| Hypopharyngeal carcinoma is a rare head and neck malignancy with an incidence of approximately 1.6 per 100,000(age-standardized incidence).Due to the lack of specific clinical manifestations and effective monitoring indicators,the early diagnosis rate of hypopharyngeal cancer is low.At the same time,the disease also has the characteristics of high invasion and metastasis rate,insensitivity to radiotherapy and chemotherapy,etc.,resulting in poor treatment effects of surgery,radiotherapy,and chemotherapy,and poor prognosis.The 5-year survival rate is only 30%-35%.Although radiotherapy,neoadjuvant chemotherapy,immunotherapy,and function-preserving surgery have made great progress in recent years,the incidence and mortality of hypopharyngeal cancer have not decreased significantly compared with 2018 and 2012.Therefore,understanding the pathogenesis of hypopharyngeal carcinoma and exploring effective prognostic indicators and drug targets are of great significance for improving hypopharyngeal carcinoma patients’ diagnosis,treatment,and prognosis.Cancer-associated fibroblasts(CAFs)are one of the most important cellular components in the tumor microenvironment.Several studies have shown that CAFs promote the metastasis of head and neck squamous cell carcinoma,increase tumor angiogenesis,regulate anti-tumor immunity,mediate resistance to cisplatin,CAFs play an important role in the occurrence and development of tumors,and have become one of the hotspots in tumor research.The collagen family is the main member of the extracellular matrix,mainly secreted by fibroblasts,which can interact with many cellular components in the TME and tumor cells and play a tumor-promoting role.As the major receptors of collagen,integrins mediate cell adhesion and transmit mechanical and chemical signals to the interior of cells through signaling pathways such as FAK,promoting tumor cell proliferation,EMT,and tumor angiogenesis.High-throughput gene chips and next-generation sequencing technologies have become important tools for screening the molecular mechanisms of multi-gene alterations in the occurrence and development of tumors,and significantly improved the screening efficiency of new cancer targets.Conjoint analysis of omics data and clinicopathological data provide opportunities for the clinical translation of basic research,targeted therapy,and combination therapy for tumors,and improve the level of accurate diagnosis and treatment of tumors.Objectives:To explore the differentially expressed genes of hypopharyngeal carcinoma cancer-associated fibroblasts(CAFs)and fibroblasts(NFs)in corresponding normal tissues by next-generation sequencing,and to investigate the effect and its mechanism of COL10A1 expressed by CAFs on the proliferation,epithelial-mesenchymal transition(EMT)and angiogenesis mimicry(VM)of hypopharyngeal carcinoma cells.Methods:(1)Following the principle of informed consent,fresh tissues from patients with hypopharyngeal carcinoma who underwent surgery in the Department of Otolaryngology and Head and Neck Surgery,First Hospital of Jilin University,were collected,and CAFs in hypopharyngeal carcinoma tissues and fibroblasts(NFs)in adjacent normal tissues were isolated and cultured.The proliferation and passage of the two were observed;the expression of cytokines was detected by immunoblotting and immunohistochemistry;the effects of CAFs and NFs on the proliferation,migration,and invasion of hypopharyngeal carcinoma cell line FaDu were observed by co-culture;Western blotting was used to examine the effect of CAFs on FaDu’s EMT.(2)The total RNA of CAFs and NFs was extracted for transcriptome sequencing,differentially expressed genes were identified,and differential gene co-expression networks were constructed;the gene modules related to clinical characteristics were identified and reclustered;GO enrichment analysis on gene modules was performed,PPI network was constructed and HUB genes were identified;key genes closely related to the survival of hypopharyngeal cancer patients were verified in the database.