牵张应力刺激介导间充质干细胞的信使RNA和长链非编码RNA表达谱变化

doi:10.3877/cma.j.issn.2095-1221.2018.05.003

目的

探讨信使RNA和长链非编码RNA在牵张应力刺激间充质干细胞（MSC）成骨分化中的作用。

方法

分离培养正常人骨髓MSC，分为应力组与无应力组，应力组通过FlexCell应力系统对其施加外源性应力（5﹪形变、频率0.1 Hz、作用时间4 h/d），无应力组不予以外源性应力刺激，通过茜素红实验和碱性磷酸酶实验检测其成骨分化能力改变。提取应力刺激下第7天RNA，进行全基因组及长链非编码芯片表达谱检测，通过生物信息学方法分析和鉴定关键长链非编码RNA。采用两样本t检验进行统计学分析。

结果

成骨第14天应力组MSC茜素红定量OD值为1.46±0.19，高于无应力组MSC 0.62±0.08，差异具有统计学意义（t?= -1.99，P < 0.05）。此外，成骨第14天应力组MSC碱性磷酸酶结果（265.3±31.2）U/L，较无应力组（121.2±21.2）U/L升高（t = -12.23，P < 0.05）。通过芯片表达谱检测，牵张应力刺激下成骨第7天共有差异表达信使RNA 598个，差异表达长链非编码RNA 329个。通过KEGG分析提示WNT信号通路是差异表达最明显的信号通路，其中WNT5a表达变化最为明显（6.74倍，P?< 0.01）。通过共表达分析提示lnc-RNA-SSR是调控WNT5a表达的主要长链非编码RNA。

结论

牵张应力刺激下MSC的信使RNA和长链非编码RNA表达谱明显改变，其中lnc-RNA-SSR可能通过WNT5a影响MSC成骨分化能力。

关键词: 间充质干细胞, 长链非编码RNA, mRNA, 牵张应力

Objective

To determine the role of mRNA and lnc-RNA in the osteogenic differentiation of MSC with strain stimulation.

Methods

MSC from healthy donors were isolated, cultured and then divided into the experimental group and the control group. The experimental group was stimulated with strain using FlexCell system (5﹪, 0.1?Hz, 4?h/d), and the control group was cultured without stimulaiton. The osteogenic differentiation ability of MSC was detected by alizarin red and alkaline phosphatase assays. RNA was extracted on day 7 of osteogenic differentiation, which was then detected using the mRNA and lnc-RNA expression microarrays. The key lnc-RNAs were identified using bioinformatics technology.

Results

On day 14 of osteogenic differentiation, the ARS quantitation results of MSC in the strain stimulation group (OD value: 1.46±0.19) were higher than those of the control group (OD value: 0.62±0.08). The difference was statistically significant (t?= -1.99, P < 0.05). Besides, the ALP activity results of MSC in the strain stimulation group (265.3±31.2)?U/L were also higher than MSC without strain stimulation (121.2±21.2)?U/L (t?= -12.23, P < 0.05) . Through microarray dection, a total of differentially expressed 598 mRNAs and 329 lnc-RNAs were identified on day 7 of osteogenic differentiation. KEGG analysis showed that WNT signal pathway was the key pathway with the most significant differences, among which WNT5a had the largest change. Co-expression analysis suggested that lnc-RNA-SSR may be the critical lnc-RNA to regulate WNT5a expression.

