| Laryngeal squamous cell carcinoma(LSCC)is the main pathological type of laryngeal cancer.Local invasion and distant organ metastasis are the main factors leading to poor curative effect and low survival rate.At present,the research of long non-coding RNAs(long nc RNAs,lnc RNAs)in malignant tumors has attracted much attention.More and more evidences showed that lnc RNAs have high tissue specificity in various tumors and play an important role in the occurrence and development of tumors.However,studies on lnc RNAs in LSCC are still relatively few.In this study,using the LSCC expression profile data in TCGA(the Cancer Genome Atlas)and GEO(Gene Expression Omnibus)databases,TM4SF19-AS1 was selected as the research object,and the biological function and mechanism of TM4SF19-AS1 were deeply analyzed.The main research is divided into the following three parts:Part One Screening and validation of laryngeal squamous cell carcinoma-related long non-coding RNAs based on TCGA and GEO databasesObjective: The lnc RNAs with significant differential expression were screened from the LSCC expression profile data of TCGA and GEO databases,and the lnc RNAs related to the development of LSCC were further mined by bioinformatics methods.The expression of the selected lnc RNAs in 36 clinical LSCC patients was verified by quantitative real-time PCR(q RT-PCR),and the relationship between these lnc RNAs and clinicopathological parameters was analyzed.Methods:1.Lnc RNA expression profile data of LSCC tissues and adjacent normal tissues from TCGA database were used for differential expression analysis using edge R software package in R software.The thresholds were set as |log2fold change(FC)|>1,with FDR value<0.01.Then,the lnc RNAs screened in TCGA database were intersected with the differentially expressed lnc RNAs of the laryngeal squamous cell carcinoma dataset(GSE137308)based on GPL16791 platform in GEO database to obtain the differentially expressed lnc RNAs in both databases.2.The receiver operating characteristic(ROC)curve was used to evaluate the sensitivity and specificity of the screened differentially expressed lnc RNAs as biomarkers in LSCC.3.The subcellular localization of the screened lnc RNAs was predicted using lnc Locator(http://www.csbio.sjtu.edu.cn),which is an online forecast database.4.Total 36 paired cancer tissue and matched adjacent non-cancer tissues were excised from surgery LSCC patients between January 2016 and November 2018 in the Second Hospital of Hebei Medical University.The expression level of the screened lnc RNAs in the 36 pairs of LSCC tissues was verified by q RT-PCR.5.The correlation between these lnc RNAs expression levels and clinicopathological parameters of LSCC patients was analyzed by independent sample t test(data with normal distribution)/Wilcoxon rank sum test(data with non-normal distribution).Results:1.Screening of differentially expressed lnc RNAs associated with laryngeal squamous cell carcinoma in TCGA and GEO databasesThe LSCC expression profile in the TCGA database were analyzed,and a total of 586 differentially expressed lnc RNAs were screened,of which 373 had high expression and 213 had low expression.To improve the credibility of lnc RNA results,we crossed the 171 up-regulated lnc RNAs in the GSE137308 dataset with 373 up-regulated lnc RNAs in the TCGA database to obtain 6up-regulated lnc RNAs(LINC01980,MSC-AS1,TM4SF19-AS1,GATA2-AS1,LBX2-AS1 and HOXA11-AS).2.Evaluation of the potential significance of differentially highly expressed lnc RNAs as molecular markersThe ROC curve was used to evaluate the sensitivity and specificity of these 6 lnc RNAs as biological indicators of LSCC.The results showed that the area under curve(AUC)values of these 6 lnc RNAs were all greater than 0.8,indicating that they could be potentially good biomarkers for the diagnosis of LSCC.3.Predicting the subcellular localization of differentially overexpressed lnc RNAsThe subcellular localization of the 6 lnc RNAs was predicted by lnc Locator,it was found that LINC01980,MSC-AS1 and TM4SF19-AS1 were localized in the cytoplasm,while GATA2-AS1,LBX2-AS1 and HOXA11-AS were localized in the cytosol.4.Expression of TM4SF19-AS1,LINC01980 and MSC-AS1 in 36 pairs of clinical LSCC cancer tissues and adjacent noncancerous tissuesThe samples of 36 clinical patients were verified by q RT-PCR.It was found that the relative expression levels of TM4SF19-AS1,LINC01980 and MSC-AS1 in LSCC cancer tissues were significantly higher than those in non-cancer tissues.The relative expression level of TM4SF19-AS1 was the highest.5.Relationship between the expression of TM4SF19-AS1,LINC01980 and MSC-AS1 and clinicopathological parameters in patients with laryngeal squamous cell carcinomaThrough the