Analysis Of Differentially Expressed LncRNA Transcripts In Human Oral Squamous Cell Carcinoma And Its Related Functional Verification

Oral squamous cell carcinoma(OSCC)has strong invasiveness and high mortality.Although the diagnosis and treatment of OSCC has achieved outstanding results,the life quality of OSCC patients in the middle and late stages is still very poor.In order to expand the treatment of OSCC,improve the survival time of OSCC patients,we will explore the molecular mechanism of OSCC and find the biomarkers and targets for diagnosis and prognosis.In recent years,the progress of genome microarray and whole genome sequencing technology has provided the genomics evidence of genes,which is conducive to the comprehensive study of genes from the whole point of view.The researchers find that non-coding RNA plays an important role in growth,development and pathology,especially long non-coding RNA(LncRNA)regulates the occurrence,metastasis and recurrence of malignant tumors,and even participates in drug resistance of tumors.Therefore,it is necessary to explore the mechanism of lncRNA in the pathogenesis of OSCC.For this purpose,lncRNA expression microarray screened the difference expression of lncRNA in OSCC and paired normal tissue samples.Discovered the node lncRNA in the pathogenesis of OSCC according to the construction of the co-expression network of differentially expressed genes,and the relationship between the node lncRNA and the clinicopathological data of OSCC was analyzed.LncRNA in co-expression network was selected for cell function test.The effects of lncRNA on proliferation,invasion and apoptosis of OSCC were observed and the possible mechanism was studied.This study will help to reveal the mechanism of the progression of OSCC,and provide an experimental basis for the exploration and development of biomarkers for the diagnosis and prognosis of OSCC and the exploration and development of targeted drugs.Part one Screening and validation of lncRNA differential expression profile in human oral squamous cell carcinomaObjective: In order to investigate the expression of lncRNA in OSCC,the expression profile of lncRNA in OSCC was detected by lncRNA expression microarray,and explored whether there are differences of lncRNA and mRNA in OSCC and paired normal tissue.Methods: The RNA was extracted from tumor tissues and paired normal tissues of 3 OSCC patients.The differential expression profiles of lncRNA and mRNA in OSCC were constructed by lncRNA expression microarray.The co-expression network of differentially expressed lncRNAs and mRNAs were established.The biological function of differentially expressed lncRNAs was predicted by GO and KEGG enrichment analyses.The target genes for differential expression of lncRNAs were predicted by regulation of cis and trans.Partial differentially expressed lncRNAs were selected to verify the microarray results by qRT-PCR technique.Results:1.The results of microarray assays showed that 2,294 differentially expressed lncRNAs and 1,938 differentially expressed mRNAs were identified.Furthermore,933 lncRNAs and 891 mRNAs were up-regulated and 1,361 lncRNAs and 1,047 mRNAs were down-regulated.2.The results of GO annotation showed that the differentially expressed genes were enriched in molecular function(MF),biological process(BP)and cellular component(CC).Furthermore,the top 3 on the list of enrichment were interleukin-1 binding(GO:0019966;Ontology: molecular function;P=0.0003),response to interferon-alpha(GO:0035455;Ontology: biological process;P=1.37E-10)and establishment of epithelial cell apical/basal polarity(GO:0045198;Ontology: biological process;P=0.0003).The results of the KEGG analysis demonstrated that differentially expressed mRNAs were mainly enriched in 38 biological pathways,including many cancer-related pathways,e.g.,“pathways in cancer”(enriched with 36 differentially expressed genes),“bladder cancer”(enriched with 8 differentially expressed genes),“metabolic pathways”(enriched with 109 differentially expressed genes),“pancreatic cancer”(enriched with 11 differentially expressed genes)and“PPAR signaling pathway”(enriched with 11 differentially expressed genes).3.The results of target gene prediction revealed that 1,470 dysregulated lncRNAs were identified to have cis or trans target genes,including 1,356 lncRNAs targeting 1,250 cis-genes,370 lncRNAs targeting 2,454 trans-genes and 256 lncRNAs targeting both cis and trans target genes.4.Through construction of co-expression network of differentially expressed lncRNAs and mRNAs,we identified 