【作者】 周斌；

【导师】 蒋国勤；

【作者基本信息】 苏州大学 ， 外科学（普外）（专业学位）， 2022， 博士

【摘要】 背景:甲状腺癌是内分泌系统常见的恶性肿瘤,近年来在全球范围内,其发病率呈明显上升的趋势。其中甲状腺乳头状癌发病率最高,约占甲状腺癌总数的80%。尽管该肿瘤生物学行为表现为惰性,患者经过常规的诊断和治疗预后良好,5年生存率可达到90%以上,但长期随访发现仍有部分患者术后出现复发和远处转移,严重影响生活质量,大幅度缩短生存时间。探索甲状腺乳头状癌发生发展的分子机制具有重要的价值。长链非编码RNA(Longnon-coding RNA,lncRNA)是指一类长度超过200个核苷酸的RNA分子,因开放阅读框架的缺失,绝大多数lncRNA难以编码蛋白,最初被认为是转录噪音,不具备生物学功能。近些年,随着高通量转录组测序的发展及普及,越来越多的lncRNA被发现鉴定,相关研究逐步深入,lncRNA因其在基因转录、翻译、剪切及修饰等生物学过程中的重要作用也备受关注。目前在甲状腺乳头状癌中已发现SLC26A4-AS1、FER1L4等lncRNA分子表达失调,调控肿瘤细胞的恶性生物学行为,这些结果补充了甲状腺乳头状癌发生发展的分子机制网络,为今后诊断治疗提供了新的策略。本研究拟从lncRNA分子着手,通过对癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中甲状腺癌lncRNA测序数据进行差异表达分析,结合组织qRT-PCR验证,筛选出甲状腺乳头状癌中差异表达显著的lncRNA,采用一系列分子生物学实验探索其对甲状腺乳头状癌细胞功能表型的影响,最后再探讨其潜在的分子作用机制。第一部分:LncRNALINC00284在甲状腺乳头状癌中的表达及临床意义目的:筛选甲状腺乳头状癌组织与癌旁组织中差异表达的lncRNA并评估其临床意义。方法:(1)利用Sangerbox3.0生信工具盒下载TCGA数据库中甲状腺癌lncRNA测序数据,通过“转录组Count数据差异分析”工具进行lncRNA差异表达分析,寻找差异显著的lncRNA,初步确定研究对象;(2)采用qRT-PCR在收集的25例甲状腺乳头状癌样本中检测所选择lncRNA的表达水平,寻找差异最为显著的lncRNA,确定为最终研究对象;(3)利用TCGA中挑选出的58例配对甲状腺癌及癌旁组织(根据TCGA中每个组织的编号进行挑选)的RNA测序数据,以及GEO数据库中5对甲状腺癌组织的RNA测序数据验证所选lncRNA的差异表达情况;(4)扩大甲状腺乳头状癌样本量至75例,采用qRT-PCR再次验证所选lncRNA的差异表达情况;(5)通过qRT-PCR在甲状腺乳头状癌细胞株和正常甲状腺滤泡上皮细胞株检测所选lncRNA的表达水平;(6)分析所选lncRNA表达与江阴市人民医院诊治的甲状腺乳头状癌患者临床病理特征的关系,包括性别、年龄、腺外侵犯、肿瘤大小、淋巴结转移、多灶性和TNM分期。结果:(1)通过TCGA数据库中甲状腺癌lncRNA差异表达分析,筛选出甲状腺癌中功能尚未研究、差异表达位于前十位的lncRNA(NPSR1-AS1、CHIAP2、LINC02738、RPSAP52、IGFL2-AS1、LINC00284、LINC01977、TMEM92-AS1、LAMP5-AS1和LINC01705);(2)在收集的25例甲状腺乳头状癌组织和癌旁组织中,利用qRT-PCR检测10个lncRNA的表达,发现LINC00284在甲状腺乳头状癌组织较癌旁组织表达升高最显著(差异倍数为3.8,P<0.0001),确定其为研究对象;(3)TCGA数据库中58例配对的甲状腺癌组织和癌旁组织的RNA测序数据进一步证实LINC00284在甲状腺癌中表达升高,相同的结果在GEO数据库中5例配对的甲状腺癌组织和癌旁组织中得到印证;(4)利用qRT-PCR在75例甲状腺乳头状癌和癌旁组织中检测LINC00284表达,发现其在癌组织中表达显著升高;(5)qRT-PCR结果显示,在甲状腺乳头状癌细胞株(IHH-4、TPC-1和K-1)中LINC00284表达均高于正常甲状腺滤泡上皮细胞株(Nthy-ori3-1);(6)LINC00284的表达与患者肿瘤大小相关,即LINC00284高表达的患者肿块体积更大(P=0.014),未发现LINC00284的表达和甲状腺乳头状癌腺外侵犯、淋巴结转移、多灶性及TNM分期存在相关性。结论:LncRNALINC00284在甲状腺乳头状癌组织及细胞株中表达显著升高,且LINC00284高表达的患者病灶体积更大,提示其与甲状腺乳头状癌的恶性生物学特性有关。第二部分:LncRNALINC00284对甲状腺乳头状癌细胞增殖能力的影响目的:研究lncRNALINC00284表达对甲状腺乳头状癌细胞体内外增殖能力及周期凋亡进程的影响。方法:(1)设计特异性干扰LINC00284表达的siRNA序列,通过瞬时转染法将siRNA转染至甲状腺乳头状癌细胞中敲低LINC00284的表达,并通过qRT-PCR验证;(2)敲低LINC00284表达后,通过CCK-8、集落形成和EdU实验检测甲状腺乳头状癌细胞增殖能力的变化;(3)下调LINC00284表达后,采用流式细胞仪检测细胞周期凋亡的变化,并通过western blot检测周期凋亡相关蛋白的表达水平;(4)敲低LINC00284表达行裸鼠皮下成瘤实验,探索其体内功能,并通过免疫组化检测裸鼠移植瘤中增殖相关蛋白Ki-67的表达。结果:(1)qRT-PCR检测显示,siRNA可以显著敲低甲状腺乳头状癌细胞TPC-1和K-1中LINC00284的表达;(2)CCK-8、集落形成及EdU实验发现,敲低LINC00284表达可以显著减弱甲状腺乳头状癌细胞的增殖能力;(3)流式细胞检测结果显示,下调LINC00284表达可导致甲状腺乳头状癌细胞周期阻滞在G1期,并且可以诱导细胞凋亡,western blot检测发现下调LINC00284表达,可显著降低G1-S期标志蛋白CDK4、Cyclin D3和抗凋亡蛋白Bcl-2的表达,上调促凋亡蛋白Bak的表达;(4)裸鼠皮下成瘤实验显示,敲低LINC00284表达的甲状腺乳头状癌细胞体内移植瘤生长能力显著减弱,免疫组化检测发现下调LINC00284表达后,Ki-67表达量显著降低。结论:敲低lncRNALINC00284的表达可显著减弱甲状腺乳头状癌细胞体内外的增殖能力,并且能够抑制细胞周期进程,诱导细胞凋亡。第三部分:LncRNALINC00284通过调控miR-3127-5p/E2F7促进甲状腺乳头状癌增殖的机制研究目的:探索LINC00284在甲状腺乳头状癌中的分子作用机制。