| Laryngeal carcinoma is one of the most common malignant tumors in department of otolaryngology.The main pathological type of it is laryngeal squamous cell carcinoma(LSCC).According to clinically,it is mainly divided into three types: glottic,supraglottic and subglottic laryngeal cancer,among which,glottic type is the most common.In 2018,there were 177,422 new cases of laryngeal cancer worldwide,accounting for about 1% of new cases of systemic malignancy,and approximate 94,771 cases died because of this disease.The incidence of laryngeal cancer is increasing every year around the world.With the continuous improvement of people's requirements for the quality of life and the development of science and technology,people have a higher pursuit for the preservation and reconstruction of laryngeal function,and the treatment plan for laryngeal cancer is also constantly improved.However,recent studies have shown that overall survival rates for patients with laryngeal cancer have not improved significantly,or even decreased,and the prognosis for advanced laryngeal cancer is still not ideal.Clinically,recurrence and metastasis are the main factors,which limit the prognosis of patients with laryngeal cancer.About 60 percent of patients are advanced when they are diagnosed.Therefore,it is very important to explore the molecular mechanism of LSCC.In recent years,with the continuous development of science and technology,researchers have found that only less than 2% of humanRNA can encode and translate into proteins,and mostRNAs are unable to encode proteins.This type of gene is called non-codingRNA(ncRNA).Long non-codingRNA(lncRNA)is a type of non-codingRNA with a length of more than 200 nucleotides,which are involved in the regulation of the occurrence and development of tumors at the level of epigenetic and post-transcriptional regulation.They are closely related to tumor invasion and metastasis.In this study,lncRNAs with different expressions in LSCC tissues and its adjacent normal tissues were screened out through the analysis of gene microarray expression profiles,among which,lncRNA RASSF8-AS1 was significantly down-regulated in laryngeal cancer tissues,which attracted our attention.MicroRNA(miRNA)is a type of non-codingRNA with about 18-25 nucleotides.By binding to the 3'UTR(3'untranslated region)of their target mRNA molecules,miRNAs directly degrade mRNA or inhibit its synthesis.They can participate in the regulation of genes at the post-transcriptional level.In recent years,the mechanisms of lncRNAs interacting with miRNAs,especially the competitive mechanism of endogenousRNA(competitive endogenousRNA ceRNA)become the research hotspot.IncRNA can act as a ceRNA molecule and competitively bind to specific miRNA or multiple miRNAs,thereby reducing the regulation of miRNAs on their downstream target genes.The objective of this study was to explore a molecular mechanism: RASSF8-AS1 regulates TLE1 expression by competitively binding miR-664b-3p and thereby affects the malignant biological behavior of laryngeal squamous cell carcinoma.The research contents about this paper were displayed as follows:Part one Screening of RASSF8-AS1 and its expression in laryngeal squamous cell carcinomaObjective: Screening of lncRNAs differentially expressed by microarray expression technique from LSCC tissues and their normal tissue adjacent.We chosen RASSF8-AS1 as the research object and analyzed the relative expression of RASSF8-AS1 in the tissue and cells of LSCC.Analysing the correlation between the relative expression of RASSF8-AS1 with clinical pathological characteristics of LSCC patients.Methods:1.LncRNAs differentially expressed in laryngeal cancer tissues and adjacent normal tissues of 4 patients with LSCC were screened by microarray analysis.2.The quantitative real-time reverse tran-polymerase chain reaction(q RT-PCR)technique was taken to find the relative expression of RASSF8-AS1 in the cancer tissues and their corresponding adjacent normal tissues of 72 patients with LSCC.3.The differential expression of RASSF8-AS1 in four LSCC cell lines TU686,TU177,TU212,AMC-HN-8 and normal control group was detected by q RT-PCR.4.Gene Expression Profiling Interactive Analysis(GEPIA)was used to analyze the differential expression of RASSF8-AS1 in various tumor tissues and its