【作者】 王迪；

【导师】 于丹；

【作者基本信息】 吉林大学 ， 耳鼻咽喉科学（专业学位）， 2024， 博士

【摘要】 第一部分应用生物信息学方法筛选HNSCC中SOX2-OT相关的ceRNA调控轴目的通过生物信息学分析和ceRNA理论构建以SOX2-OT为中心的ceRNA调控网络,筛选与预后密切相关的基因,挖掘头颈部鳞状细胞癌(Head and neck squamous cell carcinomas,HNSCC)治疗相关靶点,为治疗提供新的依据。方法下载UCSC数据库收录的来自TCGA的HNSCC基因表达谱数据和临床生存数据用作分析数据集,NCBI GEO数据库下载HNSCC基因芯片表达谱数据用来验证ceRNA网络中mRNA表达差异。通过limma包筛选得到差异表达mRNA(Differentially expressed mRNA,DEmRNA)和差异表达miRNA(Differentially expressed miRNA,DEmiRNA),使用DAVID对上述得到的DEmRNAs进行GO以及KEGG富集分析,重点关注免疫相关的通路。NCBI数据库下载SOX2-OT的RNA序列,使用Miranda筛选miRNA-lncRNA关系对,利用mi RWalk3.0数据库预测miRNA-mRNA关系对,将mRNA进一步与DEmRNAs取交集,进一步得到DEmiRNAs-DEmRNAs关系对,分析SOX2-OT与DEmRNAs的表达相关性,进一步获取lncRNA-mRNA正向共表达关系对,从而筛选lncRNA-miRNA-mRNA关系对,并进行ceRNA网络构建,通过Cytoscape软件进行,并结合STRING数据库预测分蛋白互作关系。最后,进一步找到在两套GEO数据集中均显著差异并且上下调和TCGA均保持一致的mRNA,并进行生存分析,筛选得出关键基因,并找到关键基因对应的lncRNA-miRNA-mRNA-pathway关系。结果1.共得到4433个DEmRNAs,309个DEmiRNAs,DEmRNAs显著富集了672个生物过程(Biological process,BP)、198个细胞组分(Cellular component,CC)、168个分子功能(Molecular function,MF)以及98条KEGG通路。2.预测得到了68个lncRNA-miRNA关系对,17109个DEmiRNAs-DEmRNAs关系对,1781个正向共表达的lncRNA-mRNA关系对,通过整合,最终获得了538个lncRNA-miRNA-mRNA关系,即ceRNA关系对。其中包含了我们所关注的SOX2-OT,对应65个miRNA和116个mRNA。3.对上述ceRNA网络中116个mRNA进行蛋白互作分析,一共得到了312个蛋白互作关系对,共包含97个蛋白节点。4.共筛选出20个mRNA作为关键基因,进一步找到20个关键mRNA对应的lncRNA-miRNA-mRNA-pathway关系,其中六个DEmRNAs与SOX2-OT在关键途径中相关,最后本研究选择SOX2-OT/let-7c-3p/SKP2调控轴进行深入研究。结论本研究成功构建以SOX2-OT为中心的ceRNA调控网络,并确定SOX2-OT/let-7c-3p/SKP2轴在HNSCC的发生发展中发挥调控作用,具体作用机制将在接下来的研究中进行进一步验证。第二部分SOX2-OT、let-7c-3p、SKP2在HNSCC中的表达及临床意义目的探究SOX2-OT、let-7c-3p及SKP2表达水平与HNSCC患者临床病理参数及生存预后之间的相关性。方法通过RT-qPCR检测SOX2-OT、let-7c-3p及SKP2在30对配对癌组织与癌旁组织中的表达情况。为了初步验证SOX2-OT、let-7c-3p及SKP2之间的相关性,通过Pearson相关性分析对三者表达水平进行相关性分析。进一步探讨了在不同临床分期、不同T分期及是否伴有淋巴结转移的HNSCC患者中SOX2-OT、let-7c-3p及SKP2的表达情况,明确三者在HNSCC中发挥的作用。最后,为了研究SOX2-OT、let-7c-3p及SKP2表达水平对HNSCC患者临床预后的影响,根据表达水平的中位数,所有患者被划分为低表达组与高表达组,并通过Kaplan-Meier生存分析比较两组之间总体生存率(overall survival,OS)及疾病特异性生存率(Disease-specific survival,DSS)是否存在差异。结果1.SOX2-OT在HNSCC组织中表达水平显著升高(p<0.0001),let-7c-3p在HNSCC组织中表达显著降低(p<0.0001),SKP2 mRNA在HNSCC组织中表达水平显著升高(p<0.0001),SKP2蛋白水平在HNSCC组织中同样表达显著升高(p<0.0001)。2.SOX2-OT表达水平与let-7c-3p表达水平存在负相关(r=-0.4633,p=0.0099),let-7c-3p表达水平与SKP2表达水平同样存在负相关(r=-0.5351,p=0.0023),而SOX2-OT表达水平与SKP2表达水平存在正相关(r=0.5036,p=0.0048)。3.临床分期为III+IV期患者与I+II期患者相比,SOX2-OT表达水平显著升高(p<0.01),let-7c-3p表达水平显著降低(p<0.05),SKP2表达水平显著升高(p<0.001)。淋巴结阳性患者与淋巴结阴性患者相比,SOX2-OT表达水平显著升高(p<0.05),let-7c-3p表达水平显著降低(p<0.01),SKP2表达水平显著升高(p<0.001)。T分期为T3+T4期患者与T1+T2期患者相比,SOX2-OT表达水平显著升高(p<0.05),let-7c-3p表达水平显著降低(p<0.05),SKP2表达水平显著升高(p<0.001)。4.SOX2-OT高表达组较SOX2-OT低表达组OS降低(p=0.042),DSS降低(p=0.016);let-7c-3p高表达组较let-7c-3p低表达组OS升高(p=0.038),DSS升高(p=0.047);SKP2高表达组较SKP2低表达组OS降低(p=0.020),DSS降低(p=0.023)。结论1.SOX2-OT在HNSCC组织中高表达,let-7c-3p在HNSCC组织中低表达,SKP2在HNSCC组织中高表达。2.SOX2-OT表达水平与let-7c-3p表达水平存在负相关,let-7c-3p表达水平与SKP2表达水平存在负相关,SOX2-OT表达水平与SKP2表达水平存在正相关,三者在HNSCC组织中的表达具有明显相关性。3.SOX2-OT、let-7c-3p及SKP2表达水平与HNSCC患者临床分期、淋巴结转移及T分期密切相关。4.SOX2-OT、let-7c-3p及SKP2表达水平与生存预后之间具有相关性,SOX2-OT、SKP2表达水平升高及let-7c-3p表达水平降低提示HNSCC患者预后不良。