A Novel C7 Peptide Inhibits Cervical Cancer Cell Metastasis By Blocking HGF/c-Met Pathway

Research background:Cervical cancer is one of the most commonly diagnosed malignant tumors in women.According to the GLOBOCAN statistics of 2018,the incidence and mortality of cervical cancer are at the forefront.Early treatment options include surgery,radiation,and chemotherapy.However,when advanced cancer metastasis,the patient's 5-year survival rate is very low.It is therefore desirable to inhibit tumor metastasis and improve cervical cancer treatment and prognosis.Studies have shown that c-Met(c-mesenchymal-epithelial transition)is closely related to the invasion and metastasis of various tumors.Almost all tumor tissues have high expression of c-Met receptors,and Met receptors are up-regulated in clinical cervical cancer tissues.The c-Met receptor is a heterodimeric protein encoded by the c-Met proto-oncogene,which consists of a disulfide bond from the a subunit of the extracellular domain and the subunit of the B transmembrane,and is structurally a type II tyrosine protein kinase receptor.The only effective stimulatory molecule for this receptor is the scatter factor,also known as hepatocyte growth factor(HGF).When HGF binds to c-Met,the receptor protein dimerizes,activates the intracellular kinase domain,mediates downstream PI3K/Akt and MAPK/erk signaling via the signaling adaptor protein Gab 1,and regulates tumor cell survival,proliferation,and migration.Mutant or aberrantly activated HGF/c-Met signals often result in the development of human tumors.The c-Met receptor is thus recognized as a target for anti-tumor,and it is desirable to study a targeted drug against the c-Met receptor.In the early stage,the c-Met extracellular domain protein was used as the target protein,and the phage displayed in the loop 7 peptide library was screened by phage display technology.After screening,the target phage was obtained.According to the results of its sequence determination,Shanghai Qiangyao Biotechnology Co.,Ltd.synthesized and purified the small peptide by mass spectrometry and high performance liquid chromatography(HPLC)according to the amino acid sequence provided,and named it as C7 peptide.This article focuses on the mechanism of action of C7 peptide on tumor cells in cervical cancer.Research Objective:To investigate whether the C7 peptide can down-regulate the malignant biological effects of cervical cancer cells by blocking the HGF/c-Met pathway.If it is possible,its mechanism of action is further studied.Research content:1.Firstly,the cervical cancer He La cell line was selected to establish a scatter model to explore the scatter effects of different concentrations of HGF on cervical cancer cells,in order to find out the optimal concentration of HGF and the optimal time of C7.2.The epidermal growth factor EGF was used as a control group to investigate the targeting of the C7 peptide.The experiment was divided into the following six groups:NC(negative control),C7,HGF,HGF+C7,EGF,EGF+C7.The malignant biological effects were detected by scatter experiments,migration experiments,and invasion experiments.3.In the cervical cancer He La and Si Ha cells,the protein phosphorylation level of the downstream signaling pathway was detected by Western Blot,and the molecular mechanism of the action of the C7 peptide was explored.4.The competitive inhibition of C7 peptide and HGF was detected by Western Blot.The ability of the two to compete with each other for binding to the c-Met receptor was examined by maintaining the concentration of one of the C7 peptide and HGF unchanged and gradually increasing the other concentration.5.In vivo experiments,a nude mouse lung metastasis model was established to detect the volume,weight,and metastatic nodule size of the metastatic tumor,and to make tumor sections.Research result:1.He La cells were treated with different concentrations of HGF,and the scatter effects were concentration dependent.Next,we blocked the cell surface c-Met receptor with the C7 peptide for at least 10 min to inhibit HGF-mediated cell scatter.As time goes on,the inhibition effect is more obvious.2.The C7 peptide inhibits HGF-mediated He La and Si Ha cell migration and invasion.3.The C7 peptide can inhibit HGF-mediated phosphorylation of downstream signaling molecules in He La and Si Ha cells.4.In the experiment of exploring the competing inhibition of C7 peptide and HGF,while maintaining the concentration of C7 peptide and increasing the concentration of HGF,it can be seen that the inhibitory effect of C7 peptide is gradually weakened.While the concentration of stable HGF was unchanged and the concentration of C7 peptide was gradually increased,it can be seen that the inhibition of C7 peptide was dose-dependent.5.The C7 peptide can inhibit lung metastasis of cervical cancer cells in nude mice.Conclusion:In cervical cancer cells,the C7 peptide can inhibit the malignant biological effects of cervical cancer by targeting the HGF/c-Met signaling pathway.The C7 peptide has a competing inhibitory effect with HGF.The C7 peptide can inhibit the metastasis of cervical cancer cells in nude mice.