| Laryngeal carcinoma is a high incidence and invasive malignant tumor of the head and neck.The main pathological type is laryngeal squamous cell carcinoma(LSCC).The etiology of laryngeal cancer is complicated,the early symptoms of some patients are not typical,they are often diagnosed at an advanced or metastatic stage,so the clinical prognosis is poor.At present,although a variety of intervention measures have been taken,such as early prevention,drug therapy,radiotherapy,immunotherapy,surgical resection and so on,the 5-year survival rate of LSCC patients is still not very ideal.Therefore,it is very important to reveal the pathogenesis of laryngeal cancer,especially metastatic laryngeal cancer,and to find molecular markers and therapeutic targets for early screening and prognosis.In recent years,our group has been devoted to exploring the pathogenesis of LSCC from clinical study to molecular basis.The transcripts expression profiles of 4 pairs of LSCC tissues and corresponding normal tissues were detected by high-throughput microarray analysis,and the differentially expressed mRNA in LSCC was screened.During these differentially expressed mRNA,the high expression of melanoma specific antigen(PRAME)has attracted our attention.PRAME was first found in patients with recurrent melanoma and belonged to the tumor testicular antigen family.It is a surface antigen that can be specifically recognized by cytotoxic T lymphocytes and cause tumor lysis.Through a large number of literature search,it is found that PRAME is associated with a variety of malignant tumors,such as non-small cell lung cancer,esophageal cancer,ovarian cancer and breast cancer,which plays an important role in the occurrence and development of tumors.However,there is little research in LSCC,and there is no direct evidence to confirm the biological function and potential mechanism of PRAME in LSCC.Therefore,in this study,the expression of PRAME in 57 pairs of LSCC tissues and corresponding normal tissues was detected,and the correlation between its expression level and clinicopathological parameters was analyzed;The expression of PRAME in laryngeal squamous cell carcinoma cell lines(AMC-HN-8 and TU177)was detected,and the effect of PRAME on the malignant progression of LSCC was investigated in vivo and in vitro;The possible upstream regulatory factors and downstream signal pathways were predicted,and the underlying mechanism was explored.The results of this project are divided into the following three parts:Part one The expression of PRAME in laryngeal squamous cell carcinoma and the correlation between its expression level and clinicopathological parameters of patients.Objective:To study the expression of PRAME in LSCC tissues,analyze the correlation between its expression level and clinicopathological parameters of patients,and explore its clinical significance.Methods:1.Four pairs of LSCC tissues and corresponding normal tissues were detected by microarray analysis,and the differentially expressed mRNA in LSCC were screened.2.The GEO online database(https://www.ncbi.nlm.nih.gov/gds/?term=)(GSE143224,GSE117005,GSE84957,GSE59652,GSE59102,GSE51985)was applied to obtain the gene matrix expression profiles of LSCC,All genes in each data sets were integrated using the Robust Rank Aggreg R package.3.Quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)method was used to detect the expression of PRAME in 57 pairs of LSCC tissues and corresponding normal tissues.4.GEPIA online database(http://gepia.cancer-pku.cn/)was used to search the expression of PRAME in 519 cases of head and neck squamous cell carcinoma(HNSC)and 44 cases of normal tissues.5.NCBI(https://www.ncbi.nlm.nih.gov/gene)was used to search the differential expression of PRAME in different human tissues.6.The correlation between the expression level of PRAME and the clinicopathological parameters of LSCC patients was analyzed by chi-square test and two groups of independent sample t-test,and explored the clinical significance.7.According to the qRT-PCR expression level and survival information of patients,Kaplan-Meier survival analysis was performed to analyze the correlation between high expression of PRAME and progression free survival rate(PFS)and overall survival rate(OS),explored the effects on the prognosis of patients.Results:1.Through the microarray analysis of 4 pairs of LSCC tissues and corresponding normal tissues,the differentially expressed PRAME gene was screened,and PRAME was significantly high expression in LSCC tissues.2.The GEO online database(https://www.ncbi.nlm.nih.gov/gds/?term=)(GSE143224,GSE117005,GSE84957,GSE59652,GSE59102,GSE51985)was applied to obtain the gene matrix expression profiles of LSCC,the genes with|log2foldchang(log2 FC)|>1 and adjusted P<0.05 were regard as DEGs.All genes in each data sets were sorted by log FC and integrated using the Robust Rank Aggreg R package.3.The results of qRT-PCR showed that the mRNA expression level of PRAME in 57 pairs of LSCC tissues was significantly higher than that in the corresponding normal tissues.4.GEPIA online database showed that the expression level of PRAME in519 HNSC tissue samples was significantly higher than that in 44 normal tissue samples.5.NCBI online search showed that the expression of PRAME was different in