方法:(1)通过生物信息学网站CPAT 3.0和CPC 2.0分析LINC00284是否具有编码蛋白的潜能;(2)使用在线工具“lncLocator”预测LINC00284的细胞定位,随后通过 RNA 荧光原位杂交实验(RNA fluorescenceinsituhybridization,FISH)和核质分离实验进行验证;(3)通过lncRNASNP2和RegRNA 2.0网站筛选可能与LINC00284结合的miRNAs,并通过qRT-PCR检测验证;(4)采用双荧光素酶报告基因和 RNA 结合蛋白免疫沉淀(RNA bindingproteinimmunoprecipitation,RIP)实验验证LINC00284与miRNA的结合;(5)在甲状腺乳头状癌组织和癌旁组织中,采用qRT-PCR检测miRNA的表达量,并分析LINC00284表达与miRNA表达的相关性;(6)利用Targetscan、DIANA和miRDB网站寻找miRNA下游靶基因,并结合TCGA数据库,初步筛选miRNA候选靶基因;(7)Western blot检测下调miRNA表达后靶基因表达水平的变化,并通过双荧光素酶实验验证miRNA与靶基因的结合;(8)qRT-PCR检测靶基因的表达,分析靶基因与miRNA表达以及靶基因与LINC00284表达的相关性;(9)在甲状腺乳头状癌细胞中共转染LINC00284siRNA(或miRNA靶基因siRNA)和miRNA inhibitor,进行功能挽救实验;(10)采用基因功能富集分析(Gene set enrichment analysis,GSEA)预测 LINC00284 调控的信号通路,并通过 western blot验证。结果:(1)CPAT 3.0和CPC 2.0网站结果均显示LINC00284不具备编码蛋白的潜能;(2)在线工具lncLocator预测结果、FISH和核质分离实验结果均显示LINC00284主要定位于甲状腺乳头状癌细胞的细胞浆中;(3)通过lncRNASNP2和RegRNA2.0筛选出 4 个可能与 LINC00284 结合的 miRNAs(miR-499a-3p、miR-1914-5p、miR-3127-5p 和 miR-3692-5p),qRT-PCR 检测显示敲低 LINC00284 表达后,miR-3127-5p表达显著升高,确定其为后续研究对象;(4)双荧光素酶报告基因和RIP实验显示LINC00284可与miR-3127-5p结合;(5)qRT-PCR检测显示,miR-3127-5p在甲状腺乳头状癌组织中表达较癌旁组织中降低,在甲状腺乳头状癌组织中miR-3127-5p表达和 LINC00284 表达呈负相关(R=-0.507,P<0.001);(6)通过 Targetscan、DIANA 和miRDB生物信息学工具,筛选出130个miR-3127-5p潜在的靶基因,然后结合TCGA数据库,我们筛选出7个在甲状腺癌组织中表达量明显高于癌旁组织的mRNA作为候选靶基因(TNNI1、CLIP3、ETV5、ACOT12、E2F7、FAXC 和 MISP)(fold change≥2,P<0.05);(7)Western blot检测显示,敲低miR-3127-5p表达后,E2F7表达显著上调,确定其为后续研究对象,双荧光素酶报告基因实验显示miR-3127-5p可与E2F7结合;(8)qRT-PCR检测发现,E2F7在甲状腺乳头状癌中的表达量显著高于癌旁组织,且在甲状腺乳头状癌组织中E2F7表达和miR-3127-5p表达呈负相关(R=-0.325,P=0.005),E2F7 表达和 LINC00284 表达呈正相关(R=0.620,P<0.001);(9)回复实验显示,miR-3127-5pinhibitor可部分挽救LINC00284表达下调引起的细胞增殖能力减弱,下调E2F7表达可逆转miR-3127-5p inhibitor对细胞增殖能力的促进作用;(10)GSEA结果显示与Hedgehog信号通路相关的基因在LINC00284高表达组显著富集。Western blot检测发现敲低LINC00284表达,可以下调Hedgehog信号通路相关蛋白SHH、PTCH1和GLI1的表达。结论:LncRNALINC00284可以充当分子海绵,竞争性结合miR-3127-5p,解除了部分miR-3127-5p对E2F7的抑制作用,激活Hedgehog信号通路,进而促进甲状腺乳头状癌细胞的增殖。这一发现有可能为甲状腺乳头状癌提供新的治疗靶点。总结1.LncRNALINC00284和E2F7在甲状腺乳头状癌组织和癌细胞中高表达,miR-3127-5p低表达;LINC00284高表达的患者肿块体积更大;2.LncRNALINC00284在甲状腺乳头状癌中发挥促癌作用,通过内源性竞争结合miR-3127-5p,导致下游靶基因E2F7表达升高,进而促进甲状腺乳头状癌细胞的体内外增殖,促进细胞周期进程,抑制细胞凋亡;3.LncRNA LINC00284/miR-3127-5p/E2F7 功能轴通过激活 Hedgehog 信号通路,促进甲状腺乳头状癌细胞的增殖。更多还原

【Abstract】 Background:Thyroid cancer is a common malignant tumor of endocrine system.In recent years,the incidence of thyroid cancer has been significantly increasing worldwide.Among them,papillary thyroid cancer(PTC)has the highest incidence,accounting for about 80%of the total thyroid cancer.Although the biological behavior of this tumor is inert,the patients have a good prognosis after conventional diagnosis and treatment,and the 5-year survival rate can reach more than 90%,but long-term follow-up found that some patients still have recurrence and distant metastasis after surgery,which seriously affects the quality of life and greatly reduces the survival time.Therefore,it is of great value to explore the molecular mechanism underlying the occurrence and development of PTC.Long non-coding RNA(lncRNA)refers to a class of RNA molecules with more