第三部分SOX2-OT对HNSCC细胞增殖、迁移、侵袭及细胞周期的调控作用目的探究SOX2-OT对TU686、HNE1及Fa Du细胞三种HNSCC细胞系增殖、迁移、侵袭等恶性生物学行为及细胞周期调控的影响,初步探究SOX2-OT在HNSCC发生发展中的机制。方法采用慢病毒构建SOX2-OT功能干扰细胞模型,通过CCK-8、Transwell及流式细胞术实验分析干扰SOX2-OT后对TU686、HNE1及Fa Du三种HNSCC细胞系增殖、迁移、侵袭及细胞周期的影响,以此阐明SOX2-OT在HNSCC发生发展中的作用。并进一步在体内实验进行验证,通过构建裸鼠皮下成瘤模型和肺转移模型进一步验证SOX2-OT功能。结果1.构建了SOX2-OT干扰载体并转染TU686、HNE1及Fa Du细胞后进行RT-q PCR检测,结果显示干扰SOX2-OT后显著抑制了SOX2-OT表达(p<0.001);CCK8检测发现干扰SOX2-OT后显著抑制了细胞增殖(p<0.001);Transwell细胞迁移实验检测发现干扰SOX2-OT后显著抑制了细胞迁移(p<0.001),Transwell细胞侵袭实验显示干扰SOX2-OT后显著抑制了细胞侵袭(p<0.001);流式细胞术检测细胞周期分布,结果显示,干扰SOX2-OT后G1期细胞比例显著增加,而S期细胞比例下降。2.裸鼠异种移植瘤模型实验结果表明,干扰SOX2-OT表达后,肿瘤的体积和重量显著减少(p<0.001),肿瘤组织中SOX2-OT降低,let-7c-3p表达升高,SKP2表达降低,免疫组织化学显示,Ki-67表达降低。同时,肺转移模型显示干扰SOX2-OT表达后,转移能力减弱。结论干扰SOX2-OT可以抑制HNSCC细胞增殖、迁移及侵袭,并阻滞细胞周期,SOX2-OT参与HNSCC细胞恶性生物学行为,在HNSCC中发挥癌基因的生物学功能。第四部分let-7c-3p对HNSCC细胞增殖、迁移、侵袭及细胞周期的调控作用目的探究let-7c-3p对TU686、HNE1及Fa Du细胞三种HNSCC细胞系增殖、迁移、侵袭等恶性生物学行为及细胞周期调控的影响,明确let-7c-3p在HNSCC发生发展中的作用。方法为了探究let-7c-3p对HNSCC细胞恶性生物学行为的影响,本部分研究通过对TU686、HNE1及Fa Du细胞转染let-7c-3p mimic及对照mi R-NC后,RT-q PCR确认转染效率,CCK8实验检测细胞增殖情况,Transwell实验检测细胞迁移及侵袭情况,流式细胞术检测细胞周期分布情况。结果1.转染let-7c-3p mimic后,TU686、HNE1及Fa Du三种HNSCC细胞系let-7c-3p表达水平均显著升高,转染成功,进一步用于后续实验。2.CCK8实验结果显示,转染let-7c-3p mimic后,显著抑制了TU686、HNE1及Fa Du细胞增殖(p<0.001)。Transwell实验结果显示,转染let-7c-3p mimic后,同样显著抑制了TU686、HNE1及Fa Du细胞迁移和侵袭(p<0.001)。细胞周期检测结果显示,转染let-7c-3p mimic后,G1期细胞比例显著增加,而S和G2期细胞比例下降。结论let-7c-3p抑制HNSCC细胞恶性生物学行为,在HNSCC中发挥抑癌基因的生物学功能,在细胞增殖、迁移、侵袭及细胞周期调控等过程中发挥作用。第五部分SOX2-OT通过let-7c-3p/SKP2信号轴调控HNSCC的发生发展目的探究SOX2-OT、let-7c-3p及SKP2三者之间的调控关系,验证SOX2-OT是通过let-7c-3p/SKP2轴参与调控HNSCC细胞的恶性生物学行为。方法1.SOX2-OT与let-7c-3p的调控关系研究:通过荧光素酶报告基因实验明确二者之间存在靶向结合位点,并通过RT-q PCR法检测干扰SOX2-OT后,let-7c-3p表达水平的变化,明确SOX2-OT对let-7c-3p的调控作用。2.let-7c-3p与SKP2的调控关系研究:通过荧光素酶报告基因实验明确二者之间存在靶向结合位点,并通过转染let-7c-3p mimic,检测SKP2表达水平变化,明确let-7c-3p对SKP2的调控作用。3.SOX2-OT通过竞争性结合let-7c-3p进而调控SKP2的表达研究:干扰SOX2-OT后检测SKP2表达情况,明确SOX2-OT对SKP2的调控作用,并进一步通过回复实验验证SOX2-OT对SKP2的调控作用是通过let-7c-3p/SKP2轴进行的,将细胞分组为sh-NC组、sh-SOX2-OT+NC inhibitor组及sh-SOX2-OT+let-7c-3p inhibitor组,检测不同情况下SKP2表达情况。4.SOX2-OT/let-7c-3p/SKP2轴对HNSCC细胞恶性生物学行为调控作用研究:为了验证SOX2-OT在HNSCC中的作用是通过let-7c-3p/SKP2调控轴进行的,我们通过回复实验进行进一步验证,通过对干扰SOX2-OT表达的TU686、HNE1及Fa Du细胞进行共转染let-7c-3p inhibitor或NC inhibitor,将细胞分组为sh-NC组、sh-SOX2-OT+NC inhibitor组及sh-SOX2-OT+let-7c-3p inhibitor组,CCK8实验检测细胞增殖情况,Transwell实验检测细胞迁移及侵袭情况。结果1.let-7c-3p能显著降低野生型SOX2-OT(SOX2-OT-WT)组的荧光素酶活性,而对突变型SOX2-OT(SOX2-OT-Mut)组的荧光素酶活性没有影响,差异具有统计学意义,p<0.001。RT-q PCR显示,干扰SOX2-OT的表达可以显著上调TU686、HNE1及Fa Du细胞中let-7c-3p的表达。2.let-7c-3p能够显著降低SKP2-WT的荧光素酶活性,而SKP2-Mut的荧光素酶活性未受影响,差异具有统计学意义,p<0.001。转染let-7c-3p mimic后,SKP2表达水平显著降低。3.干扰SOX2-OT表达后,SKP2表达水平显著降低,let-7c-3p inhibitor共转染使得SKP2表达水平得以恢复。4.CCK8实验结果显示,干扰SOX2-OT表达显著抑制了细胞增殖(p<0.001),而let-7c-3p inhibitor共转染后能够逆转干扰SOX2-OT所抑制的细胞增殖。Transwell实验结果显示,干扰SOX2-OT表达显著抑制了细胞迁移和侵袭(p<0.001),而let-7c-3p inhibitor共转染可以消除干扰SOX2-OT所抑制的细胞迁移和侵袭。结论1.SOX2-OT与let-7c-3p之间存在靶向结合关系,且SOX2-OT可以调控let-7c-3p表达。2.SKP2为let-7c-3p的直接靶基因,let-7c-3p可以调控SKP2表达。3.SOX2-OT可以作为ceRNA,竞争性结合let-7c-3p,进而调控SKP2的表达。4.SOX2-OT在HNSCC中的作用是通过靶向let-7c-3p/SKP2调控轴进行的,SOX2-OT/let-7c-3p/SKP2轴在HNSCC的增殖、迁移、侵袭等恶性生物学行为调控中发挥关键作用。更多还原

