| ObjectHead and neck tumors are a common group of cancer diseases whose incidence is increasing year by year.Among the many pathological types of head and neck tumors,head and neck squamous cell carcinoma(HNSCC)has the highest incidence and mortality rate.It is the combination of human living environment and family genetic factors that lead to the extremely rapid development of HNSCC.HPV infection,smoking and alcohol abuse are all risk factors for the disease.Local infiltration,lymph node metastasis and distant metastasis are the main causes of poor prognosis in HNSCC.Heterotrimeric G proteins are capable of transmitting various signals between cells,and this transmission process is regulated by G protein-coupled receptors(GPCRs).Among the different types of G proteins,guanine nucleotide-binding protein alpha subunit 13(GNA13)is most closely related to tumor progression and is involved in the development of a variety of tumors,but the expression of GNA13 in HNSCC and its effect on the biological behavior of HNSCC are unclear.One of the important ways that the exosomes secreted by living cells can be transmitted in the human media is to fuse with the target cells and release various substances inside the target cells,so that the target cells can be activated to different degrees and finally complete a series of signaling processes,becoming an important medium for cell-to-cell information transmission.The aim of this study was to explore whether GNA13 can influence the alteration of tumor microenvironmental components through exosomes and thus promote the development of HNSCC,and to make a preliminary investigation of its molecular mechanism.Materials and MethodsFirstly,the microarray data related to head and neck tumors in the GEO database were collected and the differentially expressed gene GNA13 was screened using R software.The expression levels of GNA13 in common human tumors were analyzed by using data from the Cancer Genome Atlas database(TCGA),followed by analysis of predicted GNA13 expression levels in head and neck tumors and normal head and neck tissues.Then HNSCC tissues and their paired normal paraneoplastic tissues from clinical patients were collected and the expression of GNA13 in HNSCC and normal tissues was detected using Western blotting.HNSCC Fadu cells and human normal squamous epithelial NOK cells were cultured in vitro,and the expression of GNA13 in both cells was detected using Western blotting and qRT-PCR.The expression of GNA13 in HNSCC cells was down-regulated using plasmid transfection and divided into negative control group(si-NC)and down-regulated GNA13 group(si-GNA13),and the expression of GNA13 in the cells was detected by qRT-PCR.Clone formation and CCK-8 proliferation assays examined the effect of down-regulation of GNA13 on the proliferation ability of Fadu cells.Transwell assay and cell scratch assay were performed to detect the effect of down-regulation of GNA13 on the migration and invasion ability of Fadu cells.The treated cellular exosomes were separated and purified using ultracentrifugation,and the morphology of the extracted exosomes was observed and photographed under electron microscope,and the extracted exosomes were analyzed by particle size analysis system,and finally the purified exosomes were subjected to Western blotting assay and tracer assay.After the purified exosomes and human normal fibroblasts were cultured by co-culture technique,the expression of MAFs marker proteins α-SMA and Vimentin was detected using Western blotting.Changes in fibroblast proliferation capacity were then detected by clone formation and CCK-8 proliferation assays,and changes in fibroblast migration capacity were detected by Transwell assays.Target miRNAs were predicted and screened by bioinformatic analysis software.qRT-PCR was performed to detect differentially expressed miR-26a-5p in fibroblasts after co-culture with exosomes.The downstream target genes of miR-26a-5p were predicted by online software such as Targetscan database,while the interaction of miR-26a-5p with STRADB was further validated using dual luciferase reporter gene assay and Western blotting.After overexpression of STRADB in fibroblasts,clone formation and CCK-8 proliferation assays detected changes in fibroblast proliferation ability.Transwell migration assay detected changes in fibroblast migration ability.Western blotting was performed to detect the expression of MAFs marker