| Laryngeal cancer is one of the most common malignant tumors in head and neck cancers,and more than 95% are laryngeal squamous cell carcinomas(LSCC).In 2018,there were about 177,422 new cases of laryngeal cancer,accounting for 1% of all new cases and about 94,771 patients died of laryngeal cancer worldwide,accounting for 1% of all systemic cancer deaths.The incidence of LSCC is increasing annually worldwide.Despite the remarkable achievements in diagnosis and treatment,the prognosis of advanced laryngeal cancer is still not optimistic.Recurrence and metastasis are major factors limiting their successful treatment.About 60% of patients are already in advanced stage at the time of treatment.Therefore,it is particularly important to study the mechanisms of distant invasion and metastasis of LSCC.In recent years,the roles of epithelial-mesenchymal transition(EMT)in tumor progression have gradually attracted the attention of scholars.EMT is closely related to distant metastasis and tumor dissemination,and is one of the important mechanisms of tumor invasion and metastasis.A variety of growth factors and cytokines could induce epithelial mesenchymal transition,and transforming growth factor-?(TGF-?)is the most effective stimulating factor.As a complex cytokine,TGF-? regulates the development of EMT in different biological processes such as embryonic development,tissue repair and tumor metastasis.EMT is also involved in the development of laryngeal cancer,such as miR-203,miR-205,and LINC00339 regulated the progression of LSCC.EZH2 promoted the migration and invasion of LSCC through EMT process.Long non-coding RNAs(lncRNAs)are a type of RNA molecule that is more than 200 nucleotides in length and cannot encode proteins.LncRNA isclosely associated with tumor formation,invasion,and metastasis.In the present study,the differential expression of lncRNAs was detected by microarray assays,and lncRNA miR-155 host gene(MIR155HG)was up-regulated in LSCC tissues.Other related reports indicated that MIR155 HG is a mesenchymal-related lncRNA,but its role in the carcinogenesis and development of laryngeal cancer needs further study.MicroRNAs(miRNAs)are a class of single-stranded,non-coding RNAs with a length of about 18-25 nucleotides.They regulate the expression of their targeted gene on the level of post-transcriptional by complement to the 3'UTR,which is closely related to the occurrence and development of tumors.In recent years,the research on the relationship between lncRNA and microRNA has become a hotspot;especially lncRNA severs as a host gene,regulating the expression of microRNA.MIR155 HG is located on chromosome 21 and consists of 3 exons;miR-155-5p is located in its third exon.As the host gene of miR-155,the role of MIR155 HG in lymphoma and glioma has been revealed.This study was to explore the role of TGF-? in EMT of LSCC cell line TU177 and investigate the functions and mechanisms of MIR155HG/miR-155-5p on the malignant biological behavior of LSCC cells.The results of the four parts are reported as follows:Part one Screening of differentially expressed lncRNAs in metastatic LSCC,and induction of EMT and MIR155 HG in LSCC by TGF-?Objective: Screening of differentially expressed lncRNAs in metastatic LSCC by microarray expression profile technique.The correlation between the expression level of MIR155 HG and clinicopathological characteristics in LSCC patients was analyzed,and the induction of EMT and MIR155 HG in LSCC by TGF-? was explored.Methods:1.Four metastatic LSCC tissue and corresponding normal tissues were selected for microarray expression profile and screening of differentially expressed lncRNAs.2.The expression level of MIR155 HG in 45 pairs LSCC tissues and adjacent normal tissues was detected by quantitative real-time reverse transcription polymerase chain reaction(qRT-PCR)method,the association between the expression level of MIR155 HG and clinicopathological characteristics in LSCC was analyzed.3.TU177 cells were treated with 10 ng/ ml recombinant TGF-? for 7days,the morphological changes of TU177 cells were observed under an inverted microscope.The migration and invasion ability of TU177 cells treated with TGF-? were detected by Transwell assay.4.The expression levels of EMT-related markers and MIR155 HG in TU177 cells after treated with TGF-? were detected by qRT-PCR method.Results:1.A total of 3,073 differentially expressed lncRNAs were screened by microarray expression profile,including 1,967 up-regulated lncRNAs and1,106 down-regulated lncRNAs in LSCC tissues.2.The expression level of MIR155 HG in 45 pairs LSCC tissues was significantly higher than that in