The Role Of Exosomal MiR-3529-3p From Cancer-Associated Fibroblasts In Enhancing The Malignancy Of Oral Squamous Cell Carcinoma

Objective:Cancer-associated fibroblasts(CAFs)are important stromal cells in the tumor microenvironment.Compared with normal fibroblasts,CAFs are in a functionally polarized state and are one of the key factors affecting tumor progression and therapeutic efficacy.The project team previously found that platelet derived growth factor(PDGF-BB)secreted by oral squamous cancer cells can activate human oral mucosal fibroblast cells(hOMF)into CAFs,which play a role in promoting tumor progression with unknown mechanism.Studies have shown that exosomes in the tumor microenvironment mediate the exchange of substances and information in the cells of tumor tissue and are one of the important cell-cell interactions.Exosome vesicles can deliver the beneficial regulators for tumor progression to the microenvironment,thereby promoting tumor occurrence and development.It remains to be elucidated whether the exosomes released from PDGF-BB induced CAFs can effectively promote the malignancy of oral squamous cell carcinoma(OSCC),what are the key molecules,and what are their functions and mechanisms.This study aims to explore the role and mechanism of PDGF-BB-activated CAFs in promoting the malignancy of oral squamous cell carcinoma from the perspective of exosomes,which will provide new ideas and methods for the treatment of OSCC.Methods:1.The cell lines of Human tongue squamous cell carcinoma of Cal-27,HSC-4,and SCC-6,and hOMF were routinely cultured.Cal-27 cells were cocultured with hOMFs in Transwell.CAFs were obtained by inducing hOMF with 30 ng/ml PDGFBB.Fibroblast exosomes were extracted using the exoEasy Maxi Kit.2.To clarify the effect of fibroblast exosomes on the biological characteristics of tongue squamous cell carcinoma cells,the extracted CAFs-derived exosomes(CAFsExo)or hOMF-derived exosomes(hOMF-Exo)were cocultured with tongue squamous cell carcinoma cells.The proliferation was detected by CCK8,the apoptosis was detected by Annexin-V FITC/PI,the migration was detected by wound healing assay,and the invasion ability of cancer cells was detected by Transwell assay.3.Cal-27 cells,with the most obvious reaction to exosomes,were selected for further verification.In the Transwell co-culture model,whether CAFs-Exo is effective on Cal-27 was verified by CCK8,Annexin-V FITC/PI,and wound healing assay after the inhibition of exosome secretion.4.Nude mice were transplanted subcutaneously with the tumor cells after they were treated with fibroblast exosomes,with further intermittently injecting exosomes around the tumors,and the volume and weight of tumors were measured.The protein expression in tumor tissue was detected by immunohistochemistry.5.Differentially expressed miRs in the exosomes of CAFs and hOMF were screened by high-throughput sequencing;and mRNA expression in Cal-27 cells were analyzed after exosome stimulation by high-throughput sequencing.The differential miR expression was verified in clinical samples of OSCC cancer tissues and adjacent tissues,fibroblasts,and fibroblast exosomes by qRT-PCR.6.GO analysis and KEGG biological pathway enrichment of differentially expressed molecules were analyzed by multiMiR package in R language software and DAVID online database.The interaction network of the differential genes was constructed using String and visualized by Cytoscape.MiR-3529-3p,which were differentially expressed in the exosomes of CAFs and hOMF,were selected for further study.The target genes of miR-3529-3p were predicted using TargetScan Human database and the miRDB database,and HERC5 was selected as the target gene.The substrates of HERC5 were predicted using UbiBrowser database.7.The Cal-27 cells with overexpression/knockdown of miR-3529-3p were constructed by transfection.The proliferation was detected by CCK8,the apoptosis was detected by Annexin-V FITC/PI,the migration was detected by wound healing assay,and the invasion ability of cancer cells was detected by Transwell assay.The effect of miR-3529-3p on Cal-27 cells and its involved signal pathways were clarified by examining the expression of related proteins using western blotting.The interaction and inhibition of miR-3529-3p on the target gene HERC5 was further verified by dualluciferase assay,and the effect of HERC5 on MAPK signaling pathway was verified by western blotting.8.The clinical data and paraffin tissues of OSCC patients were retrospectively collected,and the expression of HERC5 was detected by immunohistochemistry.The relevant data was downloaded from the database of TCGA,GEO,UCSC,CPTAC,and CancerMIRNome.The expression of HERC5,MAPK signaling pathway-related proteins,and miR-3529-3p was analyzed in cancer tissues and adjacent tissues