The Expression, Function, And Mechanisms Of Long Non-coding RNA LINC00668 In Laryngeal Squamous Cell Carcinoma

As an aggressive head and neck cancer,laryngeal cancer mainly consists of laryngeal squamous cell carcinoma(LSCC)(96%~98%),followed by adenocarcinoma,basal cell carcinoma,poorly differentiated carcinoma and sarcoma.In 2015,26,400 new patients nationwide were diagnosed as laryngeal cancer,which consisted of 23,700 males.Meanwhile,14,500 patients died of laryngeal cancer,which consisted of 12,600 males.This indicates that both the morbidity and mortality are predominantly male.Currently,smoking and drinking are universally recognized as the two major risk factors for laryngeal cancer.With the increasing of smoking,the incidence of laryngeal cancer has increased.Though the improvement of diagnosis and treatment of laryngeal cancer has been made,the prognosis of it remains poor,especially for advanced laryngeal cancer.During the complicated pathogenesis of laryngeal cancer,inactive tumor suppressor genes and active oncogenes play pivotal roles in the complicated biological process.Long non-coding RNA(lncRNA),a kind of transcripts longer than 200 nt,has no ability to encode proteins but has plentiful biological functions.Besides,some lncRNAs may encode small peptides and exert important biological functions through them.Compared to mRNA,lncRNA has relatively lower expression abundance and higher tissue and cell heterogeneity.LncRNA can act as signal,decoy,guide,or scaffold at transcriptional,post-transcriptional or epigenetic levels and play pivotal regulatory roles in a variety of diseases,including tumors.Many studies reported that lncRNA ectopic expressed in LSCC,head and neck squamous cell carcinoma(HNSCC),esophageal cancer and hepatic cancer,further promoted or suppressed tumor progress.However,there are few studies on lncRNAs in LSCC,especially lack of relevant mechanism studies.For exploring the potential mechanisms of oncogenesis of LSCC,the present study screened the lncRNA profile and obtained 3,073 differently expressed lncNRAs in LSCC.Furthermore,the lncRNA LINC00668(long intergenic non-protein coding RNA 668),highly expressed in LSCC,was elaborately selected for the further study.More importantly,the study of LINC00668 in LSCC has not been reported as yet.Accordingly,exploring the function and mechanisms of LINC00668 in LSCC will be of great significance for the biomarker screening,diagnosis,and targeted therapy of LSCC.Part one Screening and validation of microarray expression profile of long non-coding RNAs in laryngeal squamous cell carcinomaObjective:With the methods of microarray expression profile and qRT-PCR(quantitative real-time reverse transcription-polymerase chain reaction),this section was aimed to screen and partly validate the differentially expressed lncRNAs in LSCC tissues.Methods:1.Total 29 paired specimens were excised from surgery LSCC patients between October 2016 and June 2017.Of them,4 paired specimens were used for microarray expression profile.2.Screened out the differentially expressed lncRNAs and mRNAs in LSCC cancerous tissues according to the screening criteria(Fold change ?2 or ?0.5 and P <0.05).3.The reliability of the expression profile was verified by the randomly selected 10 lncRNAs(H19,HOTAIR,TINCR,MIR155 HG,LINC00511,LINC00520,HULC,MALAT1,MEG3,and ZNF667-AS1)with the method of qRT-PCR.Results:1.Through microarray expression profile,the differentially expressed genes were screened out based on the screening criteria(Fold change ?2 or ?0.5 and P <0.05): differentially expressed lncRNAs 3,073,including 1,967 up-regulated lncRNAs and 1,106 down-regulated lncRNAs;differentially expressed mRNAs 2,809,including 1,791 up-regulated mRNAs and 1,018 down-regulated mRNAs.2.GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analyses indicated that the differentially expressed mRNAs mainly located in DNA unwinding involved in DNA replication,condensed chromosome outer kinetochore modules,Cytokine-cytokine receptor interaction and Chemokine signaling pathway.3.The qRT-PCR results of the 10 selected lncRNAs were consistent with microarray expression profile,which further validated the well reliability of expression profile.Summary:1.Total 3,073 differentially expressed lncRNAs and 2,809 differentially expressed mRNAs in LSCC were screened out with microarray of transcriptome profile.2.The reliability of transcriptome profile was verified by qRT-PCR,and the profile could be used for subsequent research and analysis Part two Relative expression level and clinical significance of LINC0-0668 in laryngeal squamous cell carcinomaObjective:Through comprehensive analysis of the screening results of microarray,the validation results of qRT-PCR and the results of public microarray data mining,LINC00668 was selected for subsequent study.The relative expression levels of LINC00668 in 42 paired LSCC specimens were detected with the method of qRT-PCR,and the correlation between the expression levels of LINC00668 and the clinicopathological characteristics of LSCC was analyzed.Methods:1.Obtained the lncRNA expression profile related to LSCC from NCBI/GEO(National Center for Biotechnology Information/Gene Expression Omnibus)through data mining,and compared it with the microarray expression profile screened by preliminary experiment.2.Relative expression levels of LINC00668 in 42 paired LSCC tissues and matched normal tissues were detected