| ObjectiveGenomic sequencing,proteomics sequencing and clinical tissue expression detection were used to screen differential genes for hypopharyngeal carcinoma lymphatic metastasis.Combined with clinical statistical data,the influence of differential genes on the prognosis of patients was analyzed.The cell function experiment after lentivirus transfection and the foot pad tumor model in nude mice were used to explore the role of differential genes in lymphatic metastasis and its possible mechanism.MethodsTumor tissues of patients with lymphatic metastasis and non-metastatic hypopharyngeal carcinoma were examined for genomics and proteomics sequencing,and lymphatic metastatic differential genes were screened in combination with GEO database.RT-PCR,Western blot and immunohistochemistry were used to verify the expression of differential genes in a larger sample size.The clinicopathological characteristics of patients with hypopharyngeal cancer were statistically analyzed,and the relationship between differential genes and prognosis of hypopharyngeal cancer was analyzed by Kaplan-Meier survival analysis and Cox univariate and multivariate regression test.Fadu cell lines of hypopharyngeal carcinoma were constructed,and lentivirus transfection was performed to knockdown or overexpress differential genes.RT-PCR and Western blot were used to verify differential gene expression in different cell lines.Flow cytometry,scratch assay and Transwell invasion assay were used to test the effects of differential genes on cell functions,especially invasion and migration functions.The foot pad tumor model was established in nude mice,and the hypopharyngeal cancer cells were transplanted to observe the tumor growth.At the same time,RT-PCR,Western blot and immunohistochemical experiments were performed to examine the influence of the expression of differential genes on the development of primary tumor and popliteal fossa lymphatic metastasis.ResultsGene and proteomics sequencing were performed in 11 cases of clinical tissue.Combined with the results of GEO human head and neck squamous cell carcinoma database,337 differentially expressed genes were screened out,including 144 up-regulated genes and 193 down-regulated genes.Based on RT-PCR,Western bloting detection of 12 pairs fresh clinical specimens and immunohistochemical detection of 107 specimens,differential genes could be further screened to find differential gene RAF1 with significant expression differences.Kaplan-Meier survival analysis and Cox regression analysis combined with clinicopathological data showed that high expression of RAF1 was an independent risk factor for the prognosis of patients with stomach carcinoma.Fadu cell line of hypopharyngeal carcinoma was constructed,and lentivirus knockdown and overexpression of RAF1 were transfected and stable cell lines were constructed.Flow cytometry,scratch assay and Transwell invasion assay showed that the high expression of RAF1 significantly promoted the proliferation,migration and invasion ability of tumor cells and inhibited the apoptosis of tumor cells.The wildtype Fa Du cell lines of hypopharyngeal carcinoma and the stable cell lines knocked down by RAF1 were transplanted into the tumor model of the foot pad in nude mice.The tumor weight and volume of the primary pad tumor and the popliteal fossa tumor metastases were measured.Meanwhile,RT-PCR 、 Western blot 、immunohistochemical results indicated that the up-regulation of RAF1 expression could promote tumor growth and lymphatic metastasis.ConclusionThrough multi-omics sequencing combined with database analysis,we found that the lymphatic metastasis differential gene RAF1 of hypopharyngeal carcinoma is an independent risk factor for the prognosis of clinical patients,and its up-regulated expression is closely related to the development,migration,invasion and lymphatic metastasis of hypopharyngeal carcinoma.These findings suggest that RAF1 can be used as an important potential biomarker for early screening and diagnosis of hypopharyngeal carcinoma lymphatic metastasis,and provide a new direction for the treatment and prognosis improvement of hypopharyngeal carcinoma patients with lymphatic metastasis. |
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