【Abstract】 Part I Screening of SOX2-OT-related ceRNA regulatory axis in HNSCC by bioinformaticsObjectiveTo construct a SOX2-OT-centered lncRNA-miRNA-mRNA regulatory network through bioinformatics analysis and ceRNA theory,screen genes closely related to prognosis,and mine HNSCC-related targets,so as to provide a new basis for treatment.MethodsDownload the HNSCC gene expression profile data and clinical survival data from TCGA in UCSC database to analyze the data set,download HNSCC gene chip expression profile data from NCBI GEO database to verify the difference of mRNA expression in ceRNA network.Differential mRNA and miRNA were screened by limma package.DAVID was used to analyze the enrichment of differential mRNA by GO and KEGG,focusing on immune-related pathways.TheRNA sequence of lncRNA SOX2-OT is downloaded from NCBI database,miRNA-lncRNA relation pairs are screened by miranda,miRNA-mRNA relation pairs are predicted by mi RWalk3.0 database,mRNA and DEmRNAs are further intersected,DEmiRNAs-DEmRNAs relation pairs are further obtained,the expression correlation between SOX2-OT and DEmRNAs is analyzed,and lncRNA-mRNA co-expression relation pairs are obtained.using the above miRNA-mRNA and lncRNA-miRNA relation pairs,lncRNA-miRNA-mRNA relation pairs are further screened and the network is constructed.Namely ceRNA network,ceRNA regulatory network is constructed by Cytoscape software,and protein interaction is predicted by combining STRING database.Results1.A total of 4433 DEmRNAs,309 DEmiRNAs,DEmRNAs were obtained,which significantly enriched 672 GO BP,198 GO CC,168 GO MF and 98 KEGG pathways.2.68 lncRNA-miRNA relation pairs,17109 DEmiRNAs-DEmRNAs relation pairs and 1781 positive co-expressed lncRNA-mRNA relation pairs are predicted.Through integration,538 lncRNA-miRNA-mRNA relation pairs,namely ceRNA relation pairs,are obtained.It includes SOX2-OT,65 miRNA and 116 mRNA that we are concerned with.Finally,according to the further found in the two sets of GEO data sets are significant differences and up and down TCGA are consistent,and the survival analysis,screening the key genes,and find the corresponding lncRNA-miRNA-mRNA-pathway relationship of the key genes.3.A total of 312 protein interaction pairs,including 97 protein nodes,were obtained from the protein interaction analysis of 116 mRNA in the above ceRNA network.4.A total of 20 mRNA were selected as key genes.Further find 20 key mRNA to lncRNA-miRNA-mRNA-pathway relationships,Among them,six DEmRNAs and SOX2-OT are related in the key pathways.Finally,this study chooses the SOX2-OT/let-7c-3p/SKP2 control axis for in-depth study.ConclusionThis study successfully constructed a SOX2-OT-centered ceRNA regulatory network,and determined that the SOX2-OT/let-7c-3p/SKP2 regulatory axis plays a role in the occurrence and development of HNSCC,and the specific mechanism will be further verified in the following research.Part II Expression and clinical significance of SOX2-OT,let-7c-3p and SKP2 in HNSCCObjectiveTo explore the correlation