)and type Ⅰ collagen cross-linked C-terminal telopeptide type Ⅰ collagen cross-linked C-terminal telopeptide,CTX-Ⅰ were detected.Western blotting and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining were used to detect the expression of Wnt and β-catenin protein in bone tissue and cell apoptosis.Osteomeasure system and MicroCT analysis were used for bone tissue morphometric analysis.The dual luciferase reporter gene was used to verify the targeted regulation relationship between Crnde and Wnt protein.The statistical analysis was done using Student's t-test.Results The expression of CRNDE during the differentiation of osteoblasts was significantly increased in the 3 weeks after induction,and the expression of Crnde was highest at 3rd week after induction(P>0.05).The expression of Wnt-catenin and β-catenin protein in the CRNDE^(-/-)mice decreased significantly as compared with wild-type normal mice(P<0.05).Brdu immunofluorescence staining showed that there were a lot of BrdU positive cells in Tibia of normal control mice,while Crnde^(-/-)mice showed a significant decrease in BrdU positive cells.TUNEL staining showed that there was no significant difference in the number of apoptotic osteoblasts between

关 键 词：长链非编码RNA Wnt/β-连环蛋白信号通路 成骨细胞 增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]