The Expression And Function Of Transmembrane Protein 184B In Hypopharyngeal Squamous Cell Carcinoma

Background and significanceHypopharyngeal squamous cell carcinoma(hereinafter referred to as hypopharyngeal cancer)is a kind of malignancy arised from hypopharynx(also called laryngohypopharynx).The incidence,which accounts for approximately 3%of head and neck squamous cell carcinomas,has increased over the past three decades.Although hypopharyngeal carcinoma is relatively rare,it has the worst prognosis among head and neck squamous cell carcinoma.Hypopharyngeal carcinoma is characterized by extensive submucosal spread,early lymph node metastasis and distal metastasis,and most patients are in the middle and advanced stages when diagnosed.Although surgery,radiotherapy,chemotherapy,immunotherapy and other methods have been significantly improved in recent decades,the overall 5-year survival rate of hypopharyngeal cancer is only 30%-50%,which is far from satisfactory.Because the hypopharynx is anatomically adjacent to the larynx,locally advanced hypopharyngeal cancer can seriously invade and damage the structure and function of the larynx,resulting in severe damage to the physiological functions such as breathing,pronunciation and swallowing,and seriously affecting the quality of life of patients.Therefore,improving the survival rate and quality of life of patients with hypopharyngeal cancer is still an important goal of the treatment of hypopharyngeal cancer.One of the important reasons for the poor prognosis of patients with hypopharyngeal cancer is the lack of early screening and diagnostic biomarkers.Exploring new diagnostic and prognostic biomarkers will be the key to improve the survival rate and quality of life of patients with hypopharyngeal cancer.TMEM184B,also called NCD1,belongs to the Transmembrane protein family(TMEMs).TMEMs are a group of proteins located on phospholipid bilayers of cell membranes and organelles.They play different roles in a variety of tissues and cellular substructures.TMEMs are abnormally expressed in many malignant tumors.Their expression changes are significantly related to tumor prognosis,metastasis and drug resistance.In addition,these proteins are involved in different physiological stages of malignant tumors,such as tumorigenesis,growth,adhesion,invasion,apoptosis and metastasis.TMEM184B is a novel TMEM protein.We found that it was abnormally expressed in head and neck squamous cell carcinoma and hypopharyngeal squamous cell carcinoma by high-throughput sequencing technology and bioinformatics technology,which has not been found in other studies.To further clarify the expression of TMEM184B gene in hypopharyngeal carcinoma and explore its role in hypopharyngeal cancer cell function.It may be of great significance to screen molecular markers and therapeutic targets for early diagnosis of hypopharyngeal squamous cell carcinoma.This study is divided into three parts:Gene microarray and high-throughput sequencing technology were used to preliminarily investigate the abnormal expression of genes,their related pathways and cell functional enrichment in hypopharyngeal squamous cell carcinoma.The TCGA online database was used to explore the abnormal gene expression in hypopharyngeal squamous cell carcinoma and head and neck squamous cell carcinoma in a larger number of samples.TMEM184B was selected as the target gene in combination with literature search.Finally,the expression of the target gene was verified in the TCGA database and GEO database.Immunohistochemistry was used to investigate the expression of TMEM184B in 40 cases of paired hypopharyngeal squamous cell carcinoma tumor tissues and adjacent mucosa,so as to determine whether there was a difference in the expression of TMEM184B between cancer tissues and normal tissues,and to explore the correlation between the expression trend and clinical stage and tumor prognosis through statistics.Fadu cell lines with low expression of TMEM184B were constructed,and the effects of TMEM184B expression knockdown on cell proliferation,migration,invasion,apoptosis and other phenotypes of Fadu cell lines in vitro were investigated by Celigo cell count,CCK-8 assay,apoptosis assay,Transwell assay,and cell migration assay.The growth and proliferation of Fadu cells with low expression of TMEM184B in vivo were investigated by tumor formation experiment in nude mice.PART IPurposeGene microarray technology,high-throughput sequencing technology and bioinformatics technology were used to explore the gene expression profiles of hypopharyngeal squamous cell carcinoma and head and neck squamous cell carcinoma as well as their cell pathways enrichment and cell biological characteristics.At the same time,the target gene with abnormal expression pattern were searched in combination with literature search and its expression levels were verified.MethodsWhole genome sequencing was performed on 3 pairs of pathologically confirmed tumor tissues of hypopharyngeal squamous cell carcinoma and adjacent normal mucosa by highthroughput sequencing technology using gene microarray.Enrichment analysis was conducted on the sequencing results to understand the enrichment of signal pathways and cell biology related.The gene profile of abnormal expression in hypopharyngeal squamous cell carcinoma and head and neck squamous cell carcinoma were explored in a larger sample using online databases.The target genes were screened by literature search.The TCGA database and GEO database were used to verify whether the expression status of target genes in large samples of head and neck squamous cell carcinoma was consistent with those in the gene chip.40 pairs of tumor tissues and