normal control group.5.The correlation between expression level of RASSF8-AS1 in the tissues of patients with LSCC and its clinicopathological characteristics(such as TNM clinical stage,degree of pathological differentiation,cervical lymph node metastasis,age,smoking and alcohol consumption)were analysed.Results:1.3073 differentially expressed lncRNAs(Fold change?2)were screened from the microarray analysis,among which 1967 were up-regulated and 1106 were down-regulated.2.RASSF8-AS1 was selected from the above differential genes as the research object.The relative expression level of RASSF8-AS1 in 72 patients with LSCC tisssues was obviously lower than that in normal tissues(P <0.01).3.The relative expression level of RASSF8-AS1 in TU686 cell line was significantly lower than that in normal control group(P<0.01),and the same results were also obtained in TU177 cell line(P<0.01),TU212 cell line(P<0.01)and AMC-HN-8 cell line(P<0.01).4.The results of GEPIA analysis showed that the expression of RASSF8-AS1 in various tumor tissues was significantly lower than that in normal tissues.5.The low expression of RASSF8-AS1 in the cancer tissue with LSCC patients was closely related to its low differentiation,with lymph node metastasis and advanced TNM stages(P<0.05),however not related to the patient's age,drinking and smoking(P > 0.05).Part two Effects of RASSF8-AS1 on the biological characteristics ofLSCC cellsObjective: To investigate the effect of RASSF8-AS1 on the biological function of laryngeal squamous cell carcinoma cells in vitro.Methods:1.By constructing the plasmid of pc DNA3.1-RASSF8-AS1,and transfecting it and pc DNA3.1 into TU686 and TU177 cell lines respectively,the RASSF8-AS1 over-expressing cells TU686 and TU177 were made.The overexpression efficiency was detected by q RT-PCR method.2.In the cell experiment,pc DNA3.1-RASSF8-AS1 and p CDNA3.1groups were set,and Transwell chamber migration and invasion assays were used to verify the differences in the effects of the two groups on the migration and invasion ability of LSCC cell lines TU686 and TU177.3.In the cell experiment,p CDNA3.1-RASSF8-AS1 and p CDNA3.1groups were set,and MTS and clone formation assays were used to verify the differences in the effects of the two groups on the proliferation ability of LSCC cell lines TU686 and TU177.Results:1.LSCC cell lines TU686 and TU177 were respectively transfected with plasmid pc DNA3.1-RASSF8-AS1 or pc DNA3.1,and q RT-PCR result showed that the expression level of RASSF8-AS1 was greatly increased in TU686 and TU177 cell lines transfected with pc DNA3.1-RASSF8-AS1.Cell lines over-expressing RASSFF8-AS1 were established.2.In vitro cell experiment: Transwell migration and invasion assays verified that the migration and invasion abilities of TU686 and TU177 cells after transfection of pc DNA3.1-RASSF8-AS1 were significantly decreased compared with the pc DNA3.1 group,and the results were statistically different(P<0.01).3.In vitro cell experiment: MTS and clone formation assays verified that the proliferation ability of TU686 and TU177 cells after transfection of pc DNA3.1-RASSF8-AS1 were significantly reduced compared with the pc DNA3.1 group,and the results were statistically different(P<0.01).Part three Selection and identification of RASSF8-AS1 targeting miRNA and the effect of miR-664b-3p on the biological function of laryngeal squamous cell carcinoma cellsObjective: To investigate the regulatory relationship between RASSF8-AS1 and targeted miRNA(miR-664b-3p),and the effect of miR-664b-3p on the biological function of LSCC cells.Method:1.Nuclear and cytoplasmicRNAs of LSCC cell lines AMC-HN-8,TU177,TU212 and U686 were extracted by cytoplasmic and nuclearRNA isolation kits,respectively.The subcellular localization of RASSF8-AS1 was detected by q RT-PCR.2.Through biological information prediction software Target Scan(http://www.targetscan.org/)and DIANA(http://www.microrna.gr/micro TCD S)to predict RASSF8-AS1 targeted miRNA and determine their binding sites.3.The pmiRLO-RASSF8-AS1-WT and pmiRLO-RASSF8-AS1-MUT plasmids were constructed,and the targeted binding relationship between miR-664b-3p and RASSF8-AS1 was detected by dual luciferase reporter gene assay in TU177 and 293 T cells.4.RNA immunoprecipitation(RIP)based on MS2-binding sequenceMS2-binding