correlation analysis of clinicopathological parameters,it was found that the expression levels of TM4SF19-AS1,MSC-AS1 and LINC01980 were significantly correlated with tumor stage and lymph node metastasis,but not with age,smoking,alcohol consumption and degree of pathological differentiation.Conclusion: The high expression of TM4SF19-AS1 may be involved in the occurrence and progression of laryngeal squamous cell carcinoma,and is expected to be a molecular marker for the diagnosis of laryngeal squamous cell carcinoma.Part Two Effects of TM4SF19-AS1 on proliferation,invasion and migration of laryngeal squamous cell carcinoma cellsObjective: In this part,human laryngeal squamous cell carcinoma cells were cultured in vitro.Through gene transfection technology,CCK-8,Ed U and scratching and other experimental techniques,the changes of cell proliferation and migration after knockdown of TM4SF19-AS1 were observed,the role of TM4SF19-AS1 in the proliferation,migration and invasion of LSCC cells was deeply discussed,and its possible role and biological significance in the progression of laryngeal squamous cell carcinoma were preliminarily clarified.Methods:1.The expression of TM4SF19-AS1 in four LSCC cell lines(AMC-HN-8,TU177,TU212 and TU686)was detected by q RT-PCR.The top two cell lines in relative expression were used for subsequent experiments.2.Small interfering RNA(si RNA)was constructed to reduce the expression level of TM4SF19-AS1 in LSCC cell line.The knockdown efficiencies were detected by q RT-PCR.3.AMC-HN-8 and TU686 cell lines were transfected with si-TM4SF19-AS1,and the effect of TM4SF19-AS1 on the proliferation of LSCC cells was detected by CCK-8 and Ed U assays,and the effects of migration and invasion were detected by scratch assay and Transwell assay.Results:1.Expression of TM4SF19-AS1 in four laryngeal squamous cell carcinoma cell linesThrough q RT-PCR experiments,it was found that the expression levels of TM4SF19-AS1 in the four LSCC cell lines were higher than that in pool group(The average expression of non cancerous tissues adjacent to laryngeal squamous cell carcinoma in 36 cases was set as pool to represent normal laryngeal cells),and the expression levels in AMC-HN-8 and TU686 cell lines were significantly higher than that in the other two cell lines.2.Detection of knockdown efficiency of si-TM4SF19-AS1 in laryngeal squamous cell carcinoma cellsA total of two si-TM4SF19-AS1 were constructed and transfected into AMC-HN-8 and TU686 cell lines.The knockdown efficiency was verified by q RT-PCR.The results showed that compared with the negative control group(si-NC),si-TM4SF19-AS1#1 reduced the expression of TM4SF19-AS1 in cells to 56% and 48% respectively(P<0.01);si-TM4SF19-AS1#2 decreased the expression of TM4SF19-AS1 to 58% and 53%,respectively(P<0.01).Therefore,si-TM4SF19-AS1#2 with higher knockdown efficiency was selected for follow-up research.3.Effects of TM4SF19-AS1 on proliferation of laryngeal squamous cell carcinoma cellsCCk-8 and Ed U assays showed that decreasing the expression of TM4SF19-AS1 could significantly inhibit the proliferation of AMC-HN-8 and TU686 cells.4.Effects of TM4SF19-AS1 on migration and invasion of laryngeal squamous cell carcinoma cellsScratch assay and Transwell assay showed that reducing the expression of TM4SF19-AS1 could inhibit the migration and invasion abilities of AMC-HN-8 and TU686 cells.Conclusion: Reducing the expression of TM4SF19-AS1 could reduce the proliferation,migration and invasion ability of LSCC cells.It is suggested that TM4SF19-AS1 is not only differentially expressed in laryngeal squamous cell carcinoma cells,but also has certain biological functions.Part Three TM4SF19-AS1 regulates the proliferation,migration and invasion of laryngeal squamous cell carcinoma through the mi R-153-3p/ITGAV axisObjective: This part of the study took human laryngeal squamous cell carcinoma cells and surgically resected specimens of laryngeal squamous cell carcinoma as the research objects,combined with bioinformatics analysis and molecular biology techniques,to explore whether there is a regulatory relationship among TM4SF19-AS1,mi R-153-3p and ITGAV,To verify whether TM4SF19-AS1 exerts biological function by targeting ITGAV through mi R-153-3p.Methods:1.The target mi RNAs of TM4SF19-AS1 were predicted by Star Base 3.0database.The expression of these mi RNAs in LSCC samples in TCGA database and the correlation between these mi RNAs and TM4SF19-AS1 expression were analyzed to further screen mi RNAs.2.The downstream targeted m RNAs of mi RNA were preliminarily mined through Star Base 3.0 database.Combined with weighted gene co-expression network(WGCNA),GO and KEGG pathway enrichment analysis,the targeted m RNAs were further screened.3.The