CARM1 is required to maintain stemness of amniotic fluid-derived stem cells

doi:10.3969/j.issn.2095-4344.2016.36.014

Abstract

Abstract:

BACKGROUND: Studies have shown that methylation modification using CARM1-catalyzed histone H3R17/R26 can maintain the stemness of embryonic stem cells. However, mechanism underlying CARM1 effect on the stemness of amniotic fluid-derived stem cells is still unclear.
OBJECTIVE: To investigate the function and underlying molecular mechanism of CARM1 to maintain stemness in the amniotic fluid-derived stem cells.
METHODS: Amniotic fluid-derived stem cells from term pregnancy were isolated and cultured. RT-PCR was used to identify the stem cell mark and CARM1 gene expression. CARM1 expression in amniotic fluid-derived stem cells was knocked down by using two shRNA. RT-qPCR was used to detect the silencing efficiency, and western blot employed to examine the methylation level of Arginines 17 at N terminus of histone 3 (H3mR17). Moreover, the expression of embryonic stem cell markers, including OCT4, SOX2 and NANOG, were detected.
RESULTS AND CONCLUSION: Amniotic fluid-derived stem cells from term pregnancy could express CARM1 and stem cell markers, including OCT4, SOX2, Nanog and KLF4. Both of the shRNAs could knock down the expression of CARM1 efficiently. When CARM1 was knocked down, the H3mR17 level was decreased and OCT4, SOX2 expression was also reduced, but NANOG expression had no change. All these indicate that CARM1 is required for amniotic fluid-derived stem cells to maintain stemness through regulating OCT4 and SOX2 expression.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Amniotic Fluid, Pregnancy Trimester, Third, Protein-Arginine N-Methyltransferases, Lentivirus Infections, Methylation, Tissue Engineering

CLC Number:

R394.2

Wu Jing, Zhao Li-hua . CARM1 is required to maintain stemness of amniotic fluid-derived stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(36): 5412-5418.

Figures/Tables 1

2.1 妊娠晚期羊水干细胞的分离、培养和鉴定原代培养7 d后换液，倒置显微镜下观察可见成纤维细胞样细胞呈克隆样生长。继续培养至10 d进行传代培养，标记为第1代。取第2代妊娠晚期羊水干细胞，将人胚胎肝细胞作为阳性对照，293T细胞为阴性对照，RT-PCR检测干细胞标志物的表达情况。结果表明，与人胚胎干细胞相同，人妊娠晚期羊水干细胞也能够表达干细胞标志物OCT4、KLF4、SOX2和Nanog基因(图1)。 2.2 人妊娠晚期羊水干细胞能够表达CARM1基因取第2代人妊娠晚期羊水干细胞以及人胚胎干细胞，RT-qPCR检测CARM1基因的表达情况。结果发现，人妊娠晚期羊水干细胞能够表达CARM1基因，且其表达水平显著高于其在人胚胎干细胞中的表达水平(图2)。 2.3 利用慢病毒感染系统建立CARM1表达沉默的羊水干细胞系将慢病毒质粒pGIPZ-GFP-CARM1- shRNA1/2及其包装质粒psPAX2和pMD2G共转入293T细胞中，48 h后于倒置荧光显微镜下观察带有GFP的慢病毒载体的表达。结果显示，95%以上的细胞都表达绿色荧光，保证了重组病毒可被有效获得(图3)。Millipore超滤离心管对所得病毒原液进行浓缩，最终浓缩体积为200-300 μL。将6孔板中长势良好、汇合度约为50%的羊水干细胞中加入2 mL羊水干细胞完全培养基，再加入50 μL上述浓缩病毒，置于37 ℃，体积分数为5%CO2培养箱，24 h后置换新鲜培养基后继续培养，48 h后置于倒置荧光显微镜下观察绿色荧光的表达。结果显示，90%以上的羊水干细胞都表达绿色荧光(图3)。 2.4 CARM1 shRNA可显著沉默CARM1基因的表达 RT-qPCR检测shRNA的干扰效率，结果显示，2条shRNA均可有效沉默CARM1基因的表达水平(图4)。 2.5 CARM1表达沉默后，组蛋白H3R17的甲基化水平降低蛋白质免疫印迹实验检测野生型和CARM1表达沉默型人妊娠晚期羊水干细胞中CARM1的催化底物，即组蛋白H3第17号位精氨酸的甲基化水平。结果表明，CARM1表达降低后，H3R17的甲基化水平降低(图5)。 2.6 CARM1表达沉默后，干细胞标志物表达情况 RT-qPCR检测野生型羊水干细胞和CARM1表达沉默的人妊娠晚期羊水干细胞中干细胞标志物，包括OCT4，SOX2和NANOG的表达情况。结果表明，CARM1表达沉默后，OCT4和SOX2的表达明显降低，而NANOG的表达没有变化(图6)"

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