proteinsα-SMA and Vimentin in fibroblasts.Fadu cells were cultured using conditioned medium of fibroblasts overexpressing STRADB,and changes in the proliferation ability of Fadu cells were detected by clone formation and CCK-8 proliferation assays,and changes in the migration and invasion ability of Fadu cells were detected by Transwell assays.Results1.Expression prediction of GNA13 in HNSCC.Ten differentially expressed genes including GNA13 were obtained by collecting microarray data related to HNSCC from the GEO database and filtering and analyzing the data using R language.The results of predictive analysis based on TCGA bioinformatics online database showed that the expression level of GNA13 was significantly higher in head and neck tumor tissues relative to normal tissues.And the expression of GNA13 was significantly higher in HNSCC tissues compared to normal tissues.Meanwhile,GNA13 was significantly correlated with clinicopathological features such as HNSCC stage,grade and lymph node metastasis.The effect of GNA13 expression on the prognosis of HNSCC patients was analyzed by the survival analysis website,and the results of survival analysis showed that GNA13 was significantly correlated with the survival time of HNSCC patients.The results of Western blotting experiments showed that the protein expression level of GNA13 was significantly higher in HNSCC tissues relative to normal tissues adjacent to the carcinoma.2.Down-regulation of GNA13 inhibits proliferation,migration and invasion of HNSCC cells.Endogenous GNA13 was downregulated in Fadu cells by small interfering RNA(si-RNA),and the results of qRT-PCR experiments showed that the expression of GNA13 mRNA was decreased in the si-GNA13 group compared with the si-NC group.Clone formation experiments showed that the cell proliferation ability was significantly reduced in the si-GNA13 group compared to the si-NC group.CCK-8 assay showed that cell viability was significantly reduced in the si-GNA13 group compared to the si-NC group.The results of Transwell assay showed that cell migration and invasion ability were significantly decreased in the si-GNA13 group compared with the si-NC group.The results of Wound healing experiment showed that the migration distance and migration rate of si-GNA13 group were lower than those of si-NC group,and si-GNA13 group significantly inhibited the healing of Fadu cells.3.Isolation and identification of HNSCC cell-derived exosomes.TEM results showed that two groups of exosomes secreted by Fadu cells,namely the si-NC Exo group and the si-GNA13 Exo group,both had typical oval-shaped vesicles.Nanoparticle tracking analysis(NTA)to determine their size distribution showed that the majority of exosomes were concentrated in the 50-150 nm diameter range.The results of the protein assay by BCA method showed that the exosomal protein content was 0.31μg/μl in the si-NC Exo group and 0.58 μg/μl in the si-GNA13 Exo group.Western blotting showed that exosomes in the si-NC Exo group and si-GNA13 Exo group were able to express the exosome marker proteins CD9,CD63 and CD81.4.GNA13 induces fibroblast transformation through exosomes and promotes their proliferation and migration.Exosomes and fibroblasts were co-cultured in vitro for 24 hours.Confocal microscopic observation showed that exosomes in the si-NC Exo group and si-GNA13 Exo group had been effectively internalized into fibroblasts.The results of Western blotting experiments showed that the protein expression level of GNA13 was significantly reduced in the si-GNA13 Exo group compared with the si-NC Exo group.The protein expression levels of α-SMA and Vimentin were significantly reduced in the si-GNA13 Exo group compared to the si-NC Exo group.When the exosome GW4869 inhibitor was added,the protein expression levels of GNA13 were significantly reduced in the si-NC Exo+GW4869 group compared to the si-NC Exo group.The protein expression levels of α-SMA and Vimentin were significantly reduced in the si-NC Exo+GW4869 group compared to the si-NC Exo group.Clone formation experiments showed that the proliferation ability of cells in the si-GNA13 Exo group was significantly reduced compared to the si-NC Exo group.When the exosomal GW4869 inhibitor was added,the cell proliferation ability was significantly reduced in the si-NC Exo+GW4869 group compared to the si-NC Exo group.CCK-8 assay showed that cell viability was significantly reduced in the si-GNA13 Exo