| Laryngeal squamous cell carcinoma(LSCC)accounts for about 2.4%of the world’s malignant tumors,and 90%of all malignant tumors of the larynx,Its incidence rate is second in all types of head and neck squamous cell carcinoma(HNSCC).In 2018,about 95,000 people died of laryngeal cancer.The onset of LSCC is secretive,and about 60%of patients are in advanced stage at the time of diagnosis(clinical stages III and IV).At the same time,LSCC is prone to local infiltration and cervical lymph node metastasis,which seriously affects the survival rate of patients.The treatment methods of LSCC include surgery,radiotherapy,chemotherapy and immunotherapy.Unfortunately,LSCC is one of the few tumors with declining survival rate in the past 40 years.The main reasons were unknown mechanism of its occurrence and development,and limited number of confirmed functional targets and potential mechanisms.Recently,precision medicine and targeted therapy have become a topic of increasing interest.Therefore,it is urgent to reveal the pathogenesis of LSCC,determine biomarkers for early diagnosis,and explore effective new therapeutic targets.Protein coding sequences account for less than 2%of the human genome,and the two DNA strands that can be transcribed in most of the remaining regions are RNAs.However,these RNAs cannot be translated into proteins,so they are called non-coding RNAs(nc RNAs).Nc RNAs are previously considered as non-functional molecules.However,recently,more and more evidence shows that nc RNAs play an important role in regulating protein expression and various biological processes.According to the length of nc RNAs,they can be divided into two types:long non-coding RNAs(lnc RNAs)defined as molecules with more than 200 nucleotides.and small non-coding RNA.As a kind of RNA transcripts with rich protein coding ability or no protein coding ability,lnc RNAs are receiving more and more attention in the scientific community.A large number of studies have reported that lnc RNAs are involved in a variety of biological processes,including immune response,inflammation and cancer progression.Epigenetics refers to potential heritable changes in gene expression,not due to potential changes in DNA sequences.One of controlling lnc RNA expression and the most common tissue-specific is epigenetic modification change.New studies have shown that lnc RNAs can play a role in gene expression through epigenetic regulation,such as DNA methylation and histone modification.DNA methylation is the earliest and most in-depth epigenetic research.The expression of hypermethylated and hypomethylated DNA can regulate the expression of oncogenes or tumor suppressor genes.In this study,lnc RNAs differentially expressed in LSCC tissues and corresponding non-tumor tissues were analyzed and screened by microarray analysis.p-value<0.05 and fold change<0.5 was the selection criteria,and according to a large number of literatures,LINC01554,which was lowly expressed in LSCC tissues,has entered our field of vision.LINC01554 is a1931 bp polyadenylated lnc RNA,and locate on 5q15(grch 38/hg38 database,chr5:95852232-9586013,NCBI:NR＿026936.1).At the same time,through the analysis of methprimer website,it is found that LINC01554 has two large Cp G islands.The low expression of LINC01554 in esophageal squamous cell carcinoma and hepatocellular carcinoma has been reported,However,little is known about its unction and underlying mechanism in LSCC.In this study,the expression of LINC01554 in LSCC tissues and corresponding non-tumor tissues,human LSCC cell lines(TU177,AMC-HN-8),the methylation status of Cp G islands site and the correlation with clinicopathological data were detected.The effect of LINC01554 on the malignant process of LSCC was studied through cell function and in vivo experiments,and the relevant mechanism of LINC01554 was further explored.LINC01554 played a relevant role by regulating target m RNA.At the same time,the effect of LINC01554 on the drug sensitivity of LSCC cells to cisplatin was detected.The results of the four parts of this subject were reported as follows:Part One Expression and methylation status of LINC01554 in laryngealsquamous cell carcinomaObjectives:To study the expression of LINC01554 in LSCC patients and human LSCC cell lines(TU177,AMC-HN-8),the relationship between expression level of LINC01554 and the clinicopathological data of LSCC patients was analyzed.Moreover,the methylation state of LINC01554 Cp G island was further analyzed,and