Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells

Yuting HUANG; Jinling DONG; Liyuan FENG; Peng LI

doi:10.16016/j.1000-5404.201908194

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Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells

Basic Medicine | 更新时间：2026-01-09

- Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells
- Journal of Third Military Medical University Vol. 42, Issue 2, Pages: 125-132(2020)
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系生药学与中药学教研室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81472596)
- DOI：10.16016/j.1000-5404.201908194
  CLC： R329-33;R329.28;R341
- Received：27 August 2019，
  
  Revised：2019-11-20，
  
  Published：30 January 2020
- 稿件说明：
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Yuting HUANG, Jinling DONG, Liyuan FENG, et al. Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells[J]. Journal of Third Military Medical University, 2020, 42(2): 125-132.
DOI：

Yuting HUANG, Jinling DONG, Liyuan FENG, et al. Effects of endogenous nestin overexpression on proliferation, migration and MAPK signal transduction pathway in NIH3T3 cells[J]. Journal of Third Military Medical University, 2020, 42(2): 125-132. DOI： 10.16016/j.1000-5404.201908194.

摘要

目的

通过在成纤维细胞NIH3T3中内源性过表达巢蛋白(Nestin)，研究其对细胞增殖、迁移及转化生长因子(TGF-β1)作用下细胞胶原蛋白表达与MAPK相关通路的影响。

方法

通过CRISPR/SAM技术构建内源性过表达Nestin的细胞株，用MTS实验、Western blot检测细胞增殖能力，细胞划痕实验检测细胞迁移能力，经qPCR、Western blot与免疫荧光染色明确TGF-β1对其胶原蛋白与MAPK通路的影响。

结果

CRISPR/SAM技术能使内源性Nestin表达显著提高(

0.05)，而内源性过表达Nestin对NIH3T3细胞的生长、迁移有明显促进作用(

0.05)，对TGF-β1诱导的胶原蛋白表达及MAPK的激活也有明显的抑制效应(

0.01)。

结论

用CRISPR/SAM技术内源性过表达Nestin可以明显促进NIH3T3细胞的生长和迁移，但却抑制TGF-β1诱导下胶原蛋白的表达及MAPK的激活。

Abstract

Objective

To observe the effects of endogenous nestin overexpression on the proliferation

migration and transforming growth factor β (TGF-β1)-induced expression of collagen and MAPK-related pathway in NIH3T3 fibroblasts.

Methods

CRISPR/SAM technique was used to construct a nestin-overexpressing cell line in NIH3T3 fibroblasts. The effects of nestin overexpression on cell proliferation and migration were evaluated using MTS assay

immunoblotting and scratch-wound assay. Quantitative PCR

immunoblotting and immunofluorescence staining were used to determine the effect of nestin overexpression on TGF-β-induced expression of collagen and activation of MAPK pathway in the fibroblasts.

Results

CRISPR/SAM significantly increased the expression of endogenous nestin in NIH3T3 fibroblasts (

0.05)

and nestin overexpression significantly promoted the growth and migration of NIH3T3 cells (

0.05). Nestin overexpression obviously inhibited the expression of collagen and the activation of MAPK in TGF-β1-induced cells (

0.01).

Conclusion

Endogenous overexpression of nestin achieved by CRISPR/SAM technique significantly promotes the growth and migration of NIH3T3 cells and inhibits TGF-β1-induced expression of collagen and activation of MAPK.

关键词

Keywords

references

PARK D, XIANG A P, MAO FF, et al . Nestin is required for the proper self-renewal of neural stem cells[J ] . Stem Cells, 2010, 28(12): 2162-2171. DOI: 10.1002/stem.541.

BERNAL A, ARRANZ L.Nestin-expressing progenitor cells: function, identity and therapeutic implications[J]. Cell Mol Life Sci, 2018, 75(12): 2177-2195. DOI: 10.1007/s00018-018-2794-z.