Conclusion

Expression profile of mRNA and lnc-RNA in MSC significantly changed with strain stimulation. Lnc-RNA-SSR may affected MSC osteogenesis through regulating WNT5a expression with strain stimulation

Key words: Mesenchymal stem cells, Long non-coding RNA, mRNA, Tensile stress

图1 应力刺激下MSC成骨分化能力观察（茜素红染色，×40）

图2 应力刺激下MSC的mRNA和lnc-RNA表达谱

表1 差异表达的前5位的mRNA

基因名称	改变倍数	上/下调
CD1A	6.89	下调
WNT5a	6.74	上调
ANK2	6.43	上调
LAMA4	5.43	上调
E2F5	4.98	上调

表1 差异表达的前5位的mRNA

基因名称	改变倍数	上/下调
CD1A	6.89	下调
WNT5a	6.74	上调
ANK2	6.43	上调
LAMA4	5.43	上调
E2F5	4.98	上调

表2 差异表达的前5位的lnc-RNA

lnc-RNA	倍数	上/下调	染色体	正/反义链	种类
ENST00000498630.1	6.53	上调	3	+	基因间lnc-RNA
ENSG00000258602.1	5.87	上调	14	+	基因间lnc-RNA
ENSG00000259618.1	5.43	下调	15	-	双向lnc-RNA
ENSG00000246763.2	5.02	上调	5	-	内含子lnc-RNA
ENSG00000263731.1	4.87	上调	17	-	基因间lnc-RNA

表2 差异表达的前5位的lnc-RNA

lnc-RNA	倍数	上/下调	染色体	正/反义链	种类
ENST00000498630.1	6.53	上调	3	+	基因间lnc-RNA
ENSG00000258602.1	5.87	上调	14	+	基因间lnc-RNA
ENSG00000259618.1	5.43	下调	15	-	双向lnc-RNA
ENSG00000246763.2	5.02	上调	5	-	内含子lnc-RNA
ENSG00000263731.1	4.87	上调	17	-	基因间lnc-RNA

表3 KEGG分析差异表达信号通路

信号通路	差异基因数	P值	差异表达基因
WNT	11	< 0.001	WNT5a, Daam1, Axin, GBP, PS-1, TAK1, NLK, CBP, ICAT, fra-1, PLC
Cytokine-Cytokine receptor	10	0.003	LTA, EGF, IL10RA, IL22RA2, TPO, IL11, IL23R, CCR7, CCL22, LIF
Cell Cycle	8	0.009	CDK1, MCM, Cdc7, Plk1, Myt1, Mdm2, ARF, Smc1
Focal adhesion	6	0.014	FAK, Vav, Rac, DOCK1, Crk, Sos
Lipoic acid metabolism	3	0.034	LipB, Lae, LplA

表3 KEGG分析差异表达信号通路

信号通路	差异基因数	P值	差异表达基因
WNT	11	< 0.001	WNT5a, Daam1, Axin, GBP, PS-1, TAK1, NLK, CBP, ICAT, fra-1, PLC
Cytokine-Cytokine receptor	10	0.003	LTA, EGF, IL10RA, IL22RA2, TPO, IL11, IL23R, CCR7, CCL22, LIF
Cell Cycle	8	0.009	CDK1, MCM, Cdc7, Plk1, Myt1, Mdm2, ARF, Smc1
Focal adhesion	6	0.014	FAK, Vav, Rac, DOCK1, Crk, Sos
Lipoic acid metabolism	3	0.034	LipB, Lae, LplA

表4 与WNT5a共表达的lnc-RNA分析

lnc-RNA	倍数	上/下调	种类	相关系数	P值
ENSG00000237298.2	4.11	上调	基因间lnc-RNA	0.956	< 0.001
ENSG00000241956.5	3.89	上调	基因间lnc-RNA	0.910	0.013
ENSG00000187229.3	2.45	上调	基因间lnc-RNA	0.892	0.015

表4 与WNT5a共表达的lnc-RNA分析

lnc-RNA	倍数	上/下调	种类	相关系数	P值
ENSG00000237298.2	4.11	上调	基因间lnc-RNA	0.956	< 0.001
ENSG00000241956.5	3.89	上调	基因间lnc-RNA	0.910	0.013
ENSG00000187229.3	2.45	上调	基因间lnc-RNA	0.892	0.015

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Differentially expressed profile of messager RNA and long non-coding RNA in mesenchymal stem cell with strain stimulation

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Differentially expressed profile of messager RNA and long non-coding RNA in mesenchymal stem cell with strain stimulation