the effect of methylation state on gene expression as well as the relationship with the clinicopathological data of LSCC patients,was explored.Methods:1.Four metastatic LSCC tissues and corresponding non-tumor tissues were selected for microarray analysis,and differentially expressed lnc RNAs were screened by microarray analysis.2.Real time quantitative polymerase chain reaction(RT-q PCR)was used to detect the expression level of LINC01554 in LSCC tissues and corresponding non-tumor tissues of 70 patients,and the correlation between LINC01554 and clinicopathological data of LSCC patients was analyzed.And the expression level of LINC01554 in TU177 and AMC-HN-8 cells were detected.3.To identify the subcellular location,the expression level of LINC01554 were detected in RNA isolated from nucleus or cytoplasm of TU177 and AMC-HN-8 cells by RT-q PCR.At the same time,the expression of LINC01554 in TU177 and AMC-HN-8 cells were observed before and after treatment with DNA methyltransferase inhibitor 5-Aza-2’-deoxycytidine(5-Aza-d C).4.Bisulfite genomic sequencing(BGS)assay was applied to detect the methylation status of Cp G islands regions of LINC01554 in TU177 and AMC-HN-8 cells,as well as in 2 LSCC tissues and corresponding non-tumor tissues.The Cp G island of LINC01554 was divided into two regions:proximal promoter region and exon region.5.Methylation specific polymerase chain reaction(MSP)method was applied to detect the methylation status of Cp G islands regions of LINC01554in TU177 and AMC-HN-8 cells treated or untreated with 5-Aza-d C,as well as in 28 LSCC tissues and corresponding non-tumor tissues.The correlation between LINC01554 methylation status and clinicopathological data in LSCC tissues and the effect of LINC01554 methylation status on its expression were analyzed.Results:1.A total of 3,073 differentially expressed lnc RNAs were screened by microarray analysis(p-value<0.05,Fold Change≥2 or≤0.5),including1,967 up-regulated lnc RNAs and 1,106 down-regulated lnc RNAs in LSCC tissues.The expression level of LINC01554 in LSCC tissues and corresponding non-tumor tissues was in consistence with results of TCGA database.LINC01554 was used for follow-up experiments.2.The expression level of LINC01554 in LSCC tissues was obviously lower than that in corresponding non-tumor tissues.The expression level of LINC01554 were low in TU177 and AMC-HN-8 cell lines.In tumor tissues,and the decreased expression of LINC01554 was found to be negatively correlated with TNM stage and pathological differentiation.3.LINC01554 was mainly located in the nucleus of TU177 and AMC-HN-8 cells.The expression level of LINC01554 was reversed after treatment with 5-Aza-d C.4.The results of BGS showed that there was hypermethylation in the proximal promoter region of LINC01554 in TU177 and AMC-HN-8 cells and LSCC tissues,but hypomethylation in corresponding non-tumor tissues.Although the exon region of LINC01554 was also hypermethylated,there was no differential expression between LSCC tissues and corresponding non-tumor tissues.5.MSP assay showed that the proximal promoter region of LINC01554was hypermethylated in TU177 and AMC-HN-8 cells,and the methylation degree decreased after 5-Aza-d C treatment.According to the analysis of 28pairs of LSCC tissues and corresponding non-tumor tissues,most of the Cp G islands in the proximal promoter region of LINC01554 were hypermethylated in LSCC tissues,while hypomethylated in corresponding non-tumor tissues.In tumor tissues,the methylation rate of region 2 of LINC01554 was significantly associated with TNM stage and pathological differentiation.The methylation rate of LINC01554 exon region had no significant difference in LSCC tissues and corresponding non-tumor tissues,and had no significant correlation with age,smoking,drinking,lymph node metastasis,TNM stage and differentiation.The methylation degree of LINC01554 proximal promoter was negatively correlated with expression,but not with the methylation and expression of exon region.Part Two Effect of LINC01554 on malignant progression and EMTprocess of LSCC cells in vivo and in vitroObjectives:To study the changes of malignant progression and EMT process of LSCC cells after overexpression of LINC01554 in vivo and in vitro.Methods:1.By constructing the plasmid of pc DNA3.1-LINC01554 and transfecting those into TU177 and AMC-HN-8 cells respectively,the TU177and AMC-HN-8 cells of overexpressing LINC01554 were made.The transfection efficiency