lncRNA-DANCR对人脂肪间充质干细胞成脂和成骨分化能力的影响

Effects of lncRNA-DANCR on adipogenic and osteogenic differentiation potential of human adipose derived mesenchymal stromal stem cells

目的:探讨长链非编码RNA(lncRNA)-抗分化非编码RNA(DANCR)对人脂肪来源间充质干细胞(hASCs)成脂、成骨分化能力的影响。方法:收集中国医学科学院整形外科医院的3例女性患者腹部脂肪抽吸术后废弃的脂肪组织(年龄25~35岁),分离培养hASCs。构建DANCR敲减与过表达质粒,利用慢病毒感染的方式在hASCs中敲减或过表达DANCR,将实验分为5组:敲减DANCR的实验组(shDANCR1、shDANCR2)、敲减对照组(shScrmble)、过表达DANCR的实验组(pCDH-DANCR)、过表达对照组(pCDH-GFP),每组样本量为3例。RT-PCR检测上述5组细胞中DANCR的表达量。将上述5组慢病毒感染成功后的hASCs进行成脂或成骨诱导,成脂诱导7 d进行油红O染色鉴定成脂效果,成骨诱导14 d进行茜素红染色鉴定成骨效果,并应用RT-PCR检测成脂分化关键转录因子PPAR-γ、C/EBPα、FABP4、Adiponectin和脂类合成酶基因LPL、GPDH、FASN、ACC1和HSL,以及成骨分化关键转录因子RUNX2、OCN、OPN、BMP2及ALP的表达。实验数据均采用均值±标准差表示,2组间数据采用t检验(Student’s t-test)进行比较,P<0.05为差异具有统计学意义。结果:设计的2条shRNA敲减序列均可使hASCs中DANCR的表达显著减少,与对照组比较,DANCR表达量分别下降了86%和74%,差异具有统计学意义(t=84.07、P<0.001,t=75.52、P<0.001)。构建的DANCR过表达质粒可以有效增加hASCs中DANCR表达量,与对照组相比,增加了(15.067±1.986)倍,差异具有统计学意义(t=12.24,P<0.001)。成脂、成骨诱导实验显示,敲减DANCR会引起hASCs成脂诱导后脂滴数量显著多于对照组,2组敲减组与对照组相比,差异均具有统计学意义(t=17.24、P<0.001,t=30.68、P<0.001),成脂分化关键转录因子PPAR-γ、C/EBPα、FABP4、Adiponectin以及脂类合成酶的关键基因LPL、GPDH、FASN、ACC1、HSL的表达都显著高于对照组;而过表达DANCR后会抑制脂滴的形成及上述基因的表达。同样在成骨诱导中,敲减DANCR后hASCs成骨诱导14 d钙结节形成量显著多于对照组,差异具有统计学意义(t=15.25、P<0.001,t=24.61,P<0.001),成骨分化关键基因RUNX2、OCN、OPN、BMP2及ALP的表达也显著高于对照组,而过表达DANCR后成骨诱导14 d钙结节形成量较对照组显著减少(t=40.81、P<0.001),成骨分化关键转录因子表达水平也会降低。结论:lncRNA-DANCR能够抑制hASCs的成脂、成骨分化,DANCR表达量的变化影响hASCs的成脂、成骨分化效果,可以作为调控hASCs成脂、成骨定向分化的潜在靶点。

Objective To investigate the regulatory effects of anti-differetiation noncoding RNA(DANCR)on the ability of human adipose derived mesenchymal stromal stem cells(hASCs)to differentiate into adipocytes and osteoblasts.Methods hASCs was isolated from abandoned adipose tissue after liposuction.The lentivirus-based DANCR knockdown and over-expression plasmids were constructed and used to knock-down and over-express DANCR in hASCs respectively,the expression of DANCR in hASCs was detected by real time quantitative PCR.Thus,the experiment was divided into five groups:the DANCR knock-down group(shDANCR1,shDANCR2)and the control group(shScrmble);DANCR over-expression group(PCDH-DANCR)and the control group(PCDH-GFP).All the groups were cultured in the growth medium,adipogenic medium and osteogenic medium,Oil red O staining was used to characterize the adipogenic differentiation capabilities of hASCs after 7 days of adipogenic induction,and alizarin red S staining was used to assess the osteogenic differentiation capacities of hASCs after 14 days of osteogenic induction.In addition,real-time PCR analysis was used to quantify the expressions of the transcription factors that play crucial roles in adipogenic induction(PPAR-γ,C/EBPα,FABP4,Adiponectin),lipid synthase(LPL,GPDH,FASN,ACC1,HSL)and osteogenic differentiation(RUNX2,OCN,OPN,BMP2,ALP).All values are expressed as the mean±standard error of the mean.The differences between the groups were assessed using Student’s t test.The result were considered statistically significant at P<0.05.Results The expression of DANCR was correspondingly down-regulated and up-regulated in hASCs after infection of DANCR-specific lentivirus.Compared with the control group(shScramble),DANCR expression respectively decreased 86%(t=84.07,P<0.001)and 74%(t=75.52,P<0.001)in the DANCR knock-down group(shDANCR1,shDANCR2).The constructed DANCR overexpressed plasmids could effectively increase the DANCR expression,which increased 15.067±1.986 times compared with the control group(pCDH-GFP),with a statistically significant difference(t=12.24,P<0.001).To confirm adipogenesis,cells were stained with the lipid dye oil red O.Compared with the control group(shScramble)with a statistically significant difference,more lipid droplets were apparent in the DANCR knockdown group(shDANCR1,shDANCR2)following 7 d adipogenic induction(t=17.24,P<0.001,t=30.68,P<0.001),whereas the number of lipid droplets in DANCR overexpression group(pCDH-DANCR)less than the control group(pCDH-GFP).The mRNA expression levels of genes that related to adipogenic differentiation(PPAR-γ,C/EBPα,FABP4,Adiponectin)and lipid synthesis(LPL,GPDH,FASN,ACC1,HSL)were elevated in DANCR knockdown cells(shDANCR1,shDANCR2)compared with the control group(shScramble).In contrast,overexpression of DANCR significantly downregulated the expression of these genes.Osteogenesis was confirmed by alizarin red S staining.Overexpression of DANCR reduced the osteogenic differentiation as revealed by the mineralization assay of alizarin red S staining after 14 d osteogenic induction(t=40.81,P<0.001),whereas knockdown of DANCR significantly promoted the mineralization of hASCs(t=15.25,P<0.001,t=24.61,P<0.001).Consistently,DANCR overexpression downregulated several osteogenic differentiation genes(RUNX2,OCN,OPN,BMP2,ALP)at the mRNA level,whereas knockdown of DANCR significantly upregulated the expression of these genes.Conclusions The capabilities of hASCs to differentiate into adipocytes and osteoblasts could be regulated by the expression of lncRNA-DANCR.