| Research BackgroundOral squamous cell carcinoma(OSCC)is the most common oral malignant tumor with high incidence and easy to metastasize distantly.OSCC has poor overall survival rate and poor prognosis,which severely affects the quality of patients’s life.Although there are numerous treatment options available such as surgery,radiotherapy,chemotherapy,immunotherapy,nutritional therapy,cryotherapy,thermotherapy,Chinese medicine therapy,etc.The treatment of oral squamous cell carcinoma is still a major challenge in clinical practice.Moreover,previous studies have mainly focused on the relationship between genetic changes and the development of oral squamous cell carcinoma.With further study,the discovery of the critical importance of tumor microenvironment in the development and progression of oral squamous cell carcinoma may provide new ideas for the candidate diagnostic biomarkers and promising therapeutic targets for oral squamous cell carcinoma.Exosomes is one of the important components of tumor microenvironment,and many cells can secrete extracellular vesicles under normal and pathological conditions,which is an important mediator of intercellular communication.The exosomes secreted by tumor cells can carry a variety of genetic materials of tumor cells,such as microRNAs,DNA,proteins,etc.Among them,microRNAs in exosomes play an crucial role in regulating tumor growth,metastasis,angiogenesis,immune escape,tumor drug resistance.etc.Therefore,the study of factors that act as anticancer agents by regulating the exosomal composition and secretion of tumor cells is a hot topic of current research.Metformin,a drug commonly used in the treatment of diabetes,has been found to have a wide range of anticancer effects,and recent studies have shown that metformin can affect the growth of tumor cells by influencing the synthesis and secretion of exosomes in tumor cells.However,recent studies have found that another biguanide hypoglycemic agent,phenformin,has significantly better anticancer effects than metformin,and phenformin was also found to inhibit angiogenesis through the tumor microenvironment,although the exact mechanism needs to be further studied.Since previous studies have been focused on the direct role of phenformin in cancer cells,few studies on the effect of phenformin in tumor microenvironment,and the association of phenformin with Oral squamous cell carcinoma has not been studied,so the present study aims to investigate whether phenformin can affect tumor microenvironment through regulating exosome secretion of oral squamous cell carcinoma cells to affect angiogenesis of endothelial cells and to study the potential underlying molecular mechanisms.Aims(1)To investigate the role of phenformin,relative to metformin,in inhibiting the growth of OSCC cells.(2)To investigate the effects of exosomes secreted by OSCC cells stimulated by phenformin on the proliferation and migration of human umbilical vein endothelial cells(HUVECs)and in vitro angiogenesis,and to verify them by in vivo experiments.(3)To elucidate the molecular mechanisms by which the exosomes secreted by OSCC cells stimulated by phenformin affect the growth of vascular endothelial cells..Methods(1)The effects of phenformin and metformin in the proliferation of OSCC cells were measured by Cell Counting Kit-8 assay and EdU-488 Cell Proliferation Assay Kit,respectively.(2)Exosomal morphology was observed by transmission electron microscopy,exosomal diameter was analyzed by dynamic particle size analysis,exosomes were identified by Western Blot detection of their surface markers,and the exosomal uptake assay by human umbilical vein vascular endothelial cells(HUVECs)was performed by PKH67 dye labeling of exosomes to detect whether exosomes could enter into the cells.(3)OSCC cells were treated with physiological saline(PBS)and phenformin(Phen)respectively,after 48h of treatment,the above two conditioned media were collected,and exosomes were extracted from the above two conditioned media using the Exosome Extraction Kit,namely PBS-Exo groups and Phen-Exo groups.Vascular endothelial cells(HUVECs)were treated with the two groups of exosomes respectively,and then their effects on HUVECs proliferation were detected by Cell Counting Kit assay and HUVECs migration by both Transwell assay and cell scratch assay.The effect of exosomes,secreted by OSCC cells stimulated by phenformin,on angiogenesis was then analyzed in vitro by tube formation assay.(4)In vivo effects on angiogenesis of exosomes secreted by OSCC cells stimulated by phenformin were analyzed by subcutaneous angiogenesis in nude mice transplanted with HUVECs and vascularization of chick embryo chorioallantoic membrane(5)Potential key microRNA affecting angiogenesis were screened and identified by analysis of microRNA sequencing data in exosomes and validated by quantitative real-time PCR(qRT-PCR).(6)The effect of high expression and low expression of the key microRNA in HUVECs on angiogenesis was examined by in vitro tube formation assays.And the corresponding effects on downstream target genes were examined by quantitative real-time PCR(qRT-PCR)and Western Blot at the gene and protein levels.Results(1)Compared with the control group and metformin group,phenformin suppressed OSCC cells growth with higher efficiency.(2)The conditioned medium from OSCC cells stimulated by phenformin inhibited angiogenesis of HUVECs,while was abolished by the addition of exosome inhibitors.(3)The exosomes were observed by transmission electron microscopy to have a typical cup shape of exosomes,and the diameter of exosomes was between 100 nm and 150 nm by dynamic particle size analysis,and the surface markers CD81,CD63,and TSG101 were positive by Western Blot.The exosomes labeled with PKH 67 green fluorescent dye were observed in the vascular endothelial cells after incubation.(4)In contrast with the PBS-Exo group,the Phen-Exo group significantly inhibited the proliferation and migration of HUVECs,and suppressed angiogenesis in both in vivo and in vitro experiments.(5)Exosomal microRNA sequencing analysis reveals miR-1246 and miR-205,which are associated with angiogenesis,are highly expressed in the Phen-Exo group.The result also was validated by qRT-PCR analysis.(6)High expression of miR-1246 or miR-205 in HUVECs could inhibit angiogenesis together with down-regulating the expression of VEGFA,a key factor in angiogenesis,while low expression of miR-1246 and miR-205 showed the opposite results.ConclusionThe present study showed that phenformin was more effective in inhibiting the proliferation of OSCC cells,compared to that of metformin treatment.Exosomes secreted from OSCC cells stimulated by phenformin significantly suppress the proliferation and migration of human umbilical vein vascular endothelial cells.In vitro tube formation assay,the subcutaneous angiogenesis assay in nude mice,together with chick chorioallantoic membrane vascularization assay showed that exosomes secreted from OSCC cells stimulated by phenformin could significantly inhibit angiogenesis.Mechanistically,we discovered that expression of miR-1246 and miR-205 was significantly increased in exosomes secreted by OSCC cells stimulated by phenformin,while high expression of either miR-1246 or miR-205 in vascular endothelial cells could inhibited their angiogenic effects and decreased the expression of angiogenic factor VEGFA.In conclusion,this study revealed that phenformin can inhibit angiogenesis by regulating the levels of miR-1246 and miR-205 in extracellular exosomes secreted by OSCC cells,suggesting that phenformin has potential to alter the tumor microenvironment to inhibit agiogenesis resulting in antagonizing the growth of OSCC,which provides a theoretical basis for the development of a new strategy to treat oral squamous cell carcinoma. |
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