between the expression levels of SOX2-OT,let-7c-3p and SKP2 and the clinicopathological parameters and survival prognosis of patients with HNSCC.MethodsThe expressions of SOX2-OT,let-7c-3p and SKP2 in 30 pairs of cancer tissues and paracancerous tissues were detected by RT-q PCR.In order to preliminarily verify the correlation among SOX2-OT,let-7c-3p and SKP2,the expression levels of the three were analyzed by Pearson correlation analysis.To further explore the expression of SOX2-OT,let-7c-3p and SKP2 in HNSCC patients with different clinical stages,different T stages and with or without lymph node metastasis,and to clarify the role of SOX2-OT,let-7c-3p and SKP2 in HNSCC.Finally,in order to study the effects of SOX2-OT,let-7c-3p and SKP2 expression levels on the clinical prognosis of HNSCC patients,patients were divided into low expression group and high expression group according to the median expression level.Kaplan-Meier survival analysis was used to compare the difference in overall survival rate(overall survival,OS)and disease-specific survival rate(Disease-specific survival,DSS)between the two groups.Results1.The expression level of SOX2-OT in HNSCC tissues was significantly increased(p < 0.0001),the expression of let-7c-3p in HNCSS tissues was significantly decreased(p < 0.0001),the expression level of SKP2 in HNSCC tissues was significantly increased(p < 0.0001),and the expression of SKP2 protein was also significantly increased in HNSCC tissues(p < 0.0001).2.There was a negative correlation between the expression level of SOX2-OT and let-7c-3p(r = 0.4633,p = 0.0099).There was also a negative correlation between let-7c-3p expression and SKP2 expression(r = 0.5351,p = 0.0023),while there was a positive correlation between SOX2-OT expression and SKP2 expression(r = 0.5036,p = 0.0048).3.The expression level of SOX2-OT in patients with clinical stage III+IV was significantly higher than that in patients with stage I+II(p<0.01),while the expression level of let-7c-3p was significantly decreased(p<0.05),and the expression level of SKP2 was significantly increased(p<0.001).The expression level of SOX2-OT in patients with positive lymph nodes was significantly higher than that in patients with negative lymph nodes(p<0.05),while the expression level of let-7c-3p was significantly decreased(p<0.01),and the expression level of SKP2 was significantly increased(p<0.001).The level of SOX2-OT expression in patients with T stage T3+T4 was significantly higher than that in patients with T1+T2 stage(p<0.05),while the expression level of let-7c-3p was significantly decreased(p<0.05),and the expression level of SKP2 was significantly increased(p<0.001).4.Compared with SOX2-OT low expression group,OS and DSS decreased in SOX2-OT high expression group,OS and DSS increased in let-7c-3p high expression group compared with let-7c-3p low expression group,OS decreased and DSS decreased in SKP2 high expression group compared with SKP2 low expression group.Conclusions1.SOX2-OT is highly expressed