Study of Recombinant Pasteurella multocida Toxin-mediated FGFRL1-induced Proliferation in Hela Cells
LIN Zhong-Sen, ZHAO Qin, YUAN Jian-Lin, WU Rui, YAN Qi-Gui, WEN Yi-Ping, HUANG Xiao-Bo, DU Sen-Yan, CAO San-Jie*
College of Veterinary Medicine/Research Center for Swine Diseases/National Demonstration Center for Experimental Animal Education/ Sichuan Science-Observation Experimental Station of Veterinary Drugs and Veterinary Diagnostic Technique, Ministry of Agriculture and Rural Affairs, Sichuan Agricultural University, Chengdu 611130, China
Abstract：Fibroblast growth factor receptor-like 1 (FGFRL1), as a member of the fibroblast growth factor receptor family, plays a role in the regulation of cell proliferation and differentiation. Pasteurella multocida toxin (PMT) is an important virulence factor produced by P. multocida (Pm), which belongs to mitogenic protein, and causes cell proliferation on a variety of cells, leading to tissue proliferation, occupancy, and causing impair growth and death of other normal tissue cells. However, the mechanism and the factor that PMT induce a pro-proliferative effect, are currently unclear. Previous CRISPR/Cas9 whole gene library screen revealed that FGFRL1 increased the survival rate of recombinant PMT protein (rPMT) treated Hela cells. In order to investigate whether PMT affects the proliferation of Hela cells through FGFRL1 in the PI3K-AKT pathway, in the present study, normal and FGFRL1-knockout Hela cells were treated with rPMT at 0, 0.01, 0.1, and 1.0 μg/ml for 48 h. The cell proliferation rate was detected by Cell Counting Kit-8 (CCK-8) assay, cell DNA replication by EdU-488 labeling assay, and the cell migration rate by scratch assay, The results showed that cell proliferation in the FGFRL1 knockout Hela cell was significantly decreased (P<0.05) when treated with rPMT at a concentration of 0.1 μg/mL for 48 h, while the rate of scratch recovery was decreased by nearly 70% compared with that of normal Hela cell (P<0.05). It indicated that the absence of FGFRL1 diminished the pro-proliferative effect of rPMT on Hela cell. Initial validation of the pathway was performed using qPCR to detect changes in the transcriptional levels of factors upstream of the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K-AKT) pathway in Hela cells after rPMT treatment, and phosphorylation levels of AKT, a key activator of the pathway, were detected by western blot.. The results showed that the upstream factors of PI3K-AKT pathway were extremely significantly elevated after rPMT treatment (P<0.05), and the levels of AKT and phosphorylated AKT in the FGFRL1 knockout Hela cell were significantly lower than those in the normal Hela cells (P<0.01), which indicated that the activation of the PI3K-AKT pathway was inhibited. Thus, it could be preliminarily confirmed that PMT might cause Hela cell proliferation by targeting the PI3K-AKT pathway through FGFRL1. This study enriches the theoretical basis of the PMT's proliferation-promoting effect, preliminarily explores the pathway activation mode of PMT-induced cell proliferation, and provides information for further revealing the PMT's proliferation-promoting effect.
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