| Laryngeal carcinoma is a malignant neoplasm,accounting for 1%-5% of malignant tumors,with an increasing morbidity trend.Ninety percent of laryngeal carcinomas belong to laryngeal squamous cell carcinoma(LSCC).Global LSCC-related mortality has not decreased even with progressive treatment options.Statistics showed that there were 177,422 new cases and 94,771 deaths worldwide in 2018,accounting for 1% of new cancer cases and 1% of cancer deaths,respectively.It is imperative to identify effective molecular markers for LSCC.Breakthroughs have been made in the study of protein-coding genes in malignant tumors,on which the advanced targeted tumor therapies have been based,such as imatinib for Bcr-Abl leukaemia or vemurafenib for BRAFV600 E melanoma.However,coding genomes only account for less than 2% of all sequences,and a large number of non-coding genes are drivers of cancer.Long non-coding RNAs(lnc RNAs)are defined as class of functional RNA molecules with a length of more than 200 nucleotides without protein-coding ability,and the "non-coding" designation refers to a lack of open reading frames and/or codon bias in transcripts.Lnc RNAs exhibit several distinctive characteristics,namely,tissue-and cell-specific expression and spatiotemporal specificity,regulatory diversity,and low sequence conservation.In recent years,many in-depth studies have unveiled the roles of lnc RNAs,demonstrating that lnc RNAs exert critical functions by participating in diverse biological functions,including X chromosome silencing,genomic imprinting,transcriptional activation,transcriptional interference,nuclear transport,and other important regulatory processes.Epigenetic alterations play an important role in human cancers.DNA methylation driven by DNA methyltransferases(DNMTs)has become increasingly recognized as a frequent event of epigenetic alterations and one of the primary mechanisms of gene dysfunction.Aberrant methylation of many genes,such as DNA damage repair genes,oncogenes,and genes related to tumor metabolism and invasion,contributes to carcinogenesis.In many somatic tumors,promoter hypermethylation can act as a first blow followed by a second hit in the form of mutations and deletions.In our study,Agilent SBC Human(4*180K)lnc RNA Microarray(ID: 74348)was used to detect the transcript expression profiles in 4 pairs of LSCC tissues and normal tissues.A new long non-coding RNA named LINC00886 caught our attention for its low expression in tumor tissues and containing a large Cp G island.LINC00886,a 3354 bp polyadenylated lnc RNA,is located on 3q25.31.The contribution of LINC00886 to LSCC remains largely unknown.In our study,we focused on the role of LINC00886 in LSCC.The results of the three parts are shown as follows: Part one The expression,methylation status of LINC00886 in LSCC, and the correlation with clinical characteristics.Objective: The expression level and methylation status of LINC00886 in LSCC tissues,corresponding adjacent normal tissues,and laryngeal cancer cell lines were investigated.The methylation status of every Cp G site in promoter and the effect of promoter methylation on transcriptional activity of LINC00886 were explored.The correlation between the expression level of LINC00886,promoter methylation status and clinicopathological significance in LSCC patients were analyzed respectively.Methods:1.Quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR)method was applied to investigate the expression level of LINC00886 in 62 pairs LSCC tissues,adjacent normal tissues,and 3 LSCC cancer cell lines(TU177,AMC-HN-8,TU686),as well as in cancer cell lines treated or untreated with DNA methylation transferase inhibitor 5-Aza-2'-deoxycytidine(5-Aza-d C).The correlation between the expression level of LINC00886 and clinicopathological significance was analyzed.2.A bisulfite genomic sequencing(BGS)assay was used to examine the methylation status of every Cp G site of LINC00886 in two laryngeal cancer cell lines(TU177,AMC-HN-8).3.The methylation-specific polymerase chain reaction(MSP)method was performed to detect the methylation status of LINC00886 in forty matched specimens and three laryngeal cancer cell lines(TU177,AMC-HN-8,TU686)before and after 5-Aza-d C treatment.The correlation between the methylation status of LINC00886 and clinicopathological significance was analyzed.4.A luciferase reporter gene assay was performed to detect the effect of promoter methylation on transcriptional activity of LINC00886 in vitro.5.Nuclear plasma separation was performed in TU177,AMC-HN-8,and TU686 cells,the expression level LINC00886 was evaluated by q RT-PCR in RNA isolated from nucleus or cytoplasm of cancer cell lines.Results:1.LINC00886 expression was downregulated in LSCC tissues and laryngeal cancer cell lines(P<0.01).The decreased expression of LINC00886 was correlated with pathological differentiation grade and was not correlated with age,smoking,TNM stage,and lymph node metastasis(P>0.05).The expression level of LINC00886 was significantly upregulated in cancer cell lines treated with 5-Aza-d C(P<0.01).2.Methylated Cp G sites were detected by BGS in two laryngeal cancer cell lines,densely methylated Cp G sites were observed in two regions(proximal promoter region and intron region),indicating the universality of aberrant hypermethylation of LINC00886 in laryngeal cancer cells.3.The methylation status was performed by MSP method in LSCC cell lines before and after 5-Aza-d C treatment.Fully methylation of region 2 and region 3 and partial methylation of region1 were detected before 5-Aza-d C treatment and the aberrant methylation was completely reversed in region 2 and region 3 and partially reversed in region 1 after 5-Aza-d C treatment.Then,methylation frequency of the three regions was detected by MSP method in all tissues.Of LSCC samples and adjacent normal tissues,methylation was observed in 45%(18/40)and 25%(10/40)at region 1,72.5%(29/40)and 30%(12/40)at region 2,and 67.5%(27/40)and 42.5%(17/40)at region 3.The region 2 and region 3 of LINC00886 revealed frequent methylation in tumor tissues(P<0.05).However,only the region 2 methylation status