adjacent normal tissues(P<0.01).High expression level of MIR155 HG was associated with tissue differentiation,lymph node metastasis,and TNM stage and was not correlated with age,alcohol consumption or smoking(P>0.05).3.After treated with 10 ng/ ml TGF-beta for 7 days,TU177 cells gradually became spindle-shaped and slender,and showed obvious mesenchymal cell morphology.TGF-? can significantly promote the migration and invasion ability of TU177 cells.4.The mR NA expression levels of the mesenchymal-associated markers N-cadherin,Twist,Snail,Vimentin,and ZEB1 and MIR155 HG were up-regulated in TGF-? treated TU177 cells.Part two Effects of lncRNA MIR155 HG on malignant behavior of LSCC cellsObjective: To investigate the effects of MIR155 HG on the malignant biological behavior of LSCC cells in vitro and in vivo.Method:1.The expression level of MIR155 HG in different LSCC cell lines(TU177,TU686,and AMC-HN-8)was detected by qRT-PCR method.The expression level of MIR155 HG in head and neck squamous cell carcinoma tissues(HNSCC)was analyzed by online analysis software GEPIA.2.The protein coding ability of MIR155 HG was predicted by the online software Coding Potential Assessment Tool and ORF finder analysis.3.Four knockdown plasmids of sh-MIR155 HG and pcDNA3.1-MIR155 HG overexpression vector were constructed and were transfected into TU177 and AMC-HN-8 cells,respectively.The transfection efficiency was detected by qRT-PCR method.The sh-MIR155HG-1 with the best knockdown effect was selected for subsequent experiments.The proliferation,migration,and invasion ability of MIR155 HG knockdown and overexpression were detected by MTS assay,clone formation assay,and Transwell assay.4.The effect of overexpression of MIR155 HG in TU177 cells on the growth of xenografted tumor in nude mice was examined by in vivo xenograft experiments.5.The effect on the expression levels of EMT-related markers after MIR155 HG knockdown was examined by qRT-PCR method.Result:1.MIR155 HG was up-regulated in LSCC cells compared with normal cell pool(P<0.01).The results of GEPIA online analysis software showed that MIR155 HG was significantly up-regulated in head and neck squamous cell carcinoma tissues(P<0.01).2.The online software Coding Potential Assessment Tool and ORF finder demonstrate that MIR155 HG is a non-coding RNA.3.The expression level of MIR155 HG in TU177 and AMC-HN-8 cells was effectively knocked down by sh-MIR155HG-1(P<0.01).Transfection of pcDNA3.1-MIR155 HG significantly up-regulated the expression level of MIR155 HG in TU177 and AMC-HN-8 cells(P<0.01).In vitro experiments confirmed that knockdown of MIR155 HG significantly inhibited theproliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells(P<0.01).Overexpression of MIR155 HG significantly promoted the proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells(P<0.01).4.The in vivo xenograft experiments showed that the injection of stably transfected pcDNA3.1-MIR155 HG TU177 cells significantly increased the volume and weight of xenografts in vivo compared with negative control mice.5.Downregulation of MIR155 HG significantly down-regulated the mR NA expression levels of N-cadherin,Twist,Zeb1,and Vimentin detected by qRT-PCR methods(P<0.01).Part three The effect and mechanism of miR-155-5p on the malignant behavior of LSCC cellsObjective: To investigate the effect of TGF-? on the expression level of miR-155-5p and to explore the effect and mechanism of miR-155-5p on the malignant biological behavior of LSCC cells.Method:1.The expression level of miR-155-5p was detected by qRT-PCR method after treated with TGF-? in TU177 cells.2.QRT-PCR method was used to detect the expression level of miR-155-5p in 45 pairs of LSCC tissues and adjacent normal tissues.3.The association between the expression level of miR-155-5p and the clinicopathological characteristics of LSCC patients was analyzed.4.The miR-155-mimic and miR-155-inhibitor were synthesized and were transfected into TU177 and AMC-HN-8 cells,respectively,and the transfection efficiency was detected by qR T-PCR method.The MTS assay and Transwell assay were used to detect the effects of knockdown and overexpression of miR-155-5p on proliferation,migration,and invasion ability of LSCC cells.5.The effect of overexpression of miR-155-5p on the expression levels of EMT-related markers was examined by qRT-PCR method.6.SOX10 was predicted as the target gene of miR-155-5p by using bioinformatics tool TargetScan and miRwalk.The expression levels of SOX10 in 45 pairs of LSCC tissues and adjacent normal tissues were detected by qRT-PCR method.The correlation between the expression level of