Effects of TAZ, a transcriptional coactivator, on human dental pulp stem cell proliferation in vitro

doi:10.3969/j.issn.2095-4344.0479

Abstract

Abstract:

BACKGROUND: How to improve the proliferation of human dental pulp stem cells (hDPSCs) in vitro is of great importance in regenerative medicine.
OBJECTIVE: To investigate the effect of TAZ, a transcriptional coactivator, on the proliferation of hDPSCs and its potential mechanisms.
METHODS: hDPSCs were isolated from human dental pulps and cultured in vitro. The expression of TAZ in hDPSCs was detected by immunofluorescence method. Then, we knocked down the expression of TAZ in hDPSCs, and the proliferation of hDPSCs was measured by MTT and BrdU kit analysis respectively. The levels of CTGF and Cry61, which are the downstream target genes of TAZ, were detected by RT-qPCR and western blot assay. We also investigated the effect of TAZ on the levels of transforming growth factor-β (TGF-β) signaling proteins, Smad3 and Smad 4 by using western blot.
RESULTS AND CONCLUSION: TAZ protein was expressed in hDPSCs. After the transfection by siTAZ for 24 hours, the mRNA and protein levels of TAZ were both significantly decreased. After the transfection by siTAZ for 24 and 48 hours, the proliferation of hDPSCs was obviously inhibited. Meanwhile, the mRNA and protein levels of CTGF and Cry61 were decreased by siTAZ transfection. The further research showed that TAZ silence also inhibited the expression of Smad3 and Smad 4, which belonged to the TGF-β signaling pathway. To conclude, TAZ may modulate the proliferation of hDPSCs through regulating the expression of CTGF and Cry61, and TGF-β-dependent signaling pathways may be involved in this regulation.

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, Dental Pulp, Cell Proliferation, Transcriptional Activation, RNA Interference, Tissue Engineering

CLC Number:

R394.2

Zhang Wen, Luo Shi-wei, Gu Ai-ling, He Wei. Effects of TAZ, a transcriptional coactivator, on human dental pulp stem cell proliferation in vitro[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1457-1462.

Figures/Tables 2

2.1 人牙髓干细胞形态及TAZ表达通过胰蛋白酶消化法获得人原代牙髓干细胞，主要为成纤维样细胞，其形态多为梭形(图1A)。人牙髓干细胞传至3-5代后，利用细胞免疫荧光法检测TAZ蛋白表达，结果显示人牙髓干细胞TAZ蛋白表达呈阳性，且与细胞核共定位(图1B-D)，以上结果成功证实人牙髓干细胞中有TAZ蛋白表达。 2.2 TAZ靶向小干扰RNA(siTAZ)下调了人牙髓干细胞中TAZ的mRNA和蛋白表达为了探索TAZ对人牙髓干细胞体外增殖的影响，首先设计合成了干扰TAZ表达的小干扰RNA(siTAZ)。如图2所示，在成功转染siTAZ 48 h之后，RT-qPCR(图2A)和Western blot(图2B)结果均一致显示，siTAZ组的TAZ mRNA和TAZ蛋白表达水平均较对照组显著降低，提示siTAZ可以有效地沉默TAZ在人牙髓干细胞中的表达。 2.3 siTAZ抑制了人牙髓干细胞的体外增殖采用MTT法检测各组细胞的体外增殖情况，如图3A所示，在siTAZ转染后24 h，siTAZ组的细胞增殖速度已显著低于空白对照组和阴性对照组；在转染后48 h，上述抑制作用较前进一步增强。为进一步验证上述实验结果，又采用BrdU细胞增殖法检测各组细胞的体外增殖情况，与MTT法检测结果类似，siTAZ组的细胞增殖速度显著低于空白对照组和阴性对照组(图3B)。以上结果提示，沉默TAZ的表达可以显著抑制人牙髓干细胞的体外增殖。 2.4 siTAZ通过下调CTGF和Cyr61的表达而抑制人牙髓干细胞的体外增殖前期已有报道显示CTGF和Cyr61作为TAZ下游作用的重要靶蛋白，其可能与细胞的增殖、迁移和凋亡等有密切联系。因此，为进一步探索siTAZ抑制人牙髓干细胞体外增殖的潜在分子机制，实验分别检测了TAZ被沉默表达之后CTGF和Cyr61在mRNA和蛋白水平的表达变化。结果如图4所示，无论是在mRNA水平还是蛋白水平，siTAZ组CTGF和Cyr61的表达水平均较空白对照组和阴性对照组显著降低，这提示siTAZ可能是通过干扰TAZ表达，进而抑制其下游CTGF和Cyr61蛋白的表达而最终影响了人牙髓干细胞的体外增殖。 2.5 siTAZ通过TGF?β介导的信号通路来下调CTGF和Cyr61蛋白的表达有研究报道TAZ可能通过TGF?β信号通路参与对CTGF和Cyr61蛋白的调控，因此，为深入探索siTAZ下调CTGF和Cyr61蛋白表达的分子机制，进一步检测了siTAZ转染后TGF?β信号通路中重要分子Smad3和Smad4的表达变化。结果如图5所示，siTAZ组Smad3和Smad4的表达水平均较空白对照组和阴性对照组显著降低，提示TGF?β信号通路可能参与了TAZ调控CTGF和 Cyr61蛋白的表达。"

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