| BackgroundLaryngeal carcinoma is a highly invasive malignant tumor of the head and neck.Laryngeal squamous cell carcinoma(LSCC)account for more than 90%of all cases of laryngeal cancer.Adenocarcinoma,basal cell carcinoma,poorly differentiated carcinoma,lymphosarcoma and fibrosarcoma were the next most common.People who live in areas with high levels of air pollution and those who smoke and drink for a long time are at high risk.Surgical treatment of early laryngeal squamous cell carcinoma is the first choice,and carbon dioxide laser resection and partial laryngectomy under supporting laryngoscope can achieve better clinical res?lts.Although open radical surgery,chemotherapy,radiotherapy and molec?lar targeted therapy have made great progress in the treatment of laryngeal squamous cell carcinoma in recent years,unfortunately,the 5-year survival rate of advanced laryngeal squamous cell carcinoma is still low,seriously affecting the quality of life of patients.The occurrence and development of laryngeal squamous cell carcinoma is a biological process with the participation of many factors,in which the inactivation of tumor suppressor genes and the activation of proto-oncogenes may be the main factors to promote the occurrence and development of laryngeal squamous cell carcinoma.A variety of reg?latory genes and signal transduction pathways are involved,among which many coding or non-coding genes are abnormally expressed and reg?lated.In-depth study of the molec?lar mechanism and reg?lation of related genes can provide new ideas for the diagnosis and treatment of laryngeal squamous cell carcinoma and improve the prognosis of patients.According to the theory of molecular biology,the transmission of biological genetic information is carried out in the form of genetic coding according to the central law.Deoxyribonucleic acid((DNA))is a gene fragment that carries biological genetic information.Ribonucleic acid(RNA)is only used as a carrier between deoxyribonucleic acid sequence and coding protein.DNA carries genetic information and is transcribed into a part of RNA,RNA which has the function of protein coding and carries genetic information to translate and synthesize proteins,The study found that less than 2%of the genes in the human genome can encode proteins,while at least 90%of the genome sequences do not have the function of protein coding.The RNA which can not be translated into protein is called non-coding RNA(non-codingRNA,ncRNA).In recent years,with the development of large-scale high-throughput chip and second-generation sequencing technology,the relationship between lncRNA and tumor-related genes has attracted more and more attention.LncRNA is a kind of ribonucleic acid molec?le whose transcript exceeds 200bp.Like messenger RNA,they can be transcribed by RNA polymerase ?.As a non-coding RNA,lncRNA itself does not have the function of coding proteins,only a small part of them can encode some short peptides and play a role through these peptides.Previous studies have shown that lncRNAs can play a role as an oncogene or tumor suppressor gene in the occurrence and development of tumors.LncRNAs can affect tumor chromatin remodeling,transcriptional reg?lation,cell cycle reg?lation,DNA methylation,translation,histone modification and other biological functions at different levels,such as pre-transcriptional,post-transcriptional and protein translation.Researchers have found that the lncRNA of more and more tumors have these important reg?latory functions,including breast cancer,non-small cell lung cancer,pancreatic malignant tumor,osteosarcoma,hepatocell?ar carcinoma,leukemia and so on.The abnormal expression of lncRNA in a series of malignant tumors indirectly suggests that lncRNA may have both tumor-promoting and tumor-suppressing effects,but its role and mechanism are still unclear.Therefore,the study of the relationship between lncRNAs and laryngeal squamous cell carcinoma will help to better understand the molec?lar biological mechanism of laryngeal squamous cell carcinoma,develop new therapeutic targets,and provide new guiding strategies for early diagnosis and individualized treatment of laryngeal squamous cell carcinoma.LncRNA TRPM2-AS is an antisense lncRNA of TRPM2,with a full length of 875 nt,located at chr21q22.3.Some recent studies have shown that lncRNA TRPM2-AS is often up-reg?lated in several tumors,such as prostate cancer,liver cancer,and lung cancer.However,to date,the expression level and potential function of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma are still largely unresearched.Therefore,exploring the function and mechanism of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma will be of great significance for marker screening,molec?lar diagnosis and targeted therapy of laryngeal squamous cell carcinoma.This research is divided into 3 parts:(1)The relative expression of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma and corresponding paracancerous tissues was detected by qRT-PCR,and its expression level and clinical features were analyzed.