miR-155-5p and SOX10 was analyzed.Western blot and qRT-PCR method were used to confirm the regulation of miR-155-5p on the mR NA and protein expression levels of SOX10,and dual luciferase reporter assay was further used to demonstrate their targeting relationship.Results:1.The expression level of miR-155-5p was significantly increased after treated with TGF-? in TU177 cells compared with untreated group(P<0.01).2.MiR-155-5p was significantly up-regulated in LSCC tissues compared with corresponding adjacent normal tissues(P<0.01).3.High expression level of MIR155 HG was associated with tissue differentiation,lymph node metastasis,and TNM stage and were not associated with age,alcohol consumption or smoking(P>0.05).4.MiR-155-mimic can effectively upregulate the expression level of miR-155-5p in TU177 and AMC-HN-8 cells,while miR-155-inhhibitor can downregulate the expression of miR-155-5p in TU177 and AMC-HN-8 cells.In vitro experiments confirmed that overexpression of miR-155-5p significantly promoted the proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells(P<0.01).Knockdown of miR-155-5p significantly inhibited the proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells(P<0.01).5.Upregulation of miR-155-5p significantly up-regulated the mR NA expression of mesenchymal-associated markers N-cadherin,Vimentin,Twist,and Snail,but did not affect the expression level of ZEB1.However,the alteration of the mR NA expression level of epithelial marker E-cadherin was not detected.6.SOX10 has a potential binding site for miR-155-5p by searching bioinformatics website.Compared with adjacent normal tissues,SOX10 wassignificantly down-regulated in LSCC tissues(P<0.01).The results of qRT-PCR method showed that the mR NA and protein expression level of SOX10 was increased after miR-155-5p knockdown in TU177 and AMC-HN-8 cells.After overexpression of miR-155-5p,the expression level of SOX10 was decreased.The dual luciferase reporter assay showed that miR-155-5p could directly binding to the 3' untranslated region of SOX10.Part four miR-155-5p acts synergistically with MIR155 HG to promote LSCC malignant behavior and EMT processObjective: To explore the synergistic effects of miRNA-155-5p and MIR155 HG on LSCC malignant behavior and EMT process.MethodS:1.The correlation between the expression level of miRNA-155-5p and MIR155 HG was analyzed,and qRT-PCR method was used to detect the regulatory relationship between them.2.Explored the effects of co-transfection of miRNA-155-5p and MIR155 HG on proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells through MTS and Transwell assays.3.Relative expression levels of EMT marker after co-transfection of microRNA-155-5p and MIR155 HG in TU177 cells were detected by qRT-PCR method.Results:1.The expression level of miR-155-5p was significantly up-regulated after MIR155 HG overexpression in TU177 and AMC-HN-8 cells(P<0.01).The expression level of miR-155-5p was significantly down-regulated after MIR155 HG knockdown(P<0.01).The expression level of MIR155 HG was not affected after miR-155-5p knockdown or overexpression(P>0.05).2.Overexpression of miR-155-5p may abrogate the inhibitory effect of MIR155 HG knockdown on cell proliferation,migration,and invasion ability in TU177 and AMC-HN-8 cells.3.MiR-155-5p overexpression in TU177 cells partially reversed the inhibitory effect on mesenchymal markers,N-cadherin,Twist,Vimentin,exerted by MIR155 HG knockdown.Conclusions:1.TU177 cells treated with TGF-? exhibited EMT-related characteristics and promoted migration and invasion ability of TU177 cells.MIR155 HG and miR-155-5p were up-regulated by the induction of TGF-?.2.MIR155 HG was up-regulated in LSCC tissues and cells,and was associated with tissue differentiation,lymph node metastasis,and TNM stage.MIR155 HG significantly promoted the proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells in vitro,and promoted tumor growth in vivo.It is suggested that MIR155 HG was involved in the progression of LSCC.3.MiR-155-5p was up-regulated in LSCC tissues,and was associated with tissue differentiation,lymph node metastasis,and TNM stage.MiR-155-5p significantly promoted the proliferation,migration,and invasion ability of TU177 and AMC-HN-8 cells in vitro.It is suggested that MIR155 HG was involved in the progression of LSCC.4.MIR155 HG regulated the expression level of miR-155-5p and promoted proliferation,migration,and invasion ability of LSCC cells by targeting miR-155-5p/SOX10 axis.5.MIR155 HG and miR-155-5p synergistically promoted malignant behavior and EMT progression of LSCC cells. |
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