MicroRNA-203a-3p Affects Proliferation, Migration And Invasion Of Nasopharyngeal Carcinoma By Targeting VEGFC

miR-203a-3p was reported as a tumor suppressor and usually expressed in many malignancies,and its low expression is closely related to tumor biological characteristics.The purpose of this study was to investigate the effects of miR-203a-3p and its direct target gene VEGFC on the malignant biological behaviors and other related mechanisms of nasopharyngeal carcinoma cells,such as proliferation,migration,invasion,VM,EMT,and provide new targets for molecular diagnosis and treatment of nasopharyngeal carcinoma.This study was mainly divided into four parts:In the first part,the expression level of miR-203a-3p and VEGFC in 15 human NPC tissues,8 normal nasopharyngeal tissues and 2 cell lines 5-8F and C666-1was detected via real-time PCR and immunofluorescence.It was found that the level of miR-203a-3p was distinctly reduced in NPC cell lines,and the expression of VEGFC was significantly increased in NPC tissues and cells lines.In the second part,to explore the effect of miR-203a-3p on the malignant biological behavior of nasopharyngeal carcinoma cells,nasopharyngeal carcinoma cell lines 5-8F and C666-1 were transfected with miR-203a-3p mimic,miR-203a-3p inhibit and their respective negative control using Lipofectamine 2000 reagent.We examined the effect of miR-203a-3p on proliferation,invasion,migration,cell cycle,vascular mimicry and epithelial-mesenchymal transition of nasopharyngeal carcinoma cells by up-regulating or down-regulating the expression of miR-203a-3p,and nude mice subcutaneous tumors model were also constructed to study the effect of miR-203a-3p on the growth of nasopharyngeal carcinoma in vivo.The colony formation ability of nasopharyngeal carcinoma cells was detected by plate cloning assay;the cell cycle was detected by flow cytometry PI staining;the migration and invasion ability of nasopharyngeal carcinoma cells were detected by Transwell assay;vasculogenic mimicry formation assay(VM)was used to detect the ability of tube-formation of nasopharyngeal cell lines;the effect of miR-203a-3p on the growth of nasopharyngeal carcinoma in vivo was detected by establishing a subcutaneous tumor model in nude mice;the expression level of related proteins was detected by Western blot.The results showed that overexpression of miR-203a-3p can inhibit the proliferation of nasopharyngeal carcinoma cells and arrest the nasopharyngeal carcinoma cell cycle in G0/G1 phase,thereby inhibiting the growth of nasopharyngeal carcinoma cells.Meanwhile,miR-203a-3p can effectively inhibit the growth of nasopharyngeal carcinoma in vivo;miR-203a-3p can significantly inhibit the migration and invasion of nasopharyngeal carcinoma cells;while overexpression of miR-203a-3p can reduce the VM and EMT of nasopharyngeal carcinoma cells.In the third part,To explore the mechanism of miR-203a-3p to malignant biological inhibition of nasopharyngeal carcinoma cells,we examined the target gene of miR-203a-3p by searching the bioinformatics website,it was found that VEGFC can be used as a direct target gene for miR-203a-3p.The nasopharyngeal carcinoma cell lines 5-8F and C666-1 were transfected with miR-203a-3p mimic,it was found that the expression of VEGFC was significantly decreased,while transfected with miR-203a-3p inhibit,the expression of VEGFC was significantly increased,indicating the expression of the VEGFC gene is effectively inhibited miR-203a-3p.Luciferase reporter assay results showed that co-transfection of miR-203a-3p and GLA-VEGFC-3?UTR-wild plasmid significantly inhibited luciferase activity and decreased fluorescence intensity,indicating that VEGFC is a direct target of miR-203a-3p.The first part has confirmed that VEGFC is highly expressed in nasopharyngeal carcinoma tissues and cell lines,and may play a role as a tumor-promoting gene.In order to explore the mechanism of VEGFC gene in nasopharyngeal carcinoma,we further investigate the effects of VEGFC on the malignant biological characteristic of nasopharyngeal carcinoma cells by plate cloning assay,flow cytometry PI staining,Transwell assay,vasculogenic mimicry formation assay and Western blot.The results showed that the interference of VEGFC expression significantly inhibited the proliferation,VM and EMT of nasopharyngeal carcinoma cells,and had no significant effect on the migration and invasion of nasopharyngeal carcinoma cells.In the forth part,in order to further explore the regulation mechanism of miR-203a-3p/VEGFC of nasopharyngeal carcinoma,the expression of related proteins in PI3K/AKT signaling pathway was detected by Western blot.It was first verified that the expression of interfering VEGFC could down-regulate p-AKT and up-regulate AKT protein.Subsequently,it was found that overexpression of miR-203a-3p significantly down-regulated the protein levels of VEGFC and p-AKT,and up-regulated the expression of AKT protein;inhibited the expression of miR-203a-3p,up-regulated VEGFC,and down-regulated AKT and Up-regulated p-AKT protein,suggesting that miR-203a-3p can regulate the activity of PI3K/AKT signaling pathway by down-regulating the expression of VEGFC.Conclusion miR-203a-3p is low expressed in nasopharyngeal carcinoma and inhibits proliferation,migration,invasion,VM and EMT of nasopharyngeal carcinoma cells by directly binding to the 3?UTR region of VEGFC.miR-203a-3p regulates the activity of the PI3K/AKT signaling pathway by down-regulating the expression of VEGFC.