(2)To further construct an in vitro cell model,use small interfering RNA technology to influence the expression of lncRNA TRPM2-AS in laryngeal cancer cell lines,and use cell Transwell analysis and Western blotting to further study the effect of lncRNA TRPM2-AS on the biological behavior of laryngeal cancer cells influences.(3)Bioinformatics was used to predict the interaction between IncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinoma.The expression of miR-138 in laryngeal squamous cell carcinoma was detected by qRT-PCR method,the cell proliferation,Transwell migration and invasion were detected by MTT cell proliferation assay,the migration and invasion ability of cells were detected by Transwell migration and invasion assay,the expression of EMT-related proteins was detected by Westernblot,and the interaction was verified by double luciferase reporter gene analysis.PART ?The expression and the clinical significance of the IncRNA TRPM2-AS in laryngeal carcinomaObjective:To detect the relative expression of lncRNA TRPM2-AS in tumor tissues and adjacent tissues of 77 patients with laryngeal squamous cell carcinoma,and to further analyze the relationship between the expression of lncRNA TRPM2-AS and the clinical features of patients with laryngeal squamous cell carcinoma,such as tumor differentiation,lymph node metastasis and tumor stage.Methods:The tumor tissues and corresponding paracancerous tissues of 77 patients with laryngeal squamous cell carcinoma who were diagnosed and underwent radical surgery in the Department of Otorhinolaryngology,Qilu Hospital of Shandong University from October 2015 to December 2019 were collected.RNA,was extracted from tumor tissues and paracancerous tissues respectively and the relative expression of lncRNATRPM2-AS in laryngeal squamous cell carcinoma and corresponding paracancerous tissues was detected by qRT-PCR.The relationship between its expression and various clinical characteristics of tumor patients was statistically analyzed.The relationship between the expression of LncRNATRPM2-AS and clinicopathological features was evaluated by Chi-square or Fisher test.The difference of experimental data between the two groups was compared by t-test,and the difference was statistically significant(P<0.05).Results:qRT-PCR results showed that the relative expression of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma(1.8636 ±0.3957)was significantly higher than that in paracancerous tissues(0.9996±0.1479).The high expression level of lncRNA TRPM2-AS was significantly correlated with T stage(P=0.037)and later clinical stage(P=0.023).Conclusion:We found that lncRNA TRPM2-AS was highly expressed in laryngeal squamous cell carcinoma,indicating that lncRNA TRPM2-AS is an oncogene in this tumor.To further study the possible clinical value of lncRNA TRPM2-AS in patients with laryngeal squamous cell carcinoma,it was found that the expression level of lncRNA TRPM2-AS was significantly correlated with T stage and clinical stage of laryngeal squamous cell carcinoma.It is suggested that lncRNA TRPM2-AS may participate in and promote the occurrence and development of laryngeal squamous cell carcinoma.Part ?Effect of lncRNA TRPM2-AS on biological behavior of laryngeal squamous cell carcinomaObjective:To study the effects of lncRNA TRPM2-AS on the proliferation,migration and invasion of laryngeal squamous cell carcinoma cells.Methods:(1)The expression of lncRNA TRPM2-AS in different laryngeal squamous cell carcinoma cell lines AMC-HN-8 and Tu-177 and normal bronchial epithelial cell line 16HBE was detected by qRT-PCR method,and the subcellular localization of lncRNA TRPM2-AS was carried out by nuclear and plasma RNA separation test combined with qRT-PCR method.(2)The expression of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma cell line was regulated by constructing si-TRPM2-AS and pcDNA3.1-TRPM2-AS,.