| Background: Hypopharyngeal squamous cell carcinoma(HSCC)is a common type of cancer in northeastern China,accounting for 3% to 5% of head and neck cancers.Moreover,HSCC is highly susceptible to lymph node metastasis at an early stage,with a 5-year survival rate of only 30%–35%.Therefore,it is considered to have a poor prognosis.Approximately 84,254 new cases were reported globally in 2020,and the number of deaths due to HSCC was as high as 38,599.Due to the insidious onset of hypopharyngeal carcinoma,most patients are diagnosed at an advanced stage.Additionally,HSCC is highly invasive and susceptible to lymph node and distant metastases.According to the literature,approximately 60% of patients with HSCC have already developed ipsilateral cervical lymph node metastasis at their initial visit,bilateral cervical lymph node metastasis in 40% of patients,and 10% to 30% of patients with distant metastasis.Although intensive multimodality management is performed for HSCC,approximately 50% of patients still develop distant metastasis within 1 year after treatment.However,the mechanism underlying HSCC remains unclear.Tumorigenesis and development are driven by the tumor microenvironment(TME),where cells not only contact each other through “direct communication,” but also by various mediators indirectly.Tumor-derived exosomes(TDE),which are vesicle-like substances,are secreted by cancerous cells in autocrine or paracrine manners to mediate “material transportations” and “communications” among cells in TME.TDE,consisting of genetic materials,including proteins and nucleic acids,alters the physiological state of targeted cells by activating multiple signaling pathways,participating in biological processes such as tumor angiogenesis,tumor immune escape,metabolism,and facilitating tumorigenesis and development.Angiogenesis is a crucial process in tumorigenesis and development.Emerging evidence shows that TDE is involved in the exchange of genetic information between cancerous cells and vein endothelial cells,promotes angiogenesis,and aids tumor progression.Other studies have found that micro RNAs(miRNAs),circular RNAs,and long noncoding RNAs in TDE differ in their biological properties and targeting specificities and are closely related to tumorigenesis and development.Thus,the mechanism of HSCC-derived exosomal miRNAs promoting angiogenesis requires further exploration,which is of great significance for diagnosis and targeted therapy.miR-30b-5p is a member of the miR-30 b family and is formed by cleavage of the precursor of miR-30 b.As an angiogenesis-related miRNA,miR-30b-5p involves in angiogenesis in a variety of diseases.A growing number of studies have revealed a significant differential expression of miR-30b-5p in tumor tissues and adjacent tissues with the application of gene chips and high-throughput sequencing.However,miR-30b-5p is expressed differently in different tumors.miR-30b-5p is highly expressed and is an independent predictor of poor prognosis in renal clear cell carcinoma.High expression of miR-30b-5p induces tumor metastasis in triple-negative breast cancer.However,miR-30b-5p was downregulated in esophageal squamous cell carcinoma and hepatocellular carcinoma,and this low expression negatively correlated with tumor pathological stage and metastasis.High miR-30b-5p expression in HSCC can reportedly predict patient survival,but its mechanism has not yet been elucidated.Hence,further investigation into the relationship between HSCC-derived exosomal miR-30b-5p and tumor angiogenesis may provide new evidence for the pathogenesis of HSCC.Objective: To elucidate the relationship between FaDu cell-derived exosomal miR-30b-5p and tumor angiogenesis,to provide evidence that FaDu cell-derived exosomal miR-30b-5p may facilitate tumorigenesis and the development of HSCC by promoting tumor angiogenesis,and to supply potential molecular markers for diagnosis and new therapeutic targets for HSCC.Methods: 1.Effect of HSCC-derived exosomes on angiogenesis(1)Isolation and identification of the FaDu cells-derived exosomes Exosomes were isolated from the supernatant of FaDu cells using ultracentrifugation.The morphology of extracts was observed by transmission electron microscopy(TEM),the diameter of extracts was detected by NTA analysis,and markers of extracts were detected by western blot assay.