different tissues of human body.Except for testis,ovary and adrenal gland,the expression of PRAME in other normal tissues was very low or not.6.The results of chi-square test and t-test showed that the high expression of PRAME was significantly correlated with TNM stage(P<0.05)and lymph node metastasis(P<0.05)in clinicopathological parameters,but not with age,smoking and drinking.7.The results of Kaplan-Meier analysis showed that the PFS rate and OS rate of patients with high expression of PRAME were lower than those of patients with low expression of PRAME,suggesting that high level of PRAME was associated with poor prognosis of patients with LSCC.Part two The expression of PRAME in LSCC cells and the effects on cell biological behavior and epithelial-mesenchymal transformation process.Objective:To study the expression of PRAME in LSCC cell lines(AMC-HN-8 and TU177 cells),and explore the effects of PRAME on the biological behavior and epithelial-mesenchymal transformation(EMT)process of LSCC cells.Methods:1.qRT-PCR and Western Blot were used to detect the expression of PRAME at mRNA level and protein level in AMC-HN-8 and TU177 cells.2.Construction of the plasmid of pcDNA3.1-PRAME,it and pcDNA3.1empty vector were transfected into AMC-HN-8 and TU177 cells respectively.After transfection,qRT-PCR and Western Blot were used to detect the expression of PRAME at mRNA and protein levels in AMC-HN-8 and TU177cells.The knockdown plasmid of PRAME(si-PRAME)and the control plasmid(si-N)were transfected into TU177 cells respectively.The transfection efficiency was detected by qRT-PCR.3.By MTS and clone formation assays,the proliferation ability of AMC-HN-8 and TU177 cells of overexpressing PRAME was detected,the proliferation ability of TU177 cells of knockdown PRAME was detected.4.By transwell chamber migration and invasion assays and wound healing assay,the migration and invasion ability of AMC-HN-8 and TU177cells of overexpressing PRAME were detected,the migration and invasion ability of TU177 cells of knockdown PRAME were detected.5.Screening of AMC-HN-8 cell lines stably transfected with pcDNA3.1empty vector and pcDNA3.1-PRAME by G418.After the monoclonal cells were subcultured,the stable overexpression level of PRAME was detected by qRT-PCR method.AMC-HN-8 cells stably transfected with pcDNA3.1 and stably transfected with pcDNA3.1-PRAME were collected at the same time,and the AMC-HN-8 cells transiently transfected with pcDNA3.1-PRAME were collected.According to 1×10~7AMC-HN-8 cells per nude mouse+medium without fetal bovine serum100ul,mix evenly.These nude mice were injected subcutaneously under the right armpit.The tumor volume was measured with caliper every 3 days,and the formula was V=(A×B~2)/2,A means the maximum diameter and B means the diameter perpendicular to A.All these nude mice were killed 4 weeks later,and the xenografts were immediately taken and weighed.6.By qRT-PCR,the mRNA expression levels of EMT related molecules(N-cadherin,β-catenin,Vimentin,Twist,Snail and ZEB1)were detected in AMC-HN-8 and TU177 cells of overexpressing PRAME.7.By Western Blot,the protein expression levels of EMT related molecules(E-cadherin、N-cadherin、β-catenin、Vimentin、Snail and ZEB1)were detected in AMC-HN-8 and TU177 cells of overexpressing PRAME.Results:1.qRT-PCR and Western Blot methods were used to verify that the expression of PRAME in AMC-HN-8 and TU177 cells,which was higher than that in the control group at both mRNA expression level and protein expression level.2.qRT-PCR and Western Blot methods were used to verify that the expression of PRAME after transfection of pcDNA3.1-PRAME in AMC-HN-8 and TU177 cells.Compared with the pcDNA3.1 group,the expression of PRAME was significantly increased;qRT-PCR method was used to verify that the expression of PRAME after transfection of si-PRAME in TU177 cells.Compared with the si-N group,the expression of PRAME was significantly decreased.3.MTS and colony formation assays showed that the proliferation ability of AMC-HN-8 and TU177 cells was significantly enhanced after overexpression of PRAME,while that the proliferation ability of TU177 cells was significantly decreased after knockdown of PRAME.4.Transwell chamber migration and invasion assays and wound healing assay showed that the migration and invasion ability of AMC-HN-8 and TU177 cells was significantly enhanced after overexpression of PRAME,while that the migration and invasion ability of TU177 cells was significantly decreased after knockdown of PRAME.5.Screening of AMC-HN-8 cell lines stably transfected with pcDNA3.1empty vector and pcDNA3.1-PRAME by G418.By qRT-PCR,the expression level of PRAME increased significantly.It was confirmed that the cell line stably transfected with overexpressed PRAME was successfully constructed,which can be used to construct xenograft tumor model in nude mice.In the transient transfection PRAME group and stable transfection PRAME group,the volume and weight of xenograft tumor were significantly larger than those in the empty vector group,indicating that PRAME can promote the growth of transplanted tumor in vivo.6.By qRT-PCR,the mRNA expression levels of N-cadherin,β-catenin,Vimentin,Twist,Snail and ZEB1 were increased in AMC-HN-8 and TU177cells of overexpressing PRAME.7.By Western Blot,the protein