| Background:Lymphatic metastasis acts as an independent risk factor affecting the prognosis of patients with hypopharyngeal squamous cell carcinoma,and has been established as one of the primary reasons for treatment failure.This study focused on HSP90AA1,a key gene which can promote lymphatic metastasis of hypopharyngeal squamous cell carcinoma,and was initially identified from gene sequencing of the clinical specimens.Its expression and prognostic value were verified by analysis of clinical specimens and clinicopathological features,and its possible regulatory mechanism was predicted by bioinformatics analysis.Moreover,the potential role of HSP90AA1 in lymphatic metastasis of hypopharyngeal squamous cell carcinoma were confirmed by cell functional studies after lentiviral transfection and in vivo studies in xenograft tumor metastasis model.Methods:Methods: According to our previous results of the transcriptome sequencing of hypopharyngeal squamous cell carcinoma tissues from patients with lymph node metastasis and patients without lymph node metastasis,HSP90AA1,a differentially expressed gene closely related to lymphatic metastasis of hypopharyngeal squamous cell carcinoma,was selected as for analysis.RT-PCR,western-blotting and immunohistochemical staining were used to verify the expression of HSP90AA1 in patients with hypopharyngeal squamous cell carcinoma.Kaplan-Meier,log-rank test and Cox’s test were employed to analyze the prognostic value of clinicopathological features of patients with hypopharyngeal squamous cell carcinoma.Bioinformatics tools were used to analyze the HNSCC transcriptome data and the clinical data of TCGA database to explore the possible function and enrichment pathway of HSP90AA1 in promoting lymph node metastasis,and to explore the possible mechanism of HSP90AA1 in regulating lymph node metastasis of hypopharyngeal squamous cell carcinoma.The lentivirus stably transfected Fa Du cell line with HSP90AA1 gene knockdown was established.RT-PCR and Western blot were used to detect the expression of HSP90AA1,E-cadherin,N-cadherin,SNAIL,SLUG and VIMENTIN in Fa Du cells to verify the efficiency of lentivirus induced stable transfection and the potential impact of HSP90AA1 on EMT.The possible effect of HSP90AA1 on the invasion and migration of Fa Du cells was detected by flow cytometry,wound healing assay,EDU,Transwell invasion and migration assays.The xenograft tumor metastasis model of nude mice was established,and the stable transfected Fa Du cells were transplanted into the foot pads of nude mice.The successful establishment of the animal model was confirmed by in vivo fluorescence imaging.The nude mice were sacrificed,and the tumors as well as the metastatic lymph nodes were dissected.The expression of HSP90AA1,E-cadherin,N-cadherin,and SNAIL proteins was detected by Western blot and immunohistochemical staining.To investigate the effect of HSP90AA1 on the growth and lymph node metastasis of hypopharyngeal squamous cell carcinoma in vivo and its role in the regulation of EMT,the tumors as well as the metastatic lymph nodes were measured,immunohistochemical stained,Western-blotting deteced and statistical analysed.Results:Based on the transcriptome sequencing of the clinical specimens of hypopharyngeal squamous cell carcinoma in our previous study,HSP90AA1,a differentially expressed gene,which was significantly up-regulated in hypopharyngeal squamous cell carcinoma tissues with lymph node metastasis,was selected as the research object.Thereafter,q RT-PCR,Western blot and immunohistochemical staining were used to verify its expression at the tissue level.The findings of Kaplan-Meier survival analysis and COX regression analysis combined with clinicopathological characteristics indicated that high expression of HSP90AA1 was significantly correlated with lymph node metastasis of hypopharyngeal squamous cell carcinoma,and was an independent risk factor for the prognosis of patients with hypopharyngeal squamous cell carcinoma,also could be used as a reliable biological indicator for predicting the prognosis of hypopharyngeal squamous cell carcinoma patients.Bioinformatics tools were used to analyze the differential pathway of transcriptional data of 419 HNSCC samples in TCGA.It was found that HSP90AA1 may affect the tumor cell polity by regulating the encoding gene CDH1 of E-cadherin,thereby enhancing the invasion and migration of tumor cells.A stable HSP90AA1 knockdown Fa Du cell line was established,and expression of HSP90AA1 at both m RNA and protein levels was detected by RT-PCR and Western blot respectively to verify the transfection efficiency of lentivirus.In addition,results of the flow cytometry,wound healing and Transwell migration as well as invasion assays confirmed that low expression of HSP90AA1 could significantly inhibit the proliferation,migration and invasion of Fa Du cells,but promote the apoptosis of tumor cells.Moreover,it was observed that upon the knockdown of HSP90AA1 gene,the expression of E-cadherin increased.In addition,the expression of N-cadherin,SNAIL,SLUG and VIMENTIN was decreased,thus suggesting that HSP90AA1 knockdown can inhibit the occurrence of epithelial-mesenchymal transition(EMT)process.The foot pad tumor metastasis model of nude mice was designed and divided into sh-NC group and sh-HSP90AA1 group.Thereafter,Fa Du cell line of hypopharyngeal carcinoma stably transfected with blank NC and sh-HSP90AA1 stably transfected with HSP90AA1 were transplanted into nude mice,respectively.The tumor weight and volume were measured and compared between the two different groups of nude mice.The expression of HSP90AA1,E-cadherin,N-cadherin,and SNAIL proteins was detected by Western blot and immunohistochemistry.The results showed that the down-regulation of HSP90AA1 expression significantly inhibited the growth of xenograft tumor and lymph node metastasis in nude mice.Moreover,concomitant with the down-regulation of HSP90AA1 expression,the expression of E-cadherin was up-regulated,whereas that both N-cadherin and SNAIL was down-regulated,and their correlation was found to be statistically significant.This result confirmed the regulatory effect of HSP90AA1 on EMT.Conclusion:Due to the anatomic characteristics of the laryngeal region where hypopharyngeal squamous cell carcinoma occurs,hypopharyngeal squamous cell carcinoma is prone to lymph node metastasis,which greatly reduces the prognosis of patients with hypopharyngeal squamous cell carcinoma,and the up-regulated expression of HSP90AA1 increases this risk.In this study,we primarily focused on HSP90AA1,a differentially expressed gene associated with lymphatic metastasis of hypopharyngeal squamous cell carcinoma,which was identified following transcriptome sequencing,and its expression and prognostic value were further explored in hypopharyngeal squamous cell carcinoma tissues.In addition,we explored the potential mechanism of HSP90AA1 in the progression and lymph node metastasis of hypopharyngeal squamous cell carcinoma,and found that it might promote lymph node metastasis of hypopharyngeal squamous cell carcinoma by regulating EMT.Which was confirmed by lentivirus-transfected in vivo cell experiment and in vitro animal experiment of nude mouse foot pad graft tumor metastasis model.Therefore,we suggest that HSP90AA1 may regulate EMT through the reprogramming of extracellular matrix,alter cytoskeleton and cell polarity,reduce cell adhesion,and enhance its invasion and migration ability,thus promoting the entry of hypopharyngeal squamous cell carcinoma into lymphatic vessels and subsequent lymph node metastasis.Furthermore,although this study has some limitations,it is believed that HSP90AA1 is expected to be a biomarker for predicting lymph node metastasis of hypopharyngeal squamous cell carcinoma and a a potential therapeutic target for the treatment of hypopharyngeal squamous cell carcinoma after further research in the future. |
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