than 200 nucleotides in length.Due to the absence of open reading framework,most lncRNAs are difficult to encode proteins,which were initially considered as transcription noise and did not have biological functions.In recent years,with the development and popularity of high-throughput transcriptome sequencing,more and more lncRNAs have been identified,and relevant studies have been gradually deepening.LncRNAs have also attracted much attention due to their important role in gene transcription,translation,splicing,and modification.Currently,dysregulation of the expression of lncRNA molecules such as SLC26A4-AS1 and FER1L4 has been found in PTC,which regulates the malignant biological behaviors of tumor cells.These results complement the molecular mechanism network of the occurrence and development of PTC,and provide new strategies for future diagnosis and treatment.This study intends to start from the lncRNA perspective and screen out differentially expressed lncRNAs in PTC through The Cancer Genome Atlas(TCGA)database combined with qRTPCR detection in tissues.A series of biological experiments were conducted to explore its effects on of functional phenotypes of PTC cells,and finally to reveal its potential molecular mechanism.Part Ⅰ:Expression and clinical significance of lncRNA LINC00284 in PTCObjective:To screen the differentially expressed lncRNA in PTC and evaluate its clinical significance.Methods:(1)The lncRNA sequencing data of thyroid cancer from TCGA database were downloaded via Sangerbox 3.0 tool,and the differential expression analysis of lncRNA was conducted by "transcriptome Count data difference analysis" tool,to find the significantly different lncRNAs and preliminarily determine the research object.(2)qRTPCR was used to detect the expression levels of selected lncRNAs in the 25 collected PTC samples,and the lncRNA with the most significant difference was identified as the final research object.(3)The differential expression of the selected lncRNA was verified by RNA sequencing data of 58 paired thyroid cancer tissues and adjacent normal tissues(selecting according to the number of each tissue in TCGA)sorted out from TCGA,as well as RNA sequencing data of 5 pairs of tissues in GEO database.(4)The sample size of PTC was expanded to 75 cases,and the differential expression of the selected lncRNA was verified by qRT-PCR.(5)The expression levels of the selected lncRNA were detected by qRT-PCR in PTC cell lines and normal thyroid follicular epithelial cell line.(6)The relationship between the expression of the selected lncRNA and clinicopathological features of the patients with PTC diagnosed and treated in Jiangyin People’s Hospital was analyzed,including gender,age,extrathyroidal extension,tumor size,lymph node metastasis,multifocality,and TNM stage.Results:(1)Through differential expression analysis of lncRNA in thyroid cancer from TCGA database,the top 10 differentially expressed lncRNAs(NPSR1-AS1,CHIAP2,LINC02738,RPSAP52,IGFL2-AS1,LINC00284,LINC01977,TMEM92-AS1,LAMP5AS1 and LINC01705)were screened out,whose biological functions were not explored in PTC.