was detected by RT-q PCR method.2.By MTS and clone formation assays,the proliferation ability of LSCC cells of overexpressing LINC01554 was detected,respectively.3.By wound healing assay,Transwell chamber migration and invasion assays,the migration and invasion ability of TU177 and AMC-HN-8 cells with LINC01554 overexpression was evaluated,respectively.4.The human LINC01554 gene was cloned into the lentivirus vector,and the lentiviral vector was transiently transfected into 293T cells.The viral supernatant fluid was collected to infect with TU177 cells.The stable overexpression of LINC01554 was detected by RT-q PCR.It was confirmed that the construction of stably overexpressing LINC01554 cell line was successful.Total 1×10~7TU177 cells suspended in 200μl serum-free RPMI-1640 was injected subcutaneously into the right armpit of male BALB/C nude mice(5 weeks).The xenograft tumor model in nude mice was established.5.After injection,the tumor was observed to decide which was successful.After 7 days,the tumor volume was measured every 3～4 days.The tumor volume was calculated as follows:V(volume)=(length×width~2)/2.After 28days,the mice were killed,the tumors were dissected,weighed,fixed,dewaxed,embedded,sectioned,and histologically analyzed.6.By RT-q PCR,the m RNA expression levels of EMT associated markers(E-cadherin,N-cadherin,Vimentin,Snail,Twist1,Zeb1,β-catenin)were detected in TU177 and AMC-HN-8 cells of overexpressing LINC01554.7.By western blotting,the protein expression levels of EMT associated markers(E-cadherin,N-cadherin,Vimentin)were detected in TU177 and AMC-HN-8 cells of overexpressing LINC01554.Results:1.The expression level of LINC01554 was greatly increased in TU177and AMC-HN-8 cells transfected with pc DNA3.1-LINC0154,which was verified by RT-q PCR method.The cells of overexpressing LINC01554 were constructed.2.The proliferation ability was decreased in TU177 and AMC-HN-8 cells of overexpressing LINC01554 by MTS and clone formation assays.3.Wound healing assay,Transwell chamber migration and invasion assays demonstrated that the over-expression of LINC01554 could inhibit the migration and invasion ability of TU177 and AMC-HN-8 cells in vitro.4.After TU177 cells were transfected with lentivirus vector oe-LINC01554,the expression of LINC01554 was significantly increased and stably expressed by RT-q PCR.The TU177 cell line of overexpressing LINC01554 and the xenograft tumor model in nude mice was successfully constructed.5.The volume of xenograft tumor formed by TU177 cells of overexpressing LINC01554 was significantly smaller than that in the control group,and the tumor weight was also significantly lower than that in the control group.6.H&E staining showed that overexpressing LINC01554 significantly reduced the number of lesions in tumor tissues.Immunohistochemical staining and RT-q PCR showed that the expression of proliferation marker Ki67decreased in xenografts with overexpressing LINC01554,which confirmed that its proliferation ability significantly decreased.7.After overexpressing LINC01554,the m RNA expression of E-cadherin increased in TU177 and AMC-HN-8 cells.In TU177 cells,The m RNA expression of N-cadherin,Vimentin,β-catenin decreased after overexpressing LINC01554,while in AMC-HN-8 cells,the m RNA expression of N-cadherin,Vimentin,β-catenin and Twist decreased.8.After overexpressing LINC01554 in TU177 and AMC-HN-8,the protein level of E-cadherin increased after overexpressing LINC01554,N-cadherin,Vimentin decreased after overexpressing LINC01554.Part Three Prediction of LINC01554 regulatory pathways and targetedm RNAs in laryngeal squamous cell carcinomaObjective:LINC01554 related pathways and targeted regulated m RNAs were searched,and RT-q PCR was used to verify the screened target m RNA.,and the possible related pathways and targeted m RNA of LINC01554 were explored.Methods:1.Through transcriptome high-throughput sequencing,the differentially expressed m RNAs were screened out in TU177 cell line with or without overexpressing LINC01554.2.GO and KEGG pathway analyses were used for functional enrichment analyses of differentially expressed m RNAs.3.By RT-q PCR,in TU177 cells overexpressing LINC01554,the expression of some selected target m RNAs(MAPK14,YWHAE,TEAD1)was verified.4.By RT-q PCR,after verified in TU177 cells,the expression of MAPK14 and YWHAE was further conformed in LSCC tissues and corresponding non-tumor tissues.5.In vitro functional recovery experiment,clone