| Objectives: Head and neck squamous cell carcinoma(HNSCC),which originates from the mucosal epithelium of the oral cavity,pharynx,and larynx,accounts for more than 90%of head and neck tumors and is the sixth most common cancer in the world.The incidence of HNSCC varies greatly in different countries and regions.Genetic predisposition,exposure to tobacco carcinogens,excessive alcohol consumption,and human papillomavirus(HPV)infection are established risk factors for HNSCC.Although the head and neck can be directly examined,most patients with HNSCC are diagnosed at an advanced stage.More importantly,although great progress has been made in diagnosing and treating HNSCC in recent decades,the 5-year overall survival rate of HNSCC patients is less than 50%.Therefore,studying the pathogenesis of HNSCC and identifying new and reliable molecular markers for early diagnosis and treatment of HNSCC patients are of great significance to improve patient survival.Long non-coding RNAs(lnc RNAs)are a class of 200-nucleotide heterogeneous transcripts that have no or lack protein-coding ability.Increasing evidence has shown that lnc RNA dysregulation is associated with tumorigenesis and progression.In addition,lnc RNAs have been shown to regulate signaling pathways related to autophagy,apoptosis,and cancer stemness.Mechanistically,lnc RNA can affect tumor cells’ proliferation,invasion,and migration by interacting with DNA,RNA,and protein.In this study,the differentially expressed lnc RNA were screened by bioinformatics analysis,among which NUTM2A-AS1 was significantly increased in HNSCC.Micro RNAs(miRNAs)are endogenous,single-stranded,non-coding small RNAs that regulate gene expression post-transcriptionally.miRNAs negatively regulate target m RNAs by binding to their 3’untranslated regions(3’UTR),inducing the degradation of target m RNAs and interfering with translation.Clinical research evidence has found that many miRNAs are up-regulated or down-regulated in a variety of cancers,and the expression level of some miRNAs is related to the stage of the disease.Therefore,these miRNAs can be used as cancer biomarkers and play a potential role in the diagnosis and prognosis of cancer.In recent years,competing for endogenous RNAs(ce RNA)has revealed a new mechanism of interaction between Rnas.Lncrnas can regulate downstream gene expression by competitively binding to miRNAs.Lysine-specific demethylase 2B(KDM2B),a member of the Jumonji C(Jmj C)domain-containing histone demethylases(JHDM)family,Gene transcription is regulated by demethylation of dimethyl histone H3 lysine 36(H3K36me2)and trimethyl histone H3 lysine 4(H3K4me3).It is now well established that aberrant expression of KDM2 B represses tumor suppressor genes and promotes oncogene expression,which results in uncontrolled cell growth and may lead to tumorigenesis.Studies have found that KDM2 B is overexpressed in a variety of cancers,which is an oncogene mediating cancer cell proliferation,metastasis,and apoptosis.The expression of KDM2 B is increased in glioma tissues.The overexpression of KDM2 B is related to the poor prognosis of glioma and mediates the growth of glioma cells.However,the role and regulatory mechanism of KDM2 B in HNSCC remains unclear.This study aims to investigate the effect of NUTM2A-AS1 on the proliferation,invasion,and migration of HNSCC by competitively binding to miR-129-5p and regulating lysine demethylase 2B(KDM2B)expression.Methods: Part I: 1.NUTM2A-AS1 expression in HNSCC.Bioinformatics was used to analyze the differentially expressed NUTM2A-AS1 in the TCGA database,and survival analysis was performed.NUTM2A-AS1 expression was validated in clinical samples:q RT-PCR was used to detect the difference in NUTM2A-AS1 expression level between cancer and adjacent normal mucosa tissues(n=30).NUTM2A-AS1 expression was validated in HNSCC cells in vitro: q RT-PCR was used to detect the expression difference between human normal oral keratinocytes HOK and HNSCC cells CAL27,SCC9,and SCC25.2.Effect of NUTM2A-AS1 on proliferation and metastasis of HNSCC cells.Construction of NUTM2A-AS1 knockdown HNSCC stable cell lines: The si RNA interfering sequence with the highest NUTM2A-AS1 knockdown efficiency was screened by si RNA transfection in CAL27 and SCC9 cell lines.The corresponding sh RNA was designed and assembled into the lentiviral vector,and the stable cell lines with NUTM2AAS1 knockdown were screened.Experimental groups: blank