Experiment of Nesprin Protein Influence on Bone Marrow Mesenchymal Stem Cells_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

YANG Wengang , XUE Song , LIAN Feng , WANG Yongyi , HU Zhenlei , ZHU Hongsheng.

Department of Cardiovascular Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiaotong University, Shanghai 200127, P. R. China;

Corresponding author：

Keywords：

Nesprin protein; 　Bone marrow mesenchymal stem cells; 　 Cell proliferation; 　Lentivirus vector

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Abstract：　Objective　To construct a nesprin-siRNA lentiviral vector（LV-siNesprin）, transfect it into bone marrow mesenchymal stem cells （MSCs）, and observe morphology changes of MSCs. 　Methods　According to the target gene sequence of nesprin, we designed and synthesized four pairs of miRNA oligo, which were then annealed into double-strand DNA and identified by sequencing. MiRNA interference with the four kinds of plasmids （SR-1,SR-2,SR-3, and
SR-4） were transfected into rat vascular smooth muscle cells, and reverse transcriptase chain reaction(RT-PCR) and Western blotting were performed to detect the interference effects and filter out the most effective interference sequence. We used the best interference sequence carriers and pDONR221 to react together to get the entry vectors with interference sequence. Then the objective carrier pLenti6/V5-DEST expressing both entry vectors and lentiviral vectors was restructured to get lentiviral　expression vector containing interference sequence （LV-siNesprin＋green fluoresent protein （GFP））, which was packaged and the virus titer was determined. LV-siNesprin＋GFP was transfected to MSCs, and the expression of nesprin protein（LV-siNesprin＋GFP group，GFP control group and normal cell group）was detected by Western blotting. The morphology of MSCs nuclear was observed by 4’,6-diamidino-2-phenylindole （DAPI） stain. The proliferation of MSCs （LV-siNesprin＋GFP group，GFP control group and normal group） was detected by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide（MTT） after lentivirus transfected to MSCs at 24, 48, 72, and 96 hours.　Results　The four pairs of miRNA oligo were confirmed by sequencing. Successful construction of LV-siNesprin was confirmed by sequencing. The best interference with miRNA plasmid selected by RT-PCR and Western blotting was SR-3. Lentiviral was packaged, and the activity of the virus titer of the concentrated suspension was 1×106 ifu/ml. After MSCs were transfected with　LV-siNesprin, nesprin protein expression significantly decreased, and the nuclear morphology also changed including fusion　and fragmentation. The proliferation rate of MSCs in the LV-siNesprin＋GFP group was significantly slower than that of the GFP control and normal cell groups by MTT.　Conclusion　Nesprin protein plays an important role in stabilizing MSCs nuclear membrane, maintaining spatial structure of MSCs nuclear membrane，and facilitating MSCs proliferation.

Citation： YANG Wengang,XUE Song,LIAN Feng,WANG Yongyi,HU Zhenlei,ZHU Hongsheng.. Experiment of Nesprin Protein Influence on Bone Marrow Mesenchymal Stem Cells. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2012, 19(4): 408-413. doi: Copy

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13.	Zhang Q， Bethmann C， Worth NF， et al. Nesprin-1 and -2 are involved in the pathogenesis of Emery Dreifuss muscular dystrophy and are critical for nuclear envelope integrity. Hum Mol Genet， 2007， 16 （23）：2816-2833.
14.	Puckelwartz MJ， Kessler EJ， Kim G， et al. Nesprin-1 mutations in human and murine cardiomyopathy. J Mol Cell Cardiol， 2010， 48（4）：600-608.
15.	Farhana H， Mazzeo D， Patel JT， et al. Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J Biol Chem， 2010， 285 （5）：3487-3498.
16.	Zhang X， Lei K， Yuan X， et al. SUN1/2 and Syne/nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron， 2009， 64 （2）：173-187.

1. 　Chen X， Katakowski M， Li Y， et al. Human bone marrow stromal cell cultures conditioned by traumatic brain tissue extracts：growth factor production. J Neurosci Res， 2002， 69 （5）：687-691.
2. 　朱洪生，连锋，朱伟，等. 骨髓间质干细胞体外转化为心肌细胞的实验研究. 上海第二医科大学学报， 2002， 22 （5）：391-395．.
3. 　Makino S， Fukuda K， Miyoshi S， et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest， 1999， 103 （5）：697-705.
4. 　Rando OJ， Zhao K， Crabtree GR. Searching for a function for nuclear actin. Trends Cell Biol， 2000， 10 （3）：92-97.
5. 　Zhang Q， Skepper JN， Yang F， et al. Nesprins：a novel family of spectrin-repeat-containing proteins that localize to the nuclear membrane in multiple tissues. J Cell Sci， 2001， 114 （Pt 24）：4485-4498.
6. 　Zhang Q， Ragnauth CD， Skepper JN， et al. Nesprin-2 is a multi-isomeric protein that binds lamin and emerin at the nuclear envelope and forms a subcellular network in skeletal muscle. J Cell Sci， 2005， 118 （Pt 4）：673-687.
7. 　Lei K， Zhang X， Ding X， et al. SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc Natl Acad Sci U S A， 2009， 106 （25）：10207-10212.
8. 　Olins AL， Hoang TV， Zwerger M， et al. The LINC-less granulocyte nucleus. Eur J Cell Biol， 2009， 88 （4）：203-214.
9. 　Kobayashi Y， Katanosaka Y， Iwata Y， et al. Identification and characterization of GSRP-56， a novel Golgi-localized spectrin repeat-containing protein. Exp Cell Res， 2006， 312 （16）：3152-3164.
10. 　Gough LL， Beck KA. The spectrin family member Syne-1 functions in retrograde transport from Golgi to ER. Biochim Biophys Acta， 2004， 1693 （1）：29-36.
11. 　Apel ED， Lewis RM， Grady RM， et al. Syne-1， a dystrophin- and klarsicht-related protein associated with synaptic nuclei at the neuromuscular junction. J Biol Chem， 2000， 275 （41）：31986-31995.
12. 　Gruenbaum， Y GY， Meyuhas R， et al. The nuclear lamina and its functions in the nucleus. Int Rev Cytol， 2003， 226：1-62.
13. 　Zhang Q， Bethmann C， Worth NF， et al. Nesprin-1 and -2 are involved in the pathogenesis of Emery Dreifuss muscular dystrophy and are critical for nuclear envelope integrity. Hum Mol Genet， 2007， 16 （23）：2816-2833.
14. 　Puckelwartz MJ， Kessler EJ， Kim G， et al. Nesprin-1 mutations in human and murine cardiomyopathy. J Mol Cell Cardiol， 2010， 48（4）：600-608.
15. 　Farhana H， Mazzeo D， Patel JT， et al. Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes. J Biol Chem， 2010， 285 （5）：3487-3498.
16. 　Zhang X， Lei K， Yuan X， et al. SUN1/2 and Syne/nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice. Neuron， 2009， 64 （2）：173-187.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Experiment of Nesprin Protein Influence on Bone Marrow Mesenchymal Stem Cells

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