(2)qRT-PCR was used to detect the expression of 10 lncRNAs in 25 paired PTC tissues and adjacent normal tissues.The expression of LINC00284 was most significantly increased in PTC tissues compared with normal tissues(fold change:3.8,P<0.0001),which was identified as the research object.(3)Upregulation of LINC00284 expression in thyroid cancer was further confirmed by RNA sequencing data from 58 paired thyroid cancer tissues and adjacent tissues in TCGA database,and the same result was observed in 5 paired thyroid cancer tissues and adjacent tissues in GEO database.(4)qRT-PCR was used to detect the expression of LINC00284 in 75 paired PTC tissues and adjacent normal tissues,and the expression of LINC00284 in cancer tissues was significantly increased.(5)The results of qRT-PCR showed that the expression of LINC00284 in PTC cell lines(IHH-4,TPC-1,and K-1)was higher than that in normal thyroid follicular epithelial cell lines(NTHY-ORI3-1).(6)The expression of LINC00284 was correlated with tumor size,that is,patients with LINC00284 high expression had larger tumor size(P=0.014),no correlation was found between LINC00284 expression and exglandular invasion,lymph node metastasis,mulifocality,and TNM staging of PTC.Conclusions:The expression of lncRNA LINC00284 was significantly increased in PTC tissues and cell lines,and the tumor size of patients with high expression of LINC00284 was larger,suggesting that it may be related to the malignant biological characteristics of PTC.Part Ⅱ:Effects of lncRNA LINC00284 on the proliferation of PTC cellsObjective:To investigate the effects of lncRNA LINC00284 expression on proliferation in vitro and in vivo,cell cycle,and apoptosis of PTC cells.Methods:(1)The specifical siRNA sequences interfering with LINC00284 expression were designed and transfected into PTC cells by the method of transient transfection to knock down LINC00284 expression,which then was verified by qRT-PCR.(2)After the expression of LINC00284 was reduced,the proliferation ability of PTC cells was detected by CCK-8,colony formation,and EdU assays.(3)After LINC00284 expression was downregulated,cell cycle and apoptosis were detected by flow cytometry,and the expression levels of cell cycle and apoptosis-related proteins were detected by western blot.(4)A xenograft tumor model in nude mice was to explore the effects of LINC00284 expression on proliferation of PTC cells in vivo,and the expression of proliferation-related protein Ki-67 in nude mice xenografts was detected by immunohistochemistry.Results:(1)qRT-PCR detection showed that siRNA significantly downregulated the expression of LINC00284 in TPC-1 and K-1 cells.