formation,Transwell migration and invasion assays were used to verify that LINC01554 inhibited the malignant progression of laryngeal squamous cell carcinoma cells through MAPK14 and YWHAE.Results:1.A total of 205 differentially expressed m RNAs were screened out through transcriptome high-throughput sequencing after overexpressing LINC01554:102 m RNAs were positively correlated with LINC01554expression level and 103 were negatively correlated with it.2.KEGG pathway analysis suggested that LINC01554 might be involved in the regulation of MAPK signaling pathway in LSCC through the mediation of target m RNA(MAPK14,YWHAE,TEAD1).3.In TU177 cells overexpressing LINC01554,the expression of the selected three m RNAs was verified.The expression trend of two m RNA(MAPK14,YWHAE)was consistent with the results of high-throughput sequencing.4.The detection results of MAPK14 and YWHAE in LSCC tissues and corresponding non-tumor tissues were also consistent with high-throughput sequencing,suggesting that high-throughput sequencing technology has certain applicability.LINC01554 was confirmed to involve in the regulation of LSCC through MAPK signaling pathway.5.Clone formation assay confirmed that knockdown of MAPK14 or overexpression of YWHAE partially reverses the suppressive effects of pc DNA3.1-LINC01554 on TU177 cell proliferation efficiency.6.Transwell migration and invasion assays confirmed that nockdown of MAPK14 or overexpression of YWHAE partially reverses the suppressive effects of pc DNA3.1-LINC01554 on TU177 cell migration and invasion efficiency.Part Four LINC01554 affected the stem cell characteristics and chemos-ensitivity of LSCC cellsObjective:After TU177 and AMC-HN-8 cells overexpressing LINC01554,the expression of stem cell characteristic related factors NANOG and SOX2 was verified by RT-q PCR.After TU177 and AMC-HN-8 cells were treated with different concentrations of cisplatin and uniform concentrations for different times,the expression of LINC01554 was detected respectively by RT-q PCR.After overexpressing LINC01554,TU177 and AMC-HN-8 cells were treated with cisplatin to observe the effect of cisplatin on proliferation in vitro.It was preliminarily investigated that LINC01554 might regulate the stem cell characteristics and chemosensitivity of LSCC cells.Methods:1.By RT-q PCR,the expression of NANOG and SOX2 was detected in TU177 and AMC-HN-8 cells overexpressing LINC01554.2.By RT-q PCR,after TU177 and AMC-HN-8 cells treated with different concentrations of cisplatin,the expression of LINC01554 was detected.3.By RT-q PCR,after TU177 and AMC-HN-8 cells treated with cisplatin for different times,the expression of LINC01554 was detected.4.By MTS and clone formation assays,after overexpressing LINC01554,TU177 and AMC-HN-8 cells were treated with cisplatin for 48 hours,the proliferation ability was detected,respectively.Results:1.In TU177 and AMC-HN-8 cells of overexpressing LINC01554,the expression levels of NANOG and SOX2 were significantly down-regulated.2.After TU177 and AMC-HN-8 cells treated with different concentrations of cisplatin,the expression of LINC01554 increased first and then decreased by RT-q PCR,reached the highest expression level at 0.003 and0.006μmol/ml,respectively.3.After the TU177 and AMC-HN-8 cells were treated with appropriate concentrations of cisplatin,the expression of LINC01554 was detected at different stimulation time points by RT-q PCR.The expression of LINC01554increased gradually and reached the highest at 48h.4.After overexpressing LINC01554,TU177 and AMC-HN-8 cells treated with cisplatin at the same time,MTS and clone formation assays confirmed that LINC01554 improved the drug sensitivity of cisplatin in the treatment of LSCC.Conclusions:1.The expression level of LINC01554 was significantly low in LSCC tissues and LSCC cell lines,abnormal hypermethylation of its proximal promoter region led to down-regulation of its expression,and the expression level and methylation status of LINC01554 in LSCC tissues were closely related to TNM stage and differentiation degree of tumor.2.LINC01554 could inhibit the proliferation,migration,invasion and EMT process of LSCC cells in vitro and in vivo,downstream target genes MAPK14 and YWHAE were involved in the inhibitory effect of LINC01554on LSCC cells.3.LINC01554 decreased the stem cell characteristics of LSCC cells and increased the drug sensitivity of chemotherapy drug cisplatin. |
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