(2)Uptake of FaDu-Exos in human microvascular endothelial cells(HMEC-1)Exo Glow-Membrane labeled FaDu-Exos and HMEC-1 for 12 h firstly,and the uptake of FaDu-Exo by HMEC-1 cells was observed by fluorescence confocal microscopy.(3)Effect of FaDu-Exos on the functions of HMEC-1 cells After co-incubation of HMEC-1 cells with increased Exo concentration,the influence of FaDu-Exo on the concentration of vascular endothelial growth factor(VEGF)in the supernatant of HMEC-1 cell culture medium was detected by enzymelinked immunosorbent assay(ELISA).The proliferative ability of HMEC-1 cells was detected by the cell counting kit-8(CCK-8)assay,and the migratory ability of HMEC-1 cells was detected by the transwell migration assay.The ability to form tube-like structures in vitro was detected by a tube formation assay.2.The expression of miR-30b-5p in HSCC The correlation between the expression of miR-30b-5p and clinicopathological characteristics was analysed firstly.Then the Kaplan-Meier method was used to draw the patient survival curve.Finally,the expression of miR-30b-5p in HSCC tumor tissues,FaDu cells,and FaDu-Exos was detected by RT-qPCR.3.Effect of miR-30b-5p in FaDu-Exo(FaDu-Exo-miR-30b-5p)on angiogenesis(1)Effect of FaDu-Exo-miR-30b-5p on the miR-30b-5p expression in HMEC-1 cells(HMEC-1-miR-30b-5p)miR-30b-5p expression in FaDu cells and exosomes transfected with miR-30b-5p mimic or inhibitor was detected via RT-qPCR.HMEC-1 cells were co-incubated with FaDu-Exo-miR-30b-5p mimic or FaDu-Exo-miR-30b-5p inhibitor,and the miR-30b-5p expression in HMEC-1 cells(HMEC-1-miR-30b-5p)was detected via RT-qPCR.(2)FaDu-Exo-miR-30b-5p reinforces proliferation,migration and angiogenesis of HMEC-1 cells The effect of FaDu-Exo-miR-30b-5p on the concentration of VEGF in the supernatant of HMEC-1 cell culture medium was detected by ELISA;the proliferative ability of HMEC-1 cells was detected via CCK-8 assay;the migratory ability of HMEC-1 cells was detected by transwell migration assay;and the ability to form tube-like structures in vitro was detected by a tube formation assay.4.The mechanism of FaDu-Exo-miR-30b-5p regulates angiogenesis(1)PTEN is a downstream gene of miR-30b-5p The downstream genes of miR-30b-5p were predicted using the Target Scan,Star Base,DIANA,and mir DIP databases;KEGG enrichment analysis detected the downstream genes related to signaling pathways;candidate genes were further explored via interaction analysis;Target Scan database and dual-luciferase reporter assay were used to verify the specific binding sites between miR-30b-5p and mRNA of its downstream genes.(2)miR-30b-5p represses PTEN expression miR-30b-5p expression in HMEC-1 cells transfected with miR-30b-5p mimic or inhibitor was detected via RT-qPCR.The effect of HMEC-1-miR-30b-5p on PTEN expression in HMEC-1 cells(HMEC-1-PTEN)was detected via RT-qPCR and western blot assays.(3)FaDu-Exo-miR-30b-5p negatively regulates PTEN HMEC-1 cells were co-incubated with the FaDu-Exo-miR-30b-5p mimic or FaDu-Exo-miR-30b-5p inhibitor.Western blot was used to detect the influence of FaDu-Exo-miR-30b-5p on the expression of HMEC-1-PTEN.PTEN-overexpressing HMEC-1 cells(HMEC-1-PTEN mimic)were co-incubated with FaDu-Exo-miR-30b-5p mimic for 24 h.The influence of the FaDu-Exo-miR-30b-5p mimic on the expression of HMEC-1-PTEN at the mRNA and protein levels was detected using RT-qPCR and western blot assays.(4)FaDu-Exo-miR-30b-5p promotes angiogenesis by inhibiting PTEN expression Co-cultured HMEC-1-PTEN mimic with FaDu-Exo-miR-30b-5p mimic,the concentration of VEGF in the supernatant of HMEC-1-PTEN mimic cells culture medium was detected by ELISA;the proliferative ability of HMEC-1-PTEN mimic cells was detected by CCK-8 assay;migratory ability of HMEC-1-PTEN mimic cells was detected via a transwell migration experiment;the ability to form tube-like structures was detected via tube formation assay.