in HNSCC tissues,let-7c-3p is low in HNSCC tissues,and SKP2 is highly expressed in HNSCC tissues.2.There is a negative correlation between SOX2-OT expression level and let-7c-3p expression level,a negative correlation between let-7c-3p expression level and SKP2 expression level,a positive correlation between SOX2-OT expression level and SKP2 expression level,and a significant correlation between them in HNSCC tissues.3.The expression levels of SOX2-OT,let-7c-3p and SKP2 are closely related to clinical stage,lymph node metastasis and T stage in patients with HNSCC.4.The expression levels of SOX2-OT,let-7c-3p and SKP2 were correlated with survival and prognosis.The increased expression of SOX2-OT and SKP2 and the decrease of let-7c-3p expression suggested poor prognosis in patients with HNSCC.Part III Regulatory effects of SOX2-OT on proliferation,migration,invasion and cell cycle of HNSCC cellsObjectiveTo explore the effects of SOX2-OT on malignant biological behaviorssuch as proliferation,migration and invasion and cell cycle regulation of HNSCC cells TU686,HNE1 and Fa Du cells,and to explore the mechanism of SOX2-OT in the occurrence and development of HNSCC.MethodsThe functional interference cell model of SOX2-OT was constructed by lentivirus.The effects of interfering with SOX2-OT on the proliferation,migration,invasion and cell cycle of TU686,HNE1 and Fa Du HNSCC cell lines were analyzed by CCK-8,Transwell and flow cytometry,so as to clarify the role of SOX2-OT in the occurrence and development of HNSCC.Further experiments in vivo were carried out to verify the function of SOX2-OT through the establishment of subcutaneous tumor formation and lung metastasis model in nude mice.Results1.SOX2-OT interference vector was constructed and transfected into TU686,HNE1 and Fa Du cells for RT-q PCR detection,the results showed that interference with SOX2-OT significantly inhibited the expression of SOX2-OT(p < 0.001),CCK8 detection showed that interference with SOX2-OT significantly inhibited cell proliferation(p < 0.001).Transwell cell migration assay showed that interference with SOX2-OT significantly inhibited cell migration(p < 0.001),Transwell cell invasion assay showed that interference with SOX2-OT significantly inhibited cell invasion(p < 0.001),flow cytometry detected the distribution of cell cycle,the results showed that the proportion of cells in G1 phase increased significantly after interfering with SOX2-OT,while the proportion of cells in S and G2 cycle phase decreased significantly.2.In order to study the effect of SOX2-OT on the growth of HNSCC in nude mice,the xenograft tumor model of nude mice was established.The results showed that after interfering with the expression of SOX2-OT,the volume and weight of tumor decreased significantly(p < 0.001),the expression of SOX2-OT decreased,the expression of let-7c-3p increased,the expression of SKP2 decreased,and immunohistochemistry showed that the expression of Ki-67 decreased.At the same time,the lung metastasis