control group,NUTM2AAS1 empty vector group,NUTM2A-AS1 knockdown group.CCK-8 assay was used to detect the effect of NUTM2A-AS1 knockdown on the viability of HNSCC cells.Plate clone formation assay was used to detect the effect of NUTM2A-AS1 knockdown on the proliferation of HNSCC cells.The effect of NUTM2A-AS1 knockdown on the cell cycle of HNSCC cells was detected by flow cytometry.Western Blot was used to detect the expression levels of cell cycle-related proteins CDK4,CDK6,and Cyclin D1.The effect of NUTM2A-AS1 knockdown on the apoptosis level of HNSCC cells was detected by flow cytometry.The expression levels of apoptosis-related proteins Bax,Bcl2,and Caspase3 were detected by Western-Blot.Cell scratch assay was used to detect the effect of NUTM2A-AS1 knockdown on the migration ability of HNSCC cells.Transwell assay was used to detect the effect of NUTM2A-AS1 knockdown on the invasion of HNSCC cells.The expression levels of mmp2,mmp9,and timp2 were detected by Western-Blot.Western Blot was used to detect the expression levels of EMT-related proteins ECAD,NCAD,and Vimentin.3.Screening and identification of NUTM2A-AS1 downstream miRNAs.Part II: Prediction of NUTM2A-AS1 downstream target miRNAs;The correlation between NUTM2A-AS1 and miR-129-5p expression was verified: q RT-PCR was used to detect the difference in miR-129-5p expression level between cancer and adjacent normal tissues(n=30);q RT-PCR was used to detect the expression level of miR-129-5p in NUTM2A-AS1 knockdown cell lines.To verify the targeted binding of NUTM2A-AS1 miR-129-5p: Dual-luciferase reporter gene assay was used to verify the targeted binding ability of NUTM2A-AS1 and miR-129-5p.HNSCC cell lines CAL27 and SCC9 were transfected with miR-129-5p mimic and miR-129-5p inhibitor to verify the reversion effect of miR-129-5p inhibitor on HNSCC malignant changes caused by NUTM2A-AS1knockdown: Experimental groups: blank control group,NUTM2A-AS1 knockdown group,miR-129-5p inhibitor group,NUTM2A-AS1+miR-129-5p inhibitor group.Plate clone formation assay was used to detect the proliferation of HNSCC in different groups.Transwell assay was used to detect the difference in the invasion ability of HNSCC among different groups.The expression levels of ECAD,NCAD,and Vimentin proteins related to epithelial-mesenchymal transition(EMT)of HNSCC were detected by Western-Blot.4.Screening and identification of downstream m RNAs of miR-129-5p.Part III: Bioinformatics prediction of the downstream target m RNAs of miR-129-5p;To verify the correlation between miR-129-5p and KDM2 B expression: experimental groups: blank control group,scramble,miR-129-5p mimics,miR-129-5p inhibitor.q RTPCR was used to verify the expression level of miR-129-5p in each group.The expression of KDM2 B protein was detected by Western-Blot.The target binding ability of miR-129-5p and KDM2 B was verified by dual luciferase reporter gene assay.The reverse effect of KDM2 B overexpression on the malignant changes of HNSCC induced by transfection of miR-129-5p mimics was verified: experimental groups: blank control group,miR-129-5p mimics group,KDM2 B overexpression group,miR-129-5p mimics+KDM2B overexpression group.The overexpression efficiency of KDM2 B protein was detected by Western-Blot.Plate clone formation assay was used to detect the proliferation of HNSCC in different groups.Transwell assay was used to detect the difference in the invasion ability of HNSCC among different groups.The expression levels of ECAD,NCAD,and Vimentin in HNSCC were detected by Western-Blot.5,the recovery effect of NUTM2A-AS1 knockdown by overexpression of KDM2 B and the activation level of its downstream pathways.The reverse effect of KDM2 B overexpression on the malignant changes of HNSCC induced by NUTM2A-AS1 knockdown was verified in the following experimental groups: control group,NUTM2A-AS1 knockdown group,KDM2 B overexpression group,NUTM2A-AS1 knockdown +KDM2B overexpression group.Plate clone formation assay was used to detect the proliferation of HNSCC in different groups.Transwell assay was used to detect the difference in the invasion ability of HNSCC among different groups.The expression levels of ECAD,NCAD,and Vimentin in HNSCC were detected by Western-Blot.To verify the pathway link between KDM2 B downstream and EMT phenotype transition of HNSCC: Western-Blot assay was used to detect the expression levels of KDM2B/Slug/Vimentin axis-related proteins in each group.6.In