(3)The data of 78 patients with hypopharyngeal carcinoma were reviewed,and the relationship between clinicopathological characteristics and differential genes was analyzed.The CAFs transcriptome data and the secretomes were confirmed to mine the characteristics of the hypopharyngeal carcinoma CAFs secretome and identified the most differentially expressed and clinicopathologically relevant secretory protein-coding gene COL10A1.Double fluorescent staining and co-immunoprecipitation were used to detect the expression of COL10A1 and its receptors in hypopharyngeal carcinoma tissues,CAFs with high COL10A1 expression(CAF-H),and FaDu cells.(4)The COL10A1 overexpression vector was constructed and transfected into CAFs-L cells with low COL10A1 expression to construct stable expression of CAF-L/OV cells and CAF-L/Ve cells transfected with an empty vector;The COL10A1 si RNA oligonucleotide fragment(oligo)was used to transfect CAF-H cells with high COL10A1 expression..FaDu cells were treated with the culture supernatant of CAF-L/OV cells and CAF-H/si RNA cells,respectively.Cell viability was observed by CCK8 assay,and cell migration ability was detected by scratch assay.The bidirectional rescue was performed to verify the biological function of COL10A1..(5)FaDu cells were treated with CAF-L/OV cells and CAF-L/Ve cell culture supernatants,and western blotting was used to detect the expression changes of FAK protein and phosphorylated FAK protein in FaDu cells,as well as the changes of FAK downstream protein ERK and its phosphorylation.;Western blot was used to detect the expressions of E-cadherin,N-cadherin,Vimentin,and Twist related to EMT in FaDu cells;to detect the expressions of PDGFR,VEGFA and VEGFR related to angiogenesis in FaDu cells;Expression levels of vasculogenic mimicry-related MMP9,MMP14,and VE-Cadherin.Finally,a FAK inhibitor was added to the above systems to detect the changes in related indicators.(6)FaDu cells or HUVEC cells were treated with culture supernatants of CAF-L/OV cells、CAF-H/si RNA cells,and CAF-L cells alone or with FAK inhibitor,and their tube formation ability was examined by three-dimensional tube formation experiments.(7)CAF-L/OV cells and FaDu cells were co-implanted into nude mice,and the volume changes of the xenografts were observed.Immunohistochemical staining was used to detect the formation of vascular mimicry in the xenografts.Results:(1)Primary cultured CAFs express α-SMA,FAP,Vimentin,S100,CD90,and other mesenchymal cell markers;highly express IL6 and IL8,which promote the proliferation,migration,and invasion of FaDu cells in vitro,down-regulate the expression of E-cadherin,and promote FaDu cells Expression of N-cadherin,Vimentin and transcription factors Snail and Twist.(2)Transcriptome sequencing identified 2538 differentially expressed genes in CAFs and NFs and clustered them into 7 gene modules,among which M2 and M3 modules closely related to tumor T and N stages were mainly enriched in cell signaling,cytoskeleton and cell migration,angiogenesis and other biological processes related to tumor invasion and progression.Seven key genes were identified,including ACTB,FAU,ITGA8,THBS1,IL-6,RPL11,and SOCS1,which were highly correlated with the survival time of patients with head and neck squamous cell carcinoma.And in a cohort of 78 hypopharyngeal cancer cases,it was verified that the elevated expression of ITGA8 and IL6 was associated with worse T staging and worse differentiation.(3)The COL10A1 gene was screened out through CAFs transcriptome and secretome data and clinicopathological factors,and the interaction between COL10A1 and ITGA11 was confirmed by double fluorescence staining and co-immunoprecipitation.CAFs-L/OV cell promotes FaDu proliferation and migration,activates FAK/ERK phosphorylation,and further promotes the expression of EMT,angiogenesis,and vasculogenic mimicry(VM)-related factors in hypopharyngeal carcinoma.And these promotions can be reversed by FAK inhibitors.(4)CAFs highly expressing COL10A1 promote the proliferation,angiogenesis,and vasculogenic mimicry(VM)of hypopharyngeal carcinoma xenografts in vivo.Conclusions:(1)CAFs promoted FaDu cell proliferation,migration,invasion,and EMT in vitro;and promoted the proliferation,angiogenesis,and vasculogenic mimicry(VM)of hypopharyngeal carcinoma xenografts.(2)COL10A1 is the most differentially expressed secreted protein in CAFs of hypopharyngeal carcinoma and plays a biological role by binding to ITGA11 integrin of FaDu cells.(3)COL10A1 promotes EMT,angiogenesis,and vasculogenic mimicry(VM)of hypopharyngeal carcinoma cells through FAK/ERK signaling pathway. |
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