protein(MS2BS-MS2bp)was used to detect the binding relationship between RASSF8-AS1 and miR-664b-3p in LSCC cell line TU177.5.The changes of miR-664b-3p expression in LSCC cell lines TU686 and TU177 were detected by q RT-PCR after transfection of pc DNA3.1-RASSF8-AS1 and pc DNA3.1,respectively.6.Pearson correlation statistical method was used to analyze the relative expression levels of RASSF8-AS1 and miR-664b-3p in 72 patients with laryngeal cancer.7.QRT-PCR method was taken to verify the expression of miR-664b-3p in tumor tissues and their corresponding normal tissues of 72 patients with LSCC.8.The analogue of miR-664b-3p and inhibitor of miR-664b-3p were synthesized and transfected into LSCC cell lines TU686 and TU177,respectively.The expression of miR-664b-3p was detected by q RT-PCR method.9.Transwell migration and invasion assays were taken to detect the effects of up-regulation and down-regulation of miR-664b-3p on the migration and invasion ability of LSCC cells.MTS and clone formation assays were used to verify the effects of up-regulation and down-regulation of miR-664b-3p on the proliferation ability of LSCC cells.10.The effects of transfection of miR-664b-3p-mimic or co-transfection of miR-664b-3p-mimic and RASSF8-AS1 on the proliferation,migration and invasion abilities of LSCC cell lines TU686 and TU77.These were detected by MTS,clone formation,Transwell chamber migration and invasion assays.Results:1.RASSF8-AS1 was expressed in both the nucleus and cytoplasm of the four laryngeal cancer cells TU686,TU177,TU212 and AMC-HN-8.The expression level in the cytoplasm was slightly higher than that in the nucleus.2.Bioinformatics prediction showed that miR-664b-3p had potential binding sites with RASSF8-AS1,and their binding sites were analyzed.3.The dual luciferase reporter gene assay showed that compared with the NC group,the luciferase activity was significantly decreased in TU177 and293 T cells by co-transfection of pmir GLO-RASSF8-AS1-WT plasmid and miR-664b-3p-mimic(P<0.01).On the other hand,luciferase activity was significantly enhanced after co-transfection of pmir GLO-RASSFF8-AS1-WT plasmid and miR-664b-3p-inhibitor(P<0.01).There was no significant change in luciferase activity in each group after mutation of pmir GLO-RASSF8-AS1-WT(P>0.05).4.RNA immunoprecipitation(RIP)test results showed that the enrichment of miR-664b-3p in the transfected pc DNA3.1-RASSF8-AS1-MS2(12X)plasmid group was significantly higher than that in the transfected pc DNA3.1-RASSF8-AS1-MS2(12X)-MUT plasmid group(P<0.01).5.q RT-PCR result showed that the expression of miR-664b-3p in LSCC cell lines TU686 and TU177 was significantly decreased after up-regulation of RASSF8-AS1.6.Pearson analysis showed that RASSF8-AS1 was negatively correlated with the expression of miR-664b-3p in LSCC tumor tissues(r=-0.5099,P<0.01).7.The expression level of miR-664b-3p in the cancer tissues of 72 patients with LSCC was obviously higher than that in the adjacent normal tissues(t=4.542,P < 0.01).8.QRT-PCR results verified that transfection of miR-664b-3p-mimic could significantly enhance the expression level of miR-664b-3p in TU686 and TU77,while transfection of miR-664b-3p-inhibitor could significantly down-regulate the expression level of miR-664b-3p in TU686 and TU177.9.In vitro experiments confirmed that over-expression of miR-664b-3p significantly promoted the proliferation,migration and invasion abilities of LSCC cell lines TU686 and TU177(P<0.01),and knockdown of miR-664b-3p obviously inhibited the migration,invasion and proliferation abilities of TU686 and TU177 cells(P<0.01).10.The promotion effect of over-expression of miR-664b-3p on proliferation,migration and invasion of TU686 and TU177 cells was partially reversed after co-transfection of RASSF8-AS1 and miR-664b-3p-mimic(P<0.01).Part four The prediction,screening and identification of downstream target gene of miR-664b-3p in laryngeal squamous cell carcinoma and the effect of TLE1 on the biological function of laryngeal squamous cell carcinomaObjective: To investigate the regulatory relationship between TLE1 and miR-664b-3p and the effect of TLE1 on the biological function of LSCCcells.Methods:1.Through biological information prediction software Target Scan(http://www.targetscan.org/)and DIANA(http://www.microrna.gr/micro