model showed that the ability of metastasis decreased after interfering with the expression of SOX2-OT.ConclusionInterfering with SOX2-OT can inhibit the proliferation,migration and invasion of HNSCC cells,and block the cell cycle.SOX2-OT participates in the malignant biological behavior of HNSCC cells and plays the biological function of oncogenes in HNSCC.Part IV Regulatory effects of let-7c-3p on proliferation,migration,invasion and cell cycle of HNSCC cellsObjectiveTo explore the effects of let-7c-3p on the proliferation,migration,invasion and other malignant biological behaviors and cell cycle regulation of HNSCC cells,and to clarify the role of let-7c-3p in the occurrence and development of HNSCC.MethodsIn order to explore the effect of let-7c-3p on the malignant biological behavior of HNSCC cells,after transfection of let-7c-3pmimic and control mi R-NC,the transfection efficiency was confirmed by RT-q PCR,the cell proliferation was detected by CCK8 assay,the migration and invasion of cells were detected by Transwell assay,and the cell cycle distribution was detected by flow cytometry.Results1.After let-7c-3p mimic transfection,the expression of let-7c-3p in TU686,HNE1 and Fa Du HNSCC cell lines increased significantly,and the transfection was successful,which was further used in the follow-up experiment.2.The results of CCK8 assay showed that transfection of let-7c-3p mimic significantly inhibited the proliferation of TU686,HNE1 and Fa Du cells(p < 0.001).The results of Transwell assay showed that let-7c-3p mimic transfection also significantly inhibited the migration and invasion of TU686,HNE1 and Fa Du cells(p< 0.001).The results of cell cycle detection showed that after transfection of let-7c-3p mimic,the proportion of cells in G1 phase increased significantly,while the proportion of S and G2 cells phase decreased.Conclusionlet-7c-3p inhibits the malignant biological behavior of HNSCC cells,exerts the biological function of tumor suppressor genes in HNSCC,and plays a role in cell proliferation,migration,invasion and cell cycle regulation.Part V SOX2-OT regulates the occurrence and development of HNSCC through let-7c-3p/SKP2 signal axis.ObjectiveTo verify the regulatory relationship among SOX2-OT,let-7c-3p and SKP2,and to study whether SOX2-OT participates in the regulation of malignant biological behavior of HNSCC cells through the let-7c-3p/SKP2 axis.Methods1.Study on the regulatory relationship between SOX2-OT and let-7c-3p:luciferase reporter gene assay confirmed the existence of targeted binding sites between them,and detected the changes of let-7c-3p expression level after interfering with SOX2-OT by RT-q PCR method to clarify the regulatory effect of SOX2-OT on let-7c-3p.2.Study on the regulatory relationship between let-7c-3p and SKP2: luciferase reporter gene assay confirmed that there was a targeted binding site between them,and the expression level of SKP2 was detected by transfection of let-7c-3pmimic.3.SOX2-OT regulates the expression of SKP2 through