relative expression of mi R-153-3p was detected by q RT-PCR after transfection of si-TM4SF19-AS1 in AMC-HN-8 cell line.The mi R-153-3p mimic and mi R-153-3p inhibitor were constructed and transfected into AMC-HN-8 cell line respectively,and then the expression changes of TM4SF19-AS1 were detected by q RT-PCR.4.The expression of ITGAV in 36 pairs of clinical LSCC cancer tissues and adjacent non-cancer tissues was detected by q RT-PCR.,and the correlation between TM4SF19-AS1 and ITGAV expression was analyzed.The correlation between the expression levels of TM4SF19-AS1,mi R-153-3p and ITGAV at the m RNA and protein levels was detected by q RT-PCR and Western blot in AMC-HN-8 cells.5.The dual-luciferase reporter gene vectors of TM4SF19-AS1 and ITGAV were constructed respectively,and the interaction between TM4SF19-AS1,mi R-153-3p and ITGAV was verified by dual-luciferase reporter gene assay.6.In AMC-HN-8 cell line,si-TM4SF19-AS1 and pc DNA3.1-ITGAV were respectively transfected and co-transfected,and then the cell proliferation,migration and invasion abilities were detected by Ed U and Transwell assays.Results:1.TM4SF19-AS1 targets mi R-153-3p in laryngeal squamous cell carcinoma cellsAccording to the prediction of Star Base 3.0 database,a total of 5mi RNAs(mi R-153-3p,mi R-19a-3p,mi R-19b-3p,mi R-485-5p and mi R-6884-5p)may target TM4SF19-AS1.Furthermore,by analyzing the expression of mi RNAs in TCGA database,only mi R-153-3p was found to be low expressed in LSCC tissues.In AMC-HN-8 cells,when the expression of TM4SF19-AS1 was decreased,the expression of mi R-153-3p was increased,and there was a negative correlation between them.However,when the expression of mi R-153-3p was decreased or increased,the expression of TM4SF19-AS1 was not significantly changed.Dual-luciferase reporter genes assay revealed a targeted regulatory relationship between TM4SF19-AS1 and mi R-153-3p.It was speculated that there is a targeted regulatory relationship between TM4SF19-AS1 and mi R-153-3p.2.ITGAV is a target gene of mi R-153-3p in laryngeal squamous cell carcinoma cellsPredicted by the Star Base 3.0 database,2579 m RNAs may be targeted downstream of mi R-153-3p.To further narrow the screen,WGCNA screened458 m RNAs co-expressed with TM4SF19-AS1 using 2917 highly expressed m RNAs in the TCGA database(|Log2(fold chang)|>1 and p<0.05).GO and KEGG pathway enrichment analysis of these 458 m RNAs showed that in terms of cell components(CC),these m RNAs were mainly distributed in collagen-containing extracellular matrix,cell-substrate junctions and focal adhesions.In terms of molecular function(MF),these m RNAs are mainly related to the structural components of extracellular matrix,cell adhesion molecule binding and integrin binding.in terms of biological process(BP),these m RNAs are mainly involved in extracellular structure Tissue,extracellular matrix organization,and cell-substrate adhesion.KEGG pathway analysis showed that the top three enriched m RNAs were focal adhesion,PI3K-Akt signaling pathway and human papillomavirus infection,suggesting that these m RNAs may be involved in tumorigenesis and metastasis.ITGAV was located in the focal adhesion and PI3K-Akt signaling pathway.By reviewing the literature,it was found that this gene has a tumor-promoting effect in a variety of malignant tumors,so ITGAV was selected for further research.q RT-PCR was used to detect the expression of ITGAV in 36 pairs of clinical LSCC cancer tissues and adjacent noncancerous tissue samples,and it was found that its expression in laryngeal squamous cell carcinoma was significantly higher than that in adjacent noncancerous tissues.Correlation analysis found that there was a positive correlation between the expression of TM4SF19-AS1 and ITGAV.When mi R-153-3p was reduced and overexpressed,the expression of ITGAV could be increased and decreased,suggesting that the expression of mi R-153-3p was negatively correlated with ITGAV.The dual-luciferase reporter gene showed that there was a binding site between mi R-153-3p and ITGAV,thus confirming the targeted regulatory relationship between mi R-153-3p and ITGAV.3.TM4SF19-AS1 regulates ITGAV protein expression by targeting mi R-153-3p in laryngeal squamous cell carcinoma cellsAfter si-TM4SF19-AS1 and PCDNA3.1-ITGAV were respectively transfected and co-transfected into AMC-HN-8 cells,Ed U and Transwell chamber experiments showed that si-TM4SF19-AS1 could reduce the proliferation,migration and invasion of AMC-HN-8 cells.Transfection pc DNA3.1-ITGAV could reverse this effect.Conclusion: In the occurrence and progression of laryngeal squamous cell carcinoma,TM4SF19-AS1 exerts biological functions through the mi R-153-3p/ITGAV axis. |
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