Lentiviral vector overexpressing FNDC5 inhibits proliferation and migration of smooth muscle cells

doi:10.12307/2021.298

Abstract

Abstract: BACKGROUND: Fibronectin type III domain containing 5 (FNDC5), mainly expressed in skeletal muscle and other muscle tissues, is a muscle factor that affects the proliferation and migration of various cells through binding different receptors.
OBJECTIVE: To construct the lentiviral vector overexpressing FNDC5, and to obtain the smooth muscle cell line stably transfected with FNDC5.
METHODS: The extracted C57BL/6L mouse VSMCs were identified by immunofluorescence. FNDC5 gene was amplified by PCR and was then inserted to pLVX-mCMV-ZsGreen-IRES-Puro lentiviral vector. The recombinant vector was amplified in JM109 competent cells, and after sequencing and identification, the vectors were transfected into 293T cells to produce overexpression lentiviral particles. Virus titer was then measured. The recombinant lentivirus that was transfected into smooth muscle cell line, and a fluorescence microscope was used to calculate transfection efficiency. After a stably transfected cell line with FNDC5 overexpression was screened out by puromycin, the cells were divided into blank group, empty group, and FNDC5 group. RT-PCR and western blot were used to measure the mRNA and protein expression of FNDC5, and the proliferation and migration ability of smooth muscle cells were detected by cell counting kit-8 and cell scratch test.
RESULTS AND CONCLUSION: A lentiviral vector with FNDC5 overexpression was successfully constructed, and a vascular smooth muscle cell line stably transfected with FNDC5 was established. The transfection efficiency of smooth muscle cells was 86%. Compared with the blank group and empty group, the FNDC5 overexpression group had a significant increase in mRNA and the protein expression of FNDC5 (P < 0.001). Compared with blank group and empty group, the cell proliferation and migration rate of FNDC5 overexpression group decreased significantly (P < 0.05). To conclude, a vascular smooth muscle cell line stably transfected with FNDC5 is established by the lentiviral vector with FNDC5 overexpression, indicating that FNDC5 can significantly inhibit the proliferation and migration of thoracic aortic smooth muscle cells.

Key words: fibronectin type III domain containing 5, FNDC5, lentivirus vector, transfection, vascular smooth muscle cells, cell proliferation, cell migration

CLC Number:

Wang Xiang, Wei Chaojun, Wang Yao, Pan Yujia, Liu Danan. Lentiviral vector overexpressing FNDC5 inhibits proliferation and migration of smooth muscle cells[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(35): 5670-5675.

Figures/Tables 6

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