vivo experimental verification.To verify the effect of NUTM2A-AS1 knockdown on the tumorigenesis of HNSCC cells,the HNSCC cells were divided into three groups: control group,NUTM2A-AS1 empty vector group,NUTM2A-AS1 knockdown group.The changes in tumor volume and weight were recorded.KDM2 B and Slug protein expression levels in each group were detected.Immunohistochemistry was used to detect each group’s ECAD,NCAD,and Vimentin expression.Results: Part I: NUTM2A-AS1 expression was significantly higher in HNSCC than in the adjacent normal mucosa(p<0.05);Survival analysis showed that the survival time of NUTM2A-AS1 high expression patients was significantly lower than that of NUTM2AAS1 low expression patients(p=0.04).Ten pairs of HNSCC cancer and adjacent tissues were collected clinically.q RT-PCR showed that NUTM2A-AS1 expression in HNSCC cancer tissues was significantly higher than that in adjacent normal mucosa tissues.q RTPCR showed that the expression of NUTM2A-AS1 in HNSCC cell lines CAL27,SCC9,and SCC25 was significantly higher than that in human normal oral keratinocytes HOK(p<0.05);NUTM2A-AS1 knockdown inhibited the viability of HNSCC cell lines CAL27 and SCC9.NUTM2A-AS1 knockdown inhibited the proliferation of HNSCC cell lines CAL27 and SCC-9.NUTM2A-AS1 knockdown caused cell cycle arrest in HNSCC cell lines CAL27 and SCC-9.NUTM2A-AS1 knockdown inhibited the migration ability of HNSCC cell lines CAL27 and SCC-9.NUTM2A-AS1 knockdown inhibited the invasion ability of HNSCC cell lines CAL27 and SCC-9.The results of WB showed that NUTM2AAS1 knockdown significantly inhibited the expression of cycle-related proteins CDK4,CDK6,and Cyclin D1 in HNSCC cell lines CAL27 and SCC-9.The expressions of invasion-related proteins MMP2,MMP9,NCAD,and Vimentin were significantly decreased,while TIMP2 and ECAD were significantly increased.Part II: NUTM2A-AS1 was screened to target miR-129-5p by Target Scan prediction.Dual-luciferase reporter gene assay showed that NUTM2A-AS1 could directly bind to miR-129-5p.TCGA(n=546)analysis showed that the expression of miR-129-5p in HNSCC was significantly lower than that in adjacent normal tissues(p<0.05);q RT-PCR detection of 30 pairs of HNSCC cancer tissues and adjacent normal tissues showed that the relative expression of miR-129-5p in cancer tissues was significantly decreased,with statistically significant differences(P<0.01);miR-129-5p inhibitor reversed NUTM2AAS1 knockdown induced proliferation inhibition in HNSCC.Transwell assay showed that miR-129-5p inhibitor could reverse the NUTM2A-AS1 knockdown-induced invasion inhibition of HNSCC.Inhibition of miR-129-5p reversed the inhibition of EMT induced by NUTM2A-AS1 knockdown in HNSCC.Part III: Target Scan was used to predict the m RNA expression of miR-129-5p:KDM2B;Dual-luciferase reporter gene assay showed that miR-129-5p could directly bind to KDM2 B.TCGA(n=546)analysis showed that the expression of KDM2 B in HNSCC was significantly higher than that in adjacent normal tissues(p<0.05);q RT-PCR was used to verify the expression efficiency of miR-129-5p mimics and inhibitor.WB showed that miR-129-5p mimics significantly decreased KDM2 B expression,while miR-129-5p inhibitors significantly increased KDM2 B expression.Colony formation assay showed that overexpression of KDM2 B could reverse the proliferation inhibition of HNSCC cells caused by miR-129-5p overexpression.Transwell assay showed that overexpression of KDM2 B could reverse the inhibition of HNSCC cell invasion caused by miR-129-5p overexpression.WB results showed that overexpression of KDM2 B could reverse the inhibition of EMT induced by miR-129-5p mimics in HNSCC.The results of WB showed that overexpression of KDM2 B could reverse the inhibition of EMT induced by NUTM2A-AS1 knockdown in HNSCC.Knockdown of NUTM2A-AS1 significantly reduced tumor volume and weight.Immunohistochemical results showed that compared with the sh-NC group,the AOD values of KDM2 B,NCAD,Vimentin,and Slug in the shNUTM2A-AS1 group was decreased,and the AOD value of ECAD was increased.Conclusions: NUTM2A-AS1 is up-regulated in HNSCC tissues and is positively correlated with poor prognosis in HNSCC patients.Nutm2a-as1 can directly bind to miR-129-5p,and play a role through the ce RNA mechanism to regulate the expression of KDM2 B,facilitate EMT,and promote the malignant biological behavior of tumor cells in HNSCC. |
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