and their relationship with the prognosis was also analyzed.9.Statistical analysis and graphing were performed by Graphpad Prism and R software.The results of wound healing assay and immunohistochemistry were quantitatively analyzed by Image J software;For continuous variables,independent ttest was performed when there were only two groups of samples,and one-way analysis of variance was performed when there were three or more groups of samples.After the data was in statistically significance,LSD-t test or Dunnett-t was performed for pairwise comparison;chi-square test or Fisher’s exact probability were analyzed for categorical variables.Diagnostic value was measured by AUC value,and prognostic value was calculated using time-dependent ROC.The correlation between continuous variables was analyzed using the correlation coefficient r.The prognosis was analyzed using Kaplan Meier plotter.The analysis was statistically significant when the twosided α=0.05.Results:1.The results of directly stimulating Cal-27 cells with exosomes showed that,CAFs-Exo significantly promoted the proliferation,migration,and invasion of Cal-27 cells compared with hOMF-Exo.Similar results were obtained by adding exosome inhibitor in the co-culture of hOMF and Cal-27;the differences were statistically significant.The results of H2O2 or cisplatin-induced apoptosis model showed that CAFs-Exo could significantly improve the anti-apoptotic ability compared with hOMFExo.2.Three significantly elevated miRs in CAFs-Exo,miR-3074-5P,miR-374c-3p,and miR-3529-3p were screened out by analyzing high-throughput sequencing data using self-programmed R language software,and were further verified in CAFs-Exo and hOMF-Exo,exosome-stimulated Cal-27,hOMF and CAFs,as well as clinical tissues and exosomes;it was found that the expression of miR-3529-3p was highly different with statistical significance.3.Using a variety of bioinformatics analysis,differential miR and mRNA target genes was found to be enriched in MAPK pathway,and multiple MAPK genes were strongly correlated with miR-3529-3p.HERC5 is the key target gene of miR-3529-3p.There are multiple HERC5 substrates,all of which are upstream factors of the MAPK pathway.It suggested that miR-3529-3p may regulate the MAPK signaling pathway by targeting HERC5,thereby exerting the cancer-promoting function of CAFs.4.The results of the dual luciferase assay showed that miR-3529-3p targeted the 3terminal untranslated region of HERC5 mRNA and inhibit its expression;by overexpressing and knocking down miR-3529-3p,it was found that compared with the NC group,the miR-3529-3p promoted the ability of proliferation,migration,and invasion in Cal-27 cells,and improved their anti-apoptotic ability;the differences were statistically significant.5.The expression of HERC5 was detected after directly stimulating Cal-27 cells with exosomes.Compared with hOMF-Exo,CAFs-Exo reduced the expression of HERC5 and activated the MAPK/Erk signaling pathway.6.By overexpressing and knocking down miR-3529-3p,it was found that compared with the NC group,the miR-3529-3p significantly reduced the expression of HERC5,and the MAPK/ERK signaling pathway was activated;the differences were statistically significant.7.The transfection of HERC5 plasmid into HCT116 cells showed that the MAPK/Erk and MAPK/p-3 8 pathways were significantly inhibited after HERC5 overexpression compared with the control groups.8.The immunohistochemical results of tumor tissue in nude mice showed that compared with the control group,the expression of p-Erk in the CAFs-exo group was up-regulated with statistical significance,while there was no significant difference in the expression of HERC5.Immunohistochemical analysis of 68 cancer tissues and adjacent tissues showed that the expression of HERC5 in human oral squamous cell carcinoma tissues was significantly higher than that in adjacent tissues,and HERC5 had a better diagnostic value(AUC=0.76).9.By prognosis analysis of the 325 patients with oral squamous cell carcinoma,it was found that patients with low HERC5 expression had worse overall survival(HR=1.37,95%CI:0.99-1.91,P=0.060)and progression-free survival(HR=1.62,95%,CI:1.07-2.45,P=0.021).Both transcriptome and protein analysis results show that HERC5 had good diagnostic value,with the minimum AUC value above 0.75,and the highest AUC value above 0.90.Conclusions:The exosomes secreted from CAFs transformed from hOMF by activation of PDGF-BB can significantly promote the proliferation,migration,and invasion of oral squamous cell carcinoma cells and significantly improve their anti-apoptotic ability.MiR-3529-3p is the key molecule enriched in CAFs-Exo exerting tumor-promoting effects,which promotes the malignancy of OSCC by inhibiting the expression of HERC5 to activate the MAPK/Erk signaling pathway.