hMSC成骨分化相关lncRNA的筛选和初步鉴定

Screening and Primary Identification of lncRNA Involved in Osteogenic Differentiation of hMSC

【目的】筛选和初步鉴定人骨髓间充质干细胞(hMSC)成骨分化相关的长链非编码RNA(lncRNA)。【方法】利用Refseq,UCSC knowngenes,Ensembl,H-invDB 7.0,RNAdb 2.0,NRED生物软件分析成骨基因相关的lncRNA,综合获得可能的成骨相关lncRNA;3例骨髓标本来源于行腰椎手术患者,密度梯度法分离扩增hMSC后,分成骨诱导组和对照组,地塞米松、维生素C、β-甘油磷酸钠诱导hMSC成骨分化,通过ALP测定及钙结节茜素红染色进行成骨诱导鉴定;qRT-PCR验证hMSC成骨分化前、后差异表达的lncRNA,以及成骨分化前、后差异表达的基因BMP1、MSX1、Runx2、Smurf-1、ALP。【结果】通过上述生物软件综合分析发现3个成骨基因MSX1、BMP1和Smurf1周围存在相关lncRNA。4个lncRNA(AK129811、AK024937、AK096529、uc003ups)与成骨基因Smurf-1相关;2个lncRNA(AF289591和uc003xbe)与成骨基因BMP1相关;1个lncRNA(AK056311)与成骨基因MSX1相关。ALP检测示:与对照组细胞相比,成骨诱导组细胞1周ALP值最高,达到(16.82±1.64)nmol/min,随着诱导时间的延长,ALP值逐渐降低,3周降至(8.37±1.01)nmol/min。钙结节染色显示hMSC成骨诱导分化2周后胞外开始出现少量钙结节,随着诱导时间延长钙结节数量逐渐增加,成骨诱导至第3周钙结节最多,定量测定钙质量浓度达(716±49)mg/mL。RT-qPCR结果显示绝大部分lncRNA在hMSC成骨分化过程中表达均下调,其中2个与Smurf-1相关lncRNA(AK096529和uc003ups)与成骨诱导前相比,存在显著性下调。1个与MSX1相关的lncRNA(AK056311)与成骨诱导前相比,也存在显著性下调。同时,成骨分化基因Runx2、ALP表达显著性上调,MSX1、Smurf-1表达显著性下调。【结论】结合既往研究结果分析:AK096529和uc003ups可能通过正向调控Smurf1,促使Smurf1下调,减少Runx2的降解,继而促进hMSC的成骨分化。

【Objective】 To screen and primarily identify lncRNA(Long noncoding RNAs,lncRNA) involved in osteogenic differentiation of hMSC(Human marrow-derived mesenchymal stem cells,hMSC).【Methods】 Refseq,UCSC knowngenes,Ensembl,H-invDB 7.0,RNAdb 2.0 and NRED database were applied to comprehensively analyze lncRNA involved in osteogenic differentiation of hMSC.Three cases of bone marrow derived from patients with lumbar operation.hMSC were obtained by density centrifugation isolation from bone marrow,then cultured and expanded.hMSC were divided into control group and osteogenic induction group.hMSC were induced into osteoblasts with dexamethasone,vitamin C and sodium β-glycerophosphate.The phenotype of hMSC differentiating to osteoblasts was verified by the mineralized calcium node staining and alkaline phosphatase(ALP) detection.RT-qPCR assay was used to identify the differentially expressed lncRNA and osteogenic gene BMP1,MSX1,Runx2,Smurf-1 and ALP.【Results】 The analysis of comprehensive algorithms indicated that four lncRNA(AK129811,AK024937,AK096529,uc003ups) were probably involved in regulating Smurf-1,two lncRNA(AF289591 and uc003xbe) probably regulated BMP1,one lncRNA(AK056311)probably regulated MSX1.ALP detection indicated that ALP level of cells gradually increased and reached the top value(16.82 ±1.64)nmol/min after 7 days induction,but decreased to(8.37 ± 1.01) nmol/min after 21 days induction.Alizarin Red staining demonstrated that calcium nodes appeared at 2 weeks after induction,and obviously increased at 3 weeks after induction while the concentration of calcium were(716 ± 49) mg/mL.RT-qPCR analysis revealed that most lncRNA were down-expressed after hMSC osteogenic differentiation,among which two(AK096529 and uc003ups) related with Smurf-1 were significantly,one(AK056311)related with MSX1 was also significantly.Osteogenic gene Runx2 and ALP were up-expressed significantly after osteogenic induction,but Smurf-1 and MSX1 were down-expressed significantly.【Conclusion】 Base on the results from previous research,lncRNA AK096529 and uc003ups are probably involved in the hMSC osteoblast differentiation through positively regulating Smurf-1.