group compared to the si-NC Exo group.When the exosome GW4869 inhibitor was added,cell viability was significantly reduced in the si-NC Exo+GW4869 group compared to the si-NC Exo group.Transwell assays showed that cell migration ability was significantly reduced in the si-GNA13 Exo group compared to the si-NC Exo group.When the exosome GW4869 inhibitor was added,the cell migration ability was significantly reduced in the si-NC Exo+GW4869 group compared to the si-NC Exo group.5.GNA13 regulates the expression of miR-26a-5p in fibroblasts via exosomes.The possible miRNAs regulated by GNA13 were predicted by three online website databases of different predicted miRNAs.GNA13 was overexpressed in fibroblasts,and Western blotting experiments were performed to verify the efficiency of GNA13 overexpression.The differentially expressed miRNAs were verified by qRT-PCR,and miR-26a-5p was selected as the target miRNA.after co-culture of exosomes with fibroblasts,the results of qRT-PCR experiments showed that the mRNA expression level of miR-26a-5p was significantly down-regulated in the si-NC Exo group compared to the control group,while the mRNA expression level was significantly up-regulated in the si-NC Exo+GW4869 group,the mRNA expression level was significantly up-regulated.6.STRADB is the target of miR-26a-5p.Prediction analysis of possible downstream target genes regulated by miR-26a-5p was performed by three different miRNA target gene prediction sites.The results of the dual luciferase reporter gene experiment showed that the predicted target gene STRADB mRNA 3’-UTR contains a highly conserved miR-26a-5p binding site,while the results of this experiment indicated that miR-26a-5p attenuated the luciferase activity of STRADB-Wt,but had no effect on the luciferase activity of STRADB-Mut.Inhibition of miR-26a-5p expression in fibroblasts was followed by validation by qRT-PCR.The differential expression of the target gene STRADB was then verified by Western blotting.After co-culture of exosomes with fibroblasts,the results of Western blotting experiments showed that the expression level of STRADB mRNA was significantly reduced in fibroblasts from the si-GNA13 Exo group compared with fibroblasts from the si-NC Exo group.When the exosome inhibitor GW4869 was added,the expression level of STRADB mRNA was significantly reduced in fibroblasts from the si-NC Exo+GW4869 group compared with fibroblasts from the si-NC Exo group.7.Overexpression of STRADB induces fibroblast transformation and promotes proliferation,migration and invasion of HNSCC cells.The results of qRT-PCR in fibroblasts transfected with plasmids overexpressing STRADB showed that the mRNA expression level of STRADB was significantly higher in the ov-STRADB group compared with the ov-NC group.Western blotting results showed that the expression of Vimentin andα-SMA protein was significantly higher in the ov-STRADB group compared to the ov-NC group.The results of CCK-8 assay showed that fibroblasts in the ov-STRADB group had significantly higher proliferation capacity compared to those in the ov-NC group.the results of Transwell assay showed that cells in the ov-STRADB group had significantly higher migration capacity compared to those in the ov-NC group.HNSCC cells were cultured with conditioned medium of fibroblasts overexpressing STRADB.The results of clone formation and CCK-8 assay showed that the proliferation ability and cell viability of Fadu cells(si-NC/si-GNA13)in the ov-STRADB group were notably higher than those in the control group.The migration and invasion abilities of Fadu cells(si-NC/si-GNA13)were significantly higher in the ov-STRADB group compared with the control group.ConclusionGNA13 regulates the miR-26a-5p/STRADB pathway in fibroblasts through exosomes,induces fibroblast transformation in the tumor microenvironment and promotes their proliferation and migration,creating a microenvironment conducive to metastasis for tumor cells and ultimately promoting the proliferation,migration and invasion of HNSCC cells.Exploring the role and function of GNA13 as well as tumor-derived exosomes is essential for studying the tumor microenvironment.The present study provides a reliable basis for exploring the molecular mechanisms by which GNA13 promotes the proliferation and migration of HNSCC cells,and provides a new direction for the treatment of HNSCC patients who develop tumor metastasis at advanced clinical stage. |