(2)CCK-8,colony formation,and EdU assays indicated that knocking down LINC00284 expresison significantly reduced the proliferation ability of PTC cells;(3)The results of flow cytometry displayed that inhibiting LINC00284 expression could result in that cell cycle arrested in G1 phase and induce cell apoptosis.Western blot showed that reducing LINC00284 expression significantly decreased the expression of G1-S phase markers CDK4,Cyclin D3 and anti-apoptotic protein Bcl-2,and upregulated the expression of pro-apoptotic protein Bak.(4)Suppressing LINC00284 expression attenuated PTC cell proliferation in vivo.Immunohistochemical analysis showed that the expression of Ki-67 was significantly decreased after LINC00284 was downregulated.Conclusion:Downregulating the expression of lncRNA LINC00284 could significantly suppress the proliferation ability of PTC cells in vitro and in vivo,inhibit cell cycle process,and induce cell apoptosis.Part Ⅲ:The mechanism of lncRNA LINC00284 promoting the proliferation of PTC by regulating miR-3127-5p/E2F7Objective:To explore the molecular mechanism of LINC00284 in PTC.Methods:(1)Bioinformatics websites CPAT 3.0 and CPC 2.0 were used to analyze whether LINC00284 had the potential to encode proteins.(2)The cell localization of LINC00284 was predicted using the online tool "lncLocator",and subsequently verified by RNA fluorescence in situ hybridization(FISH)and subcellular fractionation assays.(3)miRNAs that may bind to LINC00284 were screened by lncRNASNP2 and RegRNA 2.0 websites,which were verified by qRT-PCR.(4)The binding of LINC00284 to miRNA was confirmed by dual luciferase reporter gene and RNA binding protein immunoprecipitation(RIP)assays.(5)The expression levels of miRNA in PTC tissues and adjacent normal tissues were detected by qRT-PCR,and the correlation between the expression of LINC00284 and miRNA was analyzed.(6)The downstream target genes of miRNA were searched via Targetscan,DIANA,and miRDB websites,and then the candidate target genes of miRNA were preliminarily selected by combining with TCGA database.(7)Western blot was used to detect the expression levels of target genes after miRNA expression was reduced,and dual luciferase reporter gene assay was adopted to verify the binding of miRNA to the target gene.(8)qRT-PCR was used to detect the expression of the target gene,and the correlation between E2F7 expression and miRNA(or LINC00284)was analyzed.(9)The function rescue experiment was conducted by co-transfection of LINC00284 siRNA(or miRNA target gene siRNA)and miRNA inhibitor into PTC cells.(10)Gene set enrichment analysis(GSEA)was used to search the signaling pathway regulated by LINC00284,which was then confirmed by western blot.Results:(1)CPAT 3.0 and CPC 2.0 websites showed that LINC00284 did not have the potential of coding protein.(2)The prediction results of online tool lncLocator,FISH,and subcellular fractionation assays indicated that LINC00284 was mainly located in the cytoplasm of PTC cells.