(3)The effects of lncRNA TRPM2-AS on the proliferation,migration and invasion of laryngeal squamous cell carcinoma cells were detected by MTT cell proliferation test,Transwell migration and invasion assay.Results:(1)The expression level of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma cell lines AMC-HN-8 and Tu-177 was higher than that in normal 16HBE cell lines,and it was mainly located in the cytoplasm of laryngeal squamous cell carcinoma cells.(2)Si-TRPM2-AS can significantly reduce the expression level of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma cell line AMC-HN-8,while overexpression vector pcDNA3.1-TRPM2-AS can significantly up-regulate the expression level of lncRNA TRPM2-AS in laryngeal squamous cell carcinoma cell line Tu-177.(3)In vitro experiments showed that knocking down lncRNA TRPM2-AS,in laryngeal squamous cell carcinoma cell line AMC-HN-8 could significantly inhibit the proliferation,migration and invasion of AMC-HN-8 cells.Overexpression of lncRNA TRPM2-AS,in laryngeal squamous cell carcinoma cell line Tu-177 can significantly promote the proliferation,migration and invasion of Tu-177 cells.Conclusion:(1)The expression of lncRNA TRPM2-AS was increased in laryngeal squamous cell carcinoma cell lines AMC-HN-8 and Tu-177,and was mainly expressed in the cytoplasm.(2)lncRNA TRPM2-AS can promote the proliferation,migration and invasion of laryngeal squamous cell carcinoma cell line Tu-177.It is suggested that lncRNA TRPM2-AS,as an oncogenic gene,may promote the proliferation,migration and invasion of laryngeal squamous cell carcinoma.Part ?LncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinomaObjective:To detect the relative expression of miR-138 in tumor tissues and paracancerous tissues of patients with laryngeal squamous cell carcinoma.The interaction between lncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinoma was analyzed.Methods:Bioinformatics was used to predict the interaction between lncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinoma.The expression of miR-138 in laryngeal squamous cell carcinoma was detected by qRT-PCR method,the cell proliferation,Transwell migration and invasion were detected by MTT cell proliferation assay,the migration and invasion ability of cells were detected by Transwell migration and invasion assay,the expression of EMT-related proteins was detected by Westernblot,and the interaction was verified by double luciferase reporter gene analysis.Results:(1)The luciferase activity of HEK293T cells transfected with TRPM2-AS-WT plasmid was significantly decreased by miR-138 mimics.The expression of miR-138 in laryngeal squamous cell carcinoma was significantly lower than that in adjacent normal tissues.There was a close negative correlation between the expression of lncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinoma(r=-0.269,P=0.018).Dual-luciferase reporter assay showed that miR-138 mimics could markedly reduce the luciferase activity of SOX4-WT plasmid in HEK293T cells.(2)The results showed that TRPM2-AS knockdown caused the reduction of SOX4 protein expression and impairment of EMT in AMC-HN-8 cells,as indicated by the increased expression of epithelial markers(E-cadherin)and decreased expression of mesenchymal markers(N-cadherin,Vimentin).Besides,the increased SOX4 protein level and enhanced EMT in TRPM2-AS-overexpressing Tu-177 cells were effectively diminished by co-transfection with miR-138 mimics.(3)Rescue experiments were then carried out,and as indicated by transwell assay,ectopic expression of miR-138 reversed the promotion effect of TRPM2-AS on Tu-177 cell migration and invasion.By MTT assay,we also noticed-that miR-138 restoration partly reversed the enhanced proliferation of TRPM2-AS-overexpressing Tu-177 cells.Conclusion:(1)The expression of miR-138 in laryngeal squamous cell carcinoma was significantly lower than that in adjacent normal tissues.There was a significant negative correlation between the expression of lncRNA TRPM2-AS and miR-138 in laryngeal squamous cell carcinoma.(2)In vitro experiments showed that the overexpression of lncRNA TRPM2-AS promoted the EMT transformation of laryngeal squamous cell carcinoma cells,while the low expression of lncRNA TRPM2-AS could inhibit the EMT of laryngeal squamous cell carcinoma cells.(3)LncRNA TRPM2-AS was confirmed to be the ceRNA of laryngeal squamous cell carcinoma cell line miR-138.LncRNA TRPM2-AS plays a carcinogenic role in laryngeal squamous cell carcinoma through the miR-138/Sox4 axis. |
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