CALDERONE A. The biological role ofnestin(+)-cells in physiological and pathological cardiovascular remodeling[J]. Front Cell Dev Biol, 2018, 6: 15. DOI: 10.3389/fcell.2018.00015.

TANG Y W, XIONG S M, YU P, et al . Direct conversion of mouse fibroblasts into neural stem cells by chemical cocktail requires stepwise activationof gro wth factors and Nup210[J ] . Cell Rep, 2018, 24(5): 1355-1362.e3. DOI: 10.1016/j.celrep.2018.06.116.

MICHALCZYK K, ZIMAN M.Nestin structure and predicted function in cellular cytoskeletal organisation[J]. Histol Histopathol, 2005, 20(2): 665-671. DOI: 10.14670/HH-20.665.

JOUNG J,KONERMANN S, GOOTENBERG J S, et al . Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening[J ] . Nat Protoc, 2017, 12(4): 828-863. DOI: 10.1038/nprot.2017.016.

CHENG L P, JIN Z G, LIU L, et al . Characterization and promoter analysis of the mousenestin gene[J ] . FEBS Lett, 2004, 565(1/2/3): 195-202. DOI: 10.1016/j.febslet.2004.03.097.

KONERMANN S, BRIGHAM M D, TREVINO A E, et al . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex[J ] . Nature, 2015, 517(7536): 583-588. DOI: 10.1038/nature14136.

KUTNER R H, ZHANG X Y, REISER J. Production, concentration and titration ofpseudotyped HIV-1-based lentiviral vectors[J]. Nat Protoc, 2009, 4(4): 495-505. DOI: 10.1038/nprot.2009.22.

DI C G, XIANG A P, JIA L, et al . Involvement of extracellular factors in maintaining self-renewal of neural stem cell bynestin[J ] . Neuroreport, 2014, 25(10): 782-787. DOI: 10.1097/WNR.0000000000000177.

NOWAK A, DZIEGIEL P. Implications ofnestin in breast cancer pathogenesis (Review)[J]. Int J Oncol, 2018, 53(2): 477-487. DOI: 10.3892/ijo.2018.4441.

HERTIG V, MATOS-NIEVES A, GARG V, et al .Nestin expression is dynamically regulated in cardiomyocytes during embryogenesis[J ] . J Cell Physiol, 2018, 233(4): 3218-3229. DOI: 10.1002/jcp.26165.

WANG J, CAI J, HUANG Y, et al .Nestin regulates proliferation and invasion of gastrointestinal stromal tumor cells by altering mitochondrial dynamics[J ] . Oncogene, 2016, 35(24): 3139-3150. DOI: 10.1038/onc.2015.370.

AKI R, AMOH Y, LI L N, et al .Nestin-expressing interfollicular blood vessel network contributes to skin transplant survival and wound healing[J ] . J Cell Biochem, 2010, 110(1): 80-86. DOI: 10.1002/jcb.22512.

LI P, LEE E H, DU F, et al .Nestin mediates hedgehog pathway tumorigenesis[J ] . Cancer Res, 2016, 76(18):5573-5583. DOI: 10.1158/0008-5472.CAN-16-1547.

COENTRO J Q, PUGLIESE E, HANLEY G, et al . Current and upcoming therapies to modulate skin scarring and fibrosis[J ] .Adv Drug Deliv Rev, 2018: S0169-S409X(18)30207-2. DOI: 10.1016/j.addr.2018.08.009.

LIANG C J, YEN Y H, HUNG L Y, et al . Thalidomide inhibitsfibronectin production in TGF-β1-treated normal and keloid fibroblasts via inhibition of the p38/Smad3 pathway[J ] . Biochem Pharmacol, 2013, 85(11): 1594-1602. DOI: 10.1016/j.bcp.2013.02.038.

HE T, BAI X Z, YANG LL, et al . Loureirin B inhibits hypertrophic scar formation via inhibition of the TGF-β1-ERK/JNK pathway[J ] . Cell Physiol Biochem, 2015, 37(2): 666-676. DOI: 10.1159/000430385.

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