(3)Four miRNAs(miR-499a-3p,miR-1914-5p,miR-3127-5p,and miR-3692-5p)that may bind to LINC00284 were screened out by lncRNASNP2 and RegRNA 2.0.qRT-PCR showed that miR-3127-5p expression was significantly increased after LINC00284 was knocked down,which was identified as the object of follow-up study.(4)The dual luciferase reporter gene and RIP assays suggested that LINC00284 could bind to miR-3127-5p.(5)qRT-PCR displayed that miR-3127-5p expression in PTC tissues was lower than that in adjacent normal tissues,and there was a negative correlation between miR3127-5p expression and LINC00284 expression in PTC tissues(R=-0.507,P<0.001).(6)130 potential target genes of miR-3127-5p were screened out by Targetscan,DIANA,and miRDB bioinformatics tools.Then combined with TCGA database,we picked out 7 mRNAs(TNNI1,CLIP3,ETV5,ACOT12,E2F7,FAXC,and MISP)as candidate target genes,whose expression levels in thyroid cancer tissues were significantly higher than those in normal tissues(fold change≥2,P<0.05).(7)Western blot analysis showed that E2F7 expression was significantly upregulated after miR-3127-5p expression was inhibited.Dual luciferase reporter gene assay indicated that miR-3127-5p could bind to E2F7.(8)qRT-PCR detection displayed that E2F7 expression in PTC tissues was significantly higher than that in adjacent normal tissues,and E2F7 expression was negatively correlated with miR-31275p expression(R=-0.325,P=0.005).E2F7 expression was positively correlated with LINC00284 expression(R=0.620,P<0.001)in PTC tissues.(9)Rescue experiments showed that miR-3127-5p inhibitor could partially rescue the the attenuation of cell proliferation arisen from LINC00284 knockdown,and downregulating E2F7 expression could reverse the promoting effect of miR-3127-5p inhibitor on cell growth.(10)Through GSEA,we observed that the related genes in Hedgehog signaling pathway were significantly enriched in the LINC00284 high expression group.Western blot revealed that inhibition of LINC00284 reduced the expression of relevant proteins SHH,PTCH1,and GLI1 in Hedgehog signaling pathway.Conclusions:LncRNA LINC00284 cound act as a molecular sponge and competitively bind with miR-3127-5p,which partially removed the inhibition of E2F7 by miR-3127-5p,activating the Hedgehog signaling pathway,in turn,promoted the proliferation of PTC cells.This discovery may provide a new therapeutic target for PTC.Conclusions1.LncRNA LINC00284 and E2F7 were upregulated in PTC tissues and cells,while miR-3127-5p was downregulated.Patients with LINC00284 high expression had larger tumor size.2.LncRNA LINC00284 played a carcinogenic role in PTC.It bound to miR-3127-5p via endogenous competition,leading to increased expression of downstream target gene E2F7,thereby promoting the proliferation of PTC cells in vitro and in vivo,promoting cell cycle progression and inhibiting apoptosis.3.LncRNA LINC00284/miR-3127-5p/E2F7 axis accelerated the proliferation of PTC cells by activating the Hedgehog signaling pathway.更多还原