competitive binding to let-7c-3p: the expression of SKP2 in cells interfered by SOX2-OT was detected by RT-q PCR and WB,and the regulatory relationship among SOX2-OT,let-7c-3p and SKP2 was verified by recovery experiment.The cells were divided into sh-NC group,sh-SOX2-OT+NC inhibitor group and sh-SOX2-OT+let-7c-3p inhibitor group,and the expression of SKP2 was detected under different conditions.4.Study on the regulatory effect of SOX2-OT/let-7c-3p/SKP2 axis on malignant biological behavior of HNSCC cells: in order to verify that the role of SOX2-OT in HNSCC is carried out through let-7c-3p/SKP2 regulatory axis,we further verified it by recovery experiment,and cotransfected let-7c-3p inhibitor or NC inhibitor into TU686,HNE1 and Fa Du cells that interfered with SOX2-OT expression.The cells were divided into sh-NC group,sh-SOX2-OT+NC inhibitor group and sh-SOX2-OT+let-7c-3p inhibitor group.The proliferation of cells was detected by CCK8 assay,the migration and invasion of cells were detected by Transwell test.Results1.let-7c-3p could significantly reduce the luciferase activity of SOX2-OT-WT group,but had no effect on the luciferase activity of SOX2-OT-Mut group,the difference was statistically significant(p < 0.001).RT-q PCR showed that interfering with the expression of SOX2-OT could significantly up-regulate the expression of let-7c-3p in TU686,HNE1 and Fa Du cells.2.let-7c-3p could significantly reduce the luciferase activity of SKP2-WT,but the luciferase activity of SKP2-Mut was not affected,the difference was statistically significant(p < 0.001).The results of RT-q PCR and WB detection showed that the expression of SKP2 mRNA and protein decreased significantly after transfection of let-7c-3p mimic.3.The results of RT-qPCR and WB showed that the expression of SKP2 mRNA and protein decreased significantly after interfering with the expression of SOX2-OT,and the expression of SKP2 mRNA and protein could be restored by let-7c-3p inhibitor co-transfection.4.The results of CCK8 assay showed that interfering with SOX2-OT expression significantly inhibited cell proliferation(p < 0.001),while let-7c-3p inhibitor co-transfection could reverse the cell proliferation inhibited by interfering SOX2-OT.The results of Transwell assay showed that interference with SOX2-OT expression significantly inhibited cell migration and invasion(p < 0.001),while let-7c-3p inhibitor co-transfection could eliminate cell migration and invasion caused by interference with SOX2-OT.Conclusions1.There is a targeted binding relationship between SOX2-OT and let-7c-3p,and SOX2-OT can regulate the expression of let-7c-3p.2.SKP2 is the direct target gene of let-7c-3p and let-7c-3p can regulate the expression of SKP2.3.SOX2-OT can be used as a ceRNA,competitively combined with let-7c-3p,and then regulate the table of SKP2.4.The role of SOX2-OT in HNSCC is carried out by targeting the let-7c-3p/SKP2 regulation axis,and the SOX2-OT/let-7c-3p/SKP2 axis plays a key role in the occurrence and development of HNSCC.更多还原