expression level of E-cadherin was decreased in AMC-HN-8 and TU177 cells of overexpressing PRAME.while,the protein expression levels of N-cadherin、β-catenin、Vimentin、Snail and ZEB1 were increased.Part three Study on upstream regulatory factors and downstream signal pathways of PRAME promoting LSCC progression.Objective:Microarray analysis was used to identify the expression of HDAC subtypes in LSCC tissues,and the correlation between the expression of PRAME and HDACs molecular subtypes was analyzed to predict the possible upstream regulatory factors that PRAME promotes the progression of LSCC.The GO enrichment analysis of differential genes was carried out by Top GO software,and the functional enrichment analysis of KEGG was carried out by Cluster Profiler to predict the possible downstream signal pathways,which were verified in vivo and in vitro experiments.Methods:1.Microarray analysis was used to identify the expression of HDAC subtypes in LSCC tissues,and the correlation between the expression of PRAME and HDACs molecular subtypes was analyzed,and the HDAC subtypes with obvious differential expression were screened.2.qRT-PCR was used to detect the expression of HDAC5 in LSCC tissues and corresponding normal tissues.3.Construction of the plasmid of pcDNA3.1-HDAC5,it and pcDNA3.1empty vector were transfected into AMC-HN-8 and TU177 cells respectively.After transfection,qRT-PCR and Western Blot were used to detect the expression of PRAME at mRNA and protein levels in AMC-HN-8 and TU177cells.4.GEPIA online database(http://gepia.cancer-pku.cn/)was used to search the expression of HDAC5 in 519 cases of head and neck squamous cell carcinoma(HNSC)and 44 cases of normal tissues.5.The GO enrichment analysis of differential genes was carried out by Top GO(version2.24.0)software(p-value<0.05),and the functional enrichment analysis of KEGG(Kyoto Encyclopedia of Genes and Genomes)was carried out by Cluster Profiler(p-value<0.05)to predict the possible downstream signal pathways that participated in the LSCC biological processes.6.Functional enrichment analysis showed that the expression of PRAME was related to PI3K/AKT/mTOR signal pathway.Therefore,we added LY294002,an inhibitor of PI3K,or rapamycin,an inhibitor of mTOR in vitro,and explored the effects on the proliferation,migration,invasion and EMT process of AMC-HN-8 and TU177 cells induced by overexpressed PRAME.In vivo experiment,LY294002 was given to nude mice after xenograft tumor formation,and explored the effect on the growth of xenograft tumor.7.Western Blot was used to verify the changes of p-AKT and p-mTOR protein levels in AMC-HN-8 and TU177 cells after adding LY294002 or rapamycin.Results:1.Microarray analysis was used to identify the expression of HDAC subtypes in LSCC tissues,and the correlation between the expression of PRAME and HDACs molecular subtypes showed that:Among the HDACs molecular subtypes,the expression of PRAME was most significantly correlated with HDAC5,and there was a significant negative correlation(COR=-0.96,P=0.00).2.The results of qRT-PCR showed that the mRNA expression level of HDAC5 in LSCC tissues was significantly lower than that in the corresponding normal tissues.3.qRT-PCR and Western Blot methods were used to verify that the expression of PRAME after transfection of pcDNA3.1-HDAC5 in AMC-HN-8and TU177 cells.Compared with the pcDNA3.1 group,the mRNA and protein expression of PRAME were significantly decreased.4.GEPIA online database showed that the expression level of HDAC5 in519 HNSC tissue samples was significantly lower than that in 44 normal tissue samples.5.The GO enrichment analysis of differential genes was carried out by Top GO(version2.24.0)software.The results showed that there were 12biological processes(BP),6 molecular functions(MF)and 2 cell components(CC)related to the overexpression of PRAME.The functional enrichment of KEGG was analyzed by Cluster Profiler(version 3.0.5).The results showed that the expression of PRAME was related to PI3K/AKT signal pathway.6.After adding LY294002 or rapamycin to AMC-HN-8 and TU177 cells,the proliferation,migration,invasion abilities and EMT markers expression of overexpressed PRAME on AMC-HN-8 and TU177 cells were partially reduced.After the intervention of LY294002 in the nude mice,the growth of xenograft tumor was effectively inhibited.7.Compared with PRAME overexpression group,the protein levels of p-AKT and p-mTOR decreased after adding LY294002 or rapamycin to AMC-HN-8 and TU177 cells by Western Blot.Conclusions:1.PRAME is highly expressed in laryngeal squamous cell carcinoma.Its expression level is closely related to TNM stage and lymph node metastasis in clinicopathological parameters,and the high level of PRAME indicates poor prognosis.2.PRAME is highly expressed in laryngeal squamous cell carcinoma cell lines(AMC-HN-8 and TU177).It can promote the proliferation,migration,invasion and EMT process of laryngeal squamous cell carcinoma cells,and promote the growth of xenografted tumors in nude mice.3.As a potential upstream regulatory factor,HDAC5 may mediate the expression of PRAME.PRAME promotes the progression of laryngeal squamous cell carcinoma at least partly depends on the activation of PI3K/AKT/mTOR signal pathway. |
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