paracancerous mucosa samples of hypopharyngeal squamous cell carcinoma were collected to explore and verify the expression of target genes at the protein level by immunohistochemical techniques.ResultA total of 2063 differentially expressed genes were detected in hypopharyngeal squamous cell carcinoma by gene microarray.A total of 20532 differentially expressed genes were screened in head and neck squamous cell carcinoma using the TCGA database.A standard of log2 |Fold Change| was set up to sort out of the top 50 differentially expressed genes in the two groups of data.Through the literature retrieval TMEM184B was screened as a target gene.The expression analysis showed that the expression of TMEM184B gene is significantly higher in the hypopharyngeal squamous cell carcinoma and head and neck squamous cell carcinoma than that in the para-carcinoma normal mucosa(p<0.05).ConclusionPreliminary studies have shown that TMEM184B gene is highly expressed in hypopharyngeal squamous cell carcinoma and head and neck squamous cell carcinoma,which may affect the function of hypopharyngeal squamous cell carcinoma as an oncogene.PART ⅡPurposeTo explore the expression of TMEM184B in clinical samples of hypopharyngeal squamous cell carcinoma and its correlation with tumor stage and prognosis.MethodsThe localization and expression of TMEM184B gene in 40 pairs of hypopharyngeal squamous cell carcinoma tumor tissues and adjacent mucosa were investigated by immunohistochemical techniques,and the differences in TMEM184B gene expression between the tumor tissues and adjacent mucosa were statistically analyzed.The tumor stage and patient survival of the samples were counted,and the correlation between the expression level of TMEM184B gene and the stage and patient survival was analyzed.Result1.TMEM184B gene was localized in cytoplasm.2.The expression level of TMEM184B gene in cancer tissues was higher than that in adjacent mucosa,and the difference was statistically significant(p<0.05).3.The expression level of TMEM184B gene was increased with the increase of tumor clinical stage,and the correlation was statistically significant(p<0.05).ConclusionThe expression of TMEM184B gene was up-regulated in clinical samples of cancer tissues,and its expression level was significantly correlated with clinical stage,suggesting that this gene may play a role as an oncogene in hypopharyngeal squamous cell carcinoma.PART ⅢPurposeThe effects of TMEM184B gene on proliferation,apoptosis,migration and invasion of hypopharyngeal squamous cell carcinoma were investigated in vitro and in vivo.MethodsLentivirus transfection technique was used to construct Fadu cell line of hypopharyngeal squamous cell carcinoma with low expression of TMEM184B.Celigo cell count,CCK-8 assay,apoptosis assay,Transwell assay,cell migration assay and tumor formation assay in nude mice were performed to explore the phenotypic changes of Fadu cell lines with low expression of TMEM184B in cell proliferation,migration,invasion and apoptosis.Result1.Lentivirus transfection technique can significantly reduce the expression of TMEM184B in Fadu cells;2.Low expression of TMEM184B can inhibit the proliferation and growth of Fadu cell lines;Knockdown of TMEM184B gene can promote apoptosis of Fadu cells.The invasion ability of Fadu cells was significantly inhibited after TMEM184B knockdown.The migration ability of Fadu cells was significantly inhibited after TMEM184B knockdown.3.The tumor volume and growth rate of nude mice in TMEM184B gene knockout group were significantly lower than those in control group(p<0.05).At the end of the experiment,the weight of tumor in the experimental group was significantly lower than that in the control group(p<0.05).ConclusionTMEM184B is an oncogene.TMEM184B gene can promote the growth,proliferation,invasion and migration of hypopharyngeal squamous cell carcinoma and inhibit its apoptosis in vitro.Knockdown of TMEM184B expression can inhibit Fadu cell growth in vivo,that is,TMEM184B can promote the growth of hypopharyngeal squamous cell carcinoma in vivo.PART ⅣPurposeTo explore and screen the interaction protein of TMEM184B.The gene of this interaction protein was overexpressed while TMEM184B gene was knocked down,so as to verify the functional recovery effect of this gene on TMEM184B gene.MethodThe interacting proteins of target gene TMEM184B was screened and selected using CoImmunoprecipitation combined with liquid chromatography and mass spectrometry(LC-MS).Fadu cell was infected by lentivirus with 3×FLAG-TMEM184B.IP was performed with AntiFlag antibody for LC-MS/MS analysis,and interacting proteins were analyzed and screened.co-IP binding was further verified by specific antibodies against candidate interaction proteins.Interacting protein overexpression vectors were constructed to infect TMEM184Bknockdown Fadu cell line.MTT and Transwell experiments were used to verify the functional recovery effect of interacting protein genes on the target genes.ResultThe interaction between STRN3 and TMEM184B in Fadu cell lines was screened by CoIP and LC-MS experiments.MTT assay showed that STRN3 was overexpressed in the same cell line when TMEM184B gene was knocked down,and the latter had a recovery effect on the proliferation function of the TMEM184B gene knockout group.Transwell assay showed that STRN3 was overexpressed in the same cell line when TMEM184B gene was knocked down,and the latter had a recovery effect on the migration function of the TMEM184B gene knockout group.ConclusionThe interacting protein STRN3 of target gene TMEM184B was screened and verified by IP-MS and Co-IP experiments.And STRN3 had a recovery function to the interference of target gene TMEM184B.