Research About Calreticulin-induced EMT Related Signal Pathway In Nasopharyngeal Carcinoma CNE2 Cells

Objective: We have found that Calreticulin(CRT)is highly expressed in nasopharyngeal carcinoma and promotes its invasion and metastasis by inducing epithelial-mesenchymal transition(EMT)in nasopharyngeal carcinoma CNE2 cells.This article aims to investigate the CRT-induced EMT-related signaling pathways in nasopharyngeal carcinoma CNE2 cells and the molecular mechanisms of promoting migration and invasion.Methods: Using bio-informatical software Ensembl online to predict the promoter region of NRP1,Jaspar was used to predict transcription factor binding sites and analysis the most likely target transcription factor of NRP1 in TGF-?/Smads signaling pathway.We transfected CNE2 cells with Si-CRT(si-NControl as a negative control),Western blot was used to detect the expression of SMAD3 and its phosphorylation and NRP1 in cells with low expression of CRT.Petreated CNE2 with SIS3,a specific phosphorylation inhibitor of SMAD3,detected the CRT and NRP1 expression after Inhibiting SMAD3 protein phosphorylation by Western blot.Detected the expression of EMT-related proteins E-cadherin and Vimentin with low expression of CRT and p-SMAD3 by Western blot.Impact of knockdowning CRT expression or inhibiting SMAD3 phosphorylation on migration and invasion of nasopharyngeal carcinoma CNE2 cells was verified by wound healing assay and Transwell assay.Rusults:(1)Ensembl obtained a base sequence of 2000 bp upstream of the NRP1 gene transcriptional starting sites as a promoter region of NRP1.Analysis by Jaspar software found that there are five binding sites for SMAD3 in the NRP1 promoter region in the TGF-?/Smads signaling pathway.(2)After knocking down the expression of CRT,NRP1 and P-SMAD3 in nasopharyngeal carcinoma CNE2 cells was significantly decreased(P=0.000),while the SMAD3 expression has no difference(P=0.240).(3)Using SIS3 to inhibite the phosphorylation of SMAD3,with down-regulation of NRP1 protein expression in nasopharyngeal carcinoma CNE2 cells(P=0.020),there was no significant difference in CRT expression(P=0.140).(4)After Knocking down the expression of CRT or inhibitting the phosphorylation of SMAD3,significantly decreased the expression of Vimentin(P<0.01),but increased the expression of E-cadherin(P<0.01).(5)Wound healing assay verified that the migration distance of nasopharyngeal carcinoma CNE2 cells after knocking down CRT expression was(13.42±3.15)?m,and the migration distance of CNE2 cells after inhibiting SMAD3 phosphorylation was(18.01±2.82)?m.There was no significant difference between the two groups(P>0.05),but significantly lower than the negative control group(60.41±4.00)?m(P<0.05).(6)Transwell migration assay verified that there were 74.03±1.00 migration cells in each high-power field after knocking down CRT expression,69.02±3.68 migration cells in each high-power field after inhibiting SMAD3 phosphorylation in CNR2 cells,There was no significant difference between the two groups(P>0.05),but significantly lower than the negative control group 124.12±5.80 migration cells in each high-power field(P<0.05).(7)Transwell invasion assay verified that there were 63.11±6.45 invasion cells in each high-power field after knocking down CRT expression,54.65±6.20 invasion cells in each high-power field after inhibiting SMAD3 phosphorylation in CNR2 cells,There was no significant difference between the two groups(P>0.05),but significantly lower than the negative control group 96.62±5.68 invasion cells in each high-power field(P<0.05).Conclusion:(1)CRT regulates the expression of NRP1 by affecting Smad3 phosphorylation in the TGF-? signaling pathway.(2)Calreticulin promotes migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing cell EMT via Smad3/NRP1 pathway.