was associated with pathological differentiation(P<0.05).Furthermore,hypermethylation status of region 2 was correlated with decreased expression of LINC00886.4.Hypermethylation of the proximal promoter region of LINC00886 leads to a significant decrease in luciferase activity.5.LINC00886 is located in the nucleus of TU177,AMC-HN-8,and TU686 cells.Part two Effects of LINC00886 on biological characteristics of LSCCcellsObjective: To detect the effects of LINC00886 on biological characteristics of LSCC cells in vitro and in vivo.Method:1.TU177 and AMC-HN-8 cells with low expression of LINC00886 were selected for subsequent experiments.The pc DNA3.1-LINC00886 over-expression vector was transfected into AMC-HN-8 and TU177 cells.Transfection efficiency was evaluated by q RT-PCR method.2.Cell proliferation activity of cancer cell lines with LINC00886 over-expression was measured by MTS and clone formation assay in vitro.3.The migration and invasion abilities in transfected cells were assessed using transwell assay in vitro.4.Flow cytometry was applied to detect the cell cycle of cancer cell lines with LINC00886 over-expression.5.Animal xenograft model was developed to evaluate the effect of LINC00886 on tumor growth in vivo.Result:1.The expression levels of LINC00886 were increased significantly in transfected TU177 and AMC-HN-8 cells than that in empty vector control group evaluated by q RT-PCR method.The LINC00886 over-expression plasmid was established successfully.2.MTS and clone formation assay demonstrated that over-expression of LINC00886 significantly suppressed cancer cell proliferation ability in AMC-HN-8 and TU177 cells.3.In vitro transwell experiments confirmed that over-expression of LINC00886 significantly inhibited the migration and invasion ability of TU177 and AMC-HN-8 cells.4.Flow cytometry-based cell cycle analysis showed that over-expression of LINC00886 resulted in an increase in the proportion of cells in G1 phase and a decline of PI index.5.The animal xenograft experiments demonstrated that injection of stably transfected of LINC00886 cells significantly suppressed tumor growth compared with the empty vector group in vivo.Part three Prediction and screening of downstream target genes andsignaling pathways that LINC00886 may participate in andresearch on regulation mechanisms of LINC00886Objective: To investigate the downstream target genes and signaling pathways that LINC00886 may participate in and to explore the effect of LINC00886 on EMT.Method:1.The pc DNA3.1-LINC00886 over-expression vector was transfected into TU177 cell.Transfection efficiency was evaluated by q RT-PCR method.High-throughput sequencing technology was used to screen out differentially expressed m RNAs after over-expression of LINC00886.2.GO and KEGG functional enrichment analyses were carried out to predict possible biological processes and signaling pathways mediated by target m RNAs.3.Six m RNAs randomly selected for compensate the deficiencies of high-throughput sequencing using q RT-PCR method.4.q RT-PCR method was used to analyze the m RNA expression levels of molecular markers(E-cadherin,N-cadherin,Vimentin,?-catenin)of EMT in AMC-HN-8 and TU177 cells transfected with pc DNA3.1-LINC00886.5.The western blot analysis was used to analyze the protein expression levels of molecular markers(E-cadherin,N-cadherin,Vimentin,?-catenin)of EMT in AMC-HN-8 and TU177 cells transfected with pc DNA3.1-LINC00886.Results:1.The expression levels of LINC00886 were increased significantly in transfected TU177 cells than that in empty vector control group.High-throughput sequencing technology revealed 429 upregulated m RNAs,suggesting that these m RNAs may be positively regulated by LINC00886.Correspondingly,255 m RNAs were downregulated,indicating that these m RNAs may be negatively regulated by LINC00886.2.GO analysis revealed that aberrantly expressed m RNAs were mainly associated with three biological process,including positive regulation of multicellular organismal process,cell surface receptor signaling pathway,positive regulation of nitrogen compound metabolic process.Pathway analysis detected that LINC00886 may participate in P53 signal pathway,amino acid biosynthesis signal pathway,Fox O signal pathway,VEGF signal pathway and TNF signal pathway.3.Six m RNAs randomly selected for compensate the deficiencies of high-throughput sequencing using q RT-PCR method.Significant change in expression of VEGFA,PIK3R2,and DAPK2 was observed due to over-expression of LINC00886 using q RT-PCR method which was in exact accordance with the result of high-throughput sequencing.VEGFA,PIK3R2,and DAPK2 were mainly enriched in VEGFA/PI3K/AKT signaling pathways.Our findings implicated LINC00886 may as a regulator of the VEGFA/PI3K/AKT signaling pathways.A review through the literature revealed VEGFA/PI3K/AKT signaling pathway promotes cancer metastasis via three important mechanisms,including angiogenesis,tumor cell proliferation and induction EMT signaling,leading to invasion and metastasis of tumor cells.Subsequently,the expression of molecular markers of EMT was evaluated in the two cell lines transfected with pc DNA3.1-LINC00886 by q RT-PCR and WB respectively.4.The relative m RNA expression levels of mesenchymal markers were found to be remarkable downregulated for N-cadherin,Vimentin,and ?-catenin and upregulated for E-cadherin in the two cell lines transfected with pc DNA3.1-LINC00886.5.The same trend was found in the protein expression levels by WB experiment.Conclusions:1.LINC00886 showed markedly low expression in LSCC tissues and cell lines.The decreased expression of LINC00886 was correlated with pathological differentiation grade.2.Aberrant promoter hypermethylation was responsible for the downregulation of LINC00886.3.Over-expression of LINC00886 dramatically mitigated cell proliferation,migration,and invasion in vitro as well as suppressed tumor growth in vivo.It is suggested that LINC00886 served as a tumor suppressor gene in LSCC.4.LINC00886 may be associated with VEGFA/PI3K/AKT signaling pathways and EMT process. |
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