by the method of qRT-PCR.3.The correlation between LINC00668 expression levels and LSCC clinicopathological features was analyzed.Results:1.The dataset GSE84957 concerning LSCC was screened out through data mining.LINC00668 was screened out through comprehensive analysis of the dataset GSE84957 and the microarray expression profile screened by preliminary experiment.2.LINC00668 was up-regulated in LSCC cancerous tissues compared to adjacent normal tissues.3.The relative expression levels of LINC00668 were associated with tumor stage,pathological differentiation degree and neck lymph node metastasis.Summary:1.Through data mining and bioinformatics analysis,combined with the lncRNA profile in the first part of the experiment,LINC00668 with high expression level in LSCC cancerous tissue was screened.2.The expression level of LINC00668 was increased in LSCC cancerous tissues and was correlated with the clinical stage,pathological differentiation degree and cervical lymph node metastasis of LSCC.Part three Effects of LINC00668 on cellular biological behaviors of laryngeal squamous cell carcinomaObjective:To study the potential effects of LINC00668 on cellular biological behaviors of LSCC cells through in vitro experiments.Methods:1.Detected the relative expression levels and the subcellular locations of LINC00668 in different LSCC cell lines(AMC-HN-8,TU212,TU177,TU686)with the method of qRT-PCR.2.Modulated the relative expression levels of LINC00668 in cells through different vectors.3.Explored the effects of LINCC00668 on proliferation,migration and invasion ability of LSCC cell lines through MTS and Transwell assays.Results:1.The relative expression levels of LINC00668 were distinct in different cell lines.LINC00668 mainly expressed in nucleus.2.The vectors could effectively knock down(ASO-LINC00668)or over express(LINC00668-pcDNA3.1(-))the relative expression levels of LINC00668 in certain cells.3.In vitro experiments proved that knockdown of LINC00668 mediated by ASO-LINC00668 could inhibit the proliferation,migration and invasion ability of TU177 and TU212 cell lines.Accordingly,overexpression of LINC00668 mediated by LINC00668-pcDNA3.1(-)could promote the proliferation,migration and invasion ability of AMC-HN-8 cell lines.Summary:1.The expression level of LINC00668 was increased in LSCC cell lines(TU177,TU212,TU686,AMC-HN-8),and mainly expressed in nucleus.2.LINC00668 could promote the proliferation,migration and invasion of LSCC cell lines TU177,TU212 and AMC-HN-8,suggesting that LINC00668 may promote the proliferation,migration and invasion of LSCC as an oncogene.Part four Prediction and screening of targeted mRNAs for LINC00668 in laryngeal squamous cell carcinomaObjective:Predicted and screened the targeted mRNAs for LINC00668 through high-throughput sequencing,and validated the reliability of the results by the method of qRT-PCR,with the purpose of exploring the potential effects of LINC00668 in LSCC.Methods:1.Screened out the differentially expressed mRNAs after LINC00668 knockdown mediated by ASO-LINC00668 in TU177 cell lines.2.GO and KEGG pathway analyses were used for functional enrichment analyses of differentially expressed mRNAs.3.Partially validated the differentially expressed mRNAs(PDCD4,ARF1,PDGFRB,PTPRB,SP100,ITGA6,DUSP5,DUSP2)in 42 paired LSCC specimens with the method of qRT-PCR.Results:1.Total 670 differentially expressed mRNAs were screened out through high-throughput sequencing after LINC00668 knockdown.Of them,166 mRNAs were positively correlated with LINC00668 expression level and 504 were negatively correlated with it.2.GO analysis indicated that the differentially expressed mRNAs mainly enriched in negative regulation of biological process,negative regulation of cellular process,intracellular,membrane-bounded organelle,DNA binding,protein dimerization activity and other functional modules.3.KEGG analysis hinted that the differentially expressed mRNAs possibly involved in Viral carcinogenesis,MAPK signaling pathway,MicroRNAs in cancer and Phospholipas D signaling pathways through SP100,DUSP5,DUSP2,PDGFRB and ARF1.4.The expression levels of randomly selected genes(SP100,DUSP5,DUSP2,PDGFRB and ARF1)were partially(5/8)consistent with high-throughput sequencing and were verified by the method of qRT-PCR,which suggested that the results of high-throughput sequencing were highly reliable.Summary:1.High-throughput transcriptome sequencing screened 670 transcripts that might be related to LINC00668.2.GO and KEGG pathway enrichment analyses showed that the mRNAs related to LINC00668 were enriched in various functional modules and signaling pathways.3.The results of high-throughput sequencing were verified by qRT-PCR with high reliability.Conclusions:Comprehensive analyses the above four parts,the following conclusions were deduced.1.Compared to adjacent normal tissues,the lncRNA profile was distinct in LSCC cancerous tissues.2.High expression levels of LINC00668 in LSCC cancerous tissues were associated with clinical stage,pathological differentiation degree and neck lymph node metastasis.3.In vitro experiments proved that LINC00668 could partly promote the proliferation,migration and invasion ability of LSCC cell lines.4.LINC00668 probably regulated Viral carcinogenesis,MAPK signaling pathway,MicroRNAs in cancer and Phospholipas D signaling pathways in LSCC by directly or indirectly interacting with targeted mRNAs(SP100,DUSP5,DUSP2,PDGFRB and ARF1).