TCD S)to predict miR-664b-3p targeted mRNA and determine their binding sites.2.Mi R-664b-3p-mimic and miR-664b-3p-inhibitor were transfected into LSCC cell lines,and then the expression level of TLE1 was detected by q RT-PCR in TU686 and TU177 cell lines.3.Western blot was used to analyze the effects of over-expression miR-664b-3p and down-regulation miR-664b-3p on TLE1 protein expression in TU177 and TU686 cell lines.4.QRT-PCR was used to detect the expression of TLE1 in carcinoma tissues and adjacent normal tissues of 72 patients with LSCC.5.Pearson correlation statistical method was used to analyze the relative expression levels of TLE1 and miR-664b-3p in 72 patients with laryngeal cancer.6.The pmiRLO-TLE1-WT and pmiRLO-TLE1-MUT plasmids were constructed,and the targeted binding relationship between miR-664b-3p and TLE1 was detected by dual luciferase reporter gene assay in TU177 and 293 T cells.7.The expression of TLE1 after up-regulation of RASSF8-AS1 was verified by q RT-PCR in LSCC cell lines TU686 and TU177.8.The protein expression of TLE1 after over-expression of RASSF8-AS1 was analyzed in LSCC cell lines TU686 and TU177 by western blot.9.The effects of transfection of pc DNA3.1-RASSF8-AS1 or co-transfection of sh-TLE1 and pc DNA3.1-RASSF8-AS1 on the proliferation,migration and invasion abilities of LSCC cell lines TU686 and TU77 were detected by MTS,clone formation,Transwell chamber migration and invasion assays.Results:1.The potential binding relationship of miR-664b-3p and TLE1 were predicted by bioinformatics,and the binding sites were analyzed.2.QRT-PCR results showed that the expression of TLE1 was significantly decreased after transfection of miR-664b-3p-mimic in LSCC cell lines TU686 and TU177 respectively,while the expression of TLE1 was significantly increased after down-regulation of miR-664b-3p compared with the normal control group(P < 0.01).3.Western blot results showed that the protein expression level of TLE1 was significantly decreased after transfection of miR-664b-3p-mimic in LSCC cell lines TU686 and TU177(P<0.01),and the protein expression level of TLE1 was significantly increased after transfection miR-664b-3p-inhibitor in LSCC cell lines TU686 and TU177(P < 0.01).4.The relative expression level of TLE1 in 72 patients with LSCC was obviously lower than that in adjacent normal tissues(P <0.01).5.Pearson analysis result showed that TLE1 was negatively correlated with the relative expression of miR-664b-3p in 72 LSCC cancer tissues(r=-0.4906,P<0.01).6.The dual luciferase reporter gene assay showed that compared with the NC group,the luciferase activity was significantly decreased in TU177 and293 T cells by co-transfection of pmir GLO-TLE1-WT plasmid and miR-664b-3p-mimic(P<0.01).On the other hand,luciferase activity was significantly enhanced after co-transfection of pmir GLO-TLE1-WT plasmid and miR-664b-3p-inhibitor(P<0.01).There was no significant change in luciferase activity in each group after mutation of pmir GLO-TLE1-WT(P>0.05).7.QRT-PCR results showed that the expression of TLE1 in LSCC cell lines TU686 and TU1771 was increased after up-regulation of RASSF8-AS1.8.Western blot results showed that TLE1 protein expression was increased after over-expression RASSF8-AS1 in LSCC cell lines TU686 and TU177.9.The inhibiting effect of over-expression of RASSF8-AS1 on proliferation,migration and invasion of TU686 and TU177 cells was partially reversed after co-transfection of pc DNA3.1-RASSF8-AS1 and sh-TLE1.Conclusions:1.The expression of RASSF8-AS1 was significantly down-regulated in LSCC samples and cells,and the expression of RASSF8-AS1 in cancer tissues was closely correlated with TNM clinical stage,degree of pathological differentiation and cervical lymph node metastasis in LSCC patients.2.Over-expression of RASSF8-AS1 could inhibit the proliferation,migration and invasion of LSCC cells,which suggested that RASSF8-AS1 is involved in the progression of laryngeal squamous cell carcinoma.3.RASSF8-AS1 was negatively correlated with miR-664b-3p,and over-expression of RASSF8-AS1 could weaken the carcinogenic effect of miR-664b-3p.4.RASSF8-AS1 regulatesd TLE1 expression through miR-664b-3p,thereby inhibiting the proliferation,migration and invasion ability of LSCC cells. |
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