(5)Influence of FaDu-Exo-miR-30b-5p on angiogenesis via PTEN/Akt signaling pathway activation Target genes of PTEN were predicted using the STRING database,and downstream signaling pathways of PTEN were predicted by KEGG enrichment analysis.Western blot assays were used to detect the expression of PTEN and its downstream pathway-related proteins in HSCC tisuses and adjacent tissues.Western blot assay was used to demonstrate the influence of HMEC-1-miR-30b-5p,FaDu-ExomiR-30b-5p,and Akt inhibitor on the expression of PTEN and its related signaling pathways.The influence of FaDu-Exo-miR-30b-5p on the VEGF concentration in the supernatant of HMEC-1-Akt inhibitor cell culture medium was detected by ELISA,the proliferative ability of HMEC-1-Akt inhibitor cells was detected by CCK-8 assay,the migratory ability of HMEC-1-Akt inhibitor cells was detected by transwell migration assay,and the ability to form tube-like structures was detected by a tube formation assay.(6)The influence of FaDu-Exo-miR-30b-5p on tumorigenesis and angiogenesis through the PTEN/Akt axis After establishing the FaDu xenograft model in nude mice,the mice were randomly divided into nine groups and treated with lentivirus to knockdown PTEN and/or intravenously injected with FaDu-Exo-miR-30b-5p agomir/FaDu-Exo-miR-30b-5p antagomir.The tumor volume was measured every three days,and the tumor growth curve was plotted.RT-qPCR was used to evaluate the expression of miR-30b-5p in the tumor tissues.The expression of PTEN and p-Akt was detected by western blotting,and angiogenesis in tumor tissue was detected by immunohistochemistry.Results: 1.Effect of HSCC-derived exosomes on angiogenesis(1)Isolation and identification of the FaDu cells-derived exosomes A population of round-shaped,small,membranous vesicles,which were clearly visible under TEM,membranous structures were observed in the periphery of vesicles and low electron density in the center.NTA analysis illustrated that the isolated vesicles,with a diameter of 50-150 nm;the markers of exosomes were expressed in the vesicles,while calnexin was not detectable.(2)Uptake of FaDu-Exos in HMEC-1 cells Laser confocal microscopy revealed that FaDu-Exos with red fluorescence were taken up by HMEC-1 cells with blue fluorescence.(3)Effect of FaDu-Exos on the functions of HMEC-1 cells Compared to the PBS group,the VEGF concentration,and the proliferative ability,migratory ability,and ability to form tube-like structures in vitro of HMEC-1 cells were obviously enhanced.2.The expression of miR-30b-5p in HSCC The expression of miR-30b-5p in HSCC tumors was significantly higher than that in non-tumor adjacent tissues;The expression of miR-30b-5p in FaDu cells was significantly higher than that in human immortalized keratinocytes(Ha Cat)cells;The expression of miR-30b-5p in FaDu-Exo was significantly higher than that in FaDu cells.3.Effect of miR-30b-5p in FaDu-Exo(FaDu-Exo-miR-30b-5p)on angiogenesis(1)Effect of FaDu-Exo-miR-30b-5p miR-30b-5p expression in HMEC-1 cells(HMEC-1-miR-30b-5p)FaDu cell lines transfected with the miR-30b-5p mimic expressed higher levels of miR-30b-5p,while those transfected with the miR-30b-5p inhibitor expressed lower levels,which demonstrated that transfections were successful.Exosomes derived from FaDu-miR-30b-5p mimic cells expressed higher levels of miR-30b-5p,while those from FaDu-miR-30b-5p inhibitor cells expressed lower levels when compared with NC group.Compared to the those in the NC group,miR-30b-5p levels were increased in HMEC-1 cells after co-culturing with the FaDu-Exo-miR-30b-5p mimic,and the inverse effect was evident after co-incubation with the FaDu-Exo-miR-30b-5p inhibitor.(2)FaDu-Exo-miR-30b-5p reinforces proliferation,migration,and angiogenesis of HMEC-1 cells Compared to the FaDu-Exo-NC mimic group,the concentration of VEGF was clearly elevated;the proliferative ability,migratory ability,and ability to form tube-like structures in vitro in HMEC-1 cells co-cultured with the FaDu-Exo-miR-30b-5p mimic was obviously enhanced.4.The mechanism of FaDu-Exo-miR-30b-5p regulates angiogenesis(1)PTEN is a downstream gene of miR-30b-5p Bioinformatic analysis predicted 1,064 target genes of miR-30b-5p,which are mainly enriched in micro RNAs in cancer-related pathways.PTEN is a core downstream gene,and miR-30b-5p specifically regulates its expression.