306 differentially expressed lncRNAs interacting with other selected mRNAs and lncRNAs.According to the results,TMPRSS11BNL?lnc-WRN-10:1 were the lncRNAs with most frequent interactions.These two lncRNAs were node genes for the entire network.5.Ten differentially expressed lncRNAs were selected for qRT-PCR verification in 72 cases of OSCC and paired normal tissues.The results showed that lncMANSC4-8:1,CXCR2P1,NRIR,lnc-CMPK2-1:3 and lnc-GLI3-4:1 were up-regulated and TMPRSS11 BNL,MEG3,lnc-WRN-10:1,DANCR and lnc-TPP2-7:2 were down-regulated in OSCC.It was suggested that the results of verification and microarray were consistent.Summary:1.A total of 2294 differentially expressed lncRNAs and 1938 differentially expressed mRNAs genes were screened,and the down-regulated expression genes were more than up-regulated genes.2.LncRNA TMPRSS11 BNL,lnc-WRN-10:1 were node genes for the entire network.3.The result of qRT-PCR was consistent with the result of microarray,which indicated that the result of microarray was reliable.Part two Expression and significance of long non-coding RNA TMPRSS-11 BNL,lnc-WRN-10:1 in human oral squamous cell carcinomaObjective: To investigate the differential expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 in OSCC and paired normal tissue,and to analyze the relationship between the expression level and clinicopathological features of OSCC.Methods: From January 2015 to October 2016,72 cases of OSCC and paired normal tissues were resected in the Fourth Hospital of Hebei Medical University.The expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 was detected by qRT-PCR method.The relationship between the expression level and clinicopathological features and prognosis of the patients was analyzed.The statistical software SPSS 22.0 was used to analyze the data,t-test was used to measure the data,Chi-square test was used to count the data,and Kaplan-Meier method and Log-rank test were used in survival analysis.The difference was statistically significant(P<0.05).Results:1.The expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 was lower in OSCC tissues.2.The expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 was not correlated with age,sex,smoking,tumor location,but correlated with clinical stage,lymph node metastasis,distant metastasis and survival status of OSCC patients.3.OSCC patients with lower expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 had shorter progression-free survival time and overall survival time.Summary: The lower expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 in OSCC tissues was associated with clinical stage,lymph node metastasis,distant metastasis and survival status.OSCC patients with lower expression of lncRNA TMPRSS11 BNL,lnc-WRN-10:1 had shorter progression-free survival time and overall survival time.Part three Effect of long non-coding RNA MEG3 on biological function of Tca8113 cells in oral squamous cell carcinomaObjective: To investigate the effect of lncRNA MEG3 on the biological function of OSCC Tca8113 cells.Methods: LncRNA MEG3 was overexpressed in OSCC Tca8113 cells by lipofectamine transfection.MTT,Transwell migration and invasion assay,and flow cytometry was used to detect the changes of proliferation,migration,invasion and apoptosis of OSCC Tca8113 cells.Western blot and qRT-PCR were used to observe the effect of lncRNA MEG3 overexpression on p53 protein expression.Results:1.The expression of lncRNA MEG3 was lower in OSCC Tca8113 cells.2.Overexpression of lncRNA MEG3 could significantly inhibit the proliferation,migration and invasion of OSCC Tca8113 cells,lead to apoptosis and promote the expression of p53 protein.Summary: The overexpression of lncRNA MEG3 in OSCC Tca8113 cells could significantly inhibit the biological behavior of OSCC Tca8113 cells.Conclusion:1.The 2294 differentially expressed lncRNAs and 1938 differentially expressed mRNAs were screened by microarray.The down-regulated expression genes were more than the up-regulated expression genes,which indicated that the down-regulated expression genes might play a more important role in the pathogenesis of OSCC.2.The result of qRT-PCR was consistent with the result of microarray,which indicated that the result of microarray was reliable.3.LncRNA TMPRSS11 BNL,lnc-WRN-10:1 might be a key node gene in the pathogenesis of OSCC,and were closely related to clinical stage,lymph node metastasis,distant metastasis and survival status..4.LncRNA MEG3 played an anti-oncogene role and could significantly inhibit the biological activity of OSCC Tca8113 cells.