(2)miR-30b-5p represses PTEN expression RT-qPCR results revealed that HMEC-1 cell lines transfected with miR-30b-5p mimic expressed a higher level of miR-30b-5p,while those transfected with miR-30b-5p inhibitor expressed a lower level of miR-30b-5p,demonstrating that transfections were successful.Compared to that in the NC group,the expression of HMEC-1-PTEN was downregulated by gain-of-function of miR-30b-5p,but notably increased by the loss-of-function of miR-30b-5p in HMEC-1 cells at the mRNA and protein levels.(3)FaDu-Exo-miR-30b-5p negatively regulates PTEN The results revealed that the PTEN level was reduced in HMEC-1 cells coincubated with FaDu-Exo-miR-30b-5p mimic and increased in response to coincubation with FaDu-Exo-miR-30b-5p inhibitor when compared with NC group;FaDu-Exo-miR-30b-5p mimic repressed the PTEN expression in the HMEC-1-PTEN mimic cells at both mRNA and protein levels.(4)FaDu-Exo-miR-30b-5p promotes angiogenesis by inhibiting PTEN expression In response to the PTEN overexpression in HMEC-1 cells,the VEGF concentration,proliferation,migration,and in vitro angiogenic abilities were considerably impeded,whereas co-incubation with the FaDu-Exo-miR-30b-5p mimic reversed the abovementioned impeded abilities.(5)Influence of FaDu-Exo-miR-30b-5p on angiogenesis via PTEN/Akt signaling pathway activation The results revealed that PTEN can interact with Akt1;PTEN was predicted to modulate the PI3K/Akt pathway in tumors;Compared with adjacent tissues,the expression of PTEN was significantly down-regulated in HSCC tissues,and the expressions of p-Akt/Akt and VEGF-A were significantly up-regulated,especially in HSCC tissues with high expression of miR-30b-5p.The HMEC-1-p-Akt/Akt upregulation by HMEC-1-miR-30b-5p-mimic was rescued by PTEN;PTEN expression in the HMEC-1 cells was notably reduced,while that of p-Akt/Akt was dramatically increased upon co-incubation with FaDu-Exo-miR-30b-5p mimic compared to that in the control group;Akt inhibitor treatment repressed the p-Akt/Akt level in HMEC-1 cells,which could be remarkably rescued by co-incubation with FaDu-Exo-miR-30b-5p mimic.Compared to that in the control group,the VEGF concentration in HMEC-1 cells co-incubated with GDC0068 clearly declined;the proliferative ability,migratory ability,and ability to form tube-like structures in vitro were obviously reduced.After coincubation with the FaDu-Exo-miR-30b-5p mimic,the inhibitory effects of GDC0068 on the function of HMEC-1 cells were rescued.(6)The influence of FaDu-Exo-miR-30b-5p on tumorigenesis and angiogenesis through the PTEN/Akt axis The results revealed that tumor volumes were significantly increased in mice following PTEN knockdown,while the FaDu-Exo-miR-30b-5p antagomir counteracted these changes.Compared with the control group,FaDu-Exo-miR-30b-5p agomir significantly up-regulated the expression of PTEN and down-regulated the expression level of p-Akt/Akt in tumor tissue,while FaDu-Exo-miR-30b-5p agomir significantly down-regulated the expression of PTEN and up-regulated the expression level of pAkt/Akt in tumor tissue;PTEN knockdown led to an increase in active p-Akt levels in the tumor tissues,while this enhancement was appreciably reversed by FaDu-Exo-miR-30b-5p antagomir.Microvascular density was elevated in the tumor tissues of mice in response to PTEN deficiency or FaDu-Exo-miR-30b-5p agomir,but decreased after the loss of miR-30b-5p induced by FaDu-Exo-miR-30b-5p antagomir compared to that of FaDu-Exo-miR-30b-5p agomir.Conclusions: 1.FaDu-Exo reinforces the proliferation,migration and angiogenesis of HMEC-1 cells;2.miR-30b-5p expression is upregulated in HSCC,FaDu,and FaDu-Exo cells,it correlates significantly with T stage,lymph node metastasis,and clinical stage in the HSCC patients,and increased risk death in the HSCC patients;3.FaDu-Exo-miR-30b-5p promotes angiogenesis and tumorigenesis via the PTEN/Akt axis. |
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