【作者】 陈晓；

【导师】 田兴松；

【作者基本信息】 山东大学 ， 外科学（普通外科）（专业学位）， 2020， 博士

【摘要】 研究背景和意义甲状腺癌是世界上常见的恶性肿瘤之一,过去的几十年中,甲状腺癌的发病率在世界范围内都呈现明显上升的趋势。在我国,2015年甲状腺癌的发病率达到女性恶性肿瘤发病率的第四位,占女性恶性肿瘤的8.49%。临床工作中常见的甲状腺癌病理类型有四种:乳头状癌、髓样癌、滤泡状癌以及未分化癌。其中乳头状癌是最为常见的病理类型,约占全部甲状腺癌的88%。有研究认为甲状腺乳头状癌发病率的上升导致了甲状腺癌发病率的整体上升。手术治疗为甲状腺乳头状癌的主要治疗手段之一,手术治疗效果良好。据报道,甲状腺乳头状癌术后20年生存率可达99%。尽管甲状腺乳头状癌术后生存率很高,但长期随访结果显示,其复发率高达25%。甲状腺乳头状癌不但给患者带来了手术治疗的身体的创伤,同时也因对疾病复发恐惧,带来了沉重的心理负担。因此,进行相关的基础研究,阐明甲状腺乳头状癌的发生、发展的分子机制具有重要的价值。MicroRNA(miRNA)是一类非编码单链小RNA分子,一般是由内源基因编码的,长度约为19-25个核苷酸。MiRNA可以通过识别靶基因转录后的目的mRNA的3’端非编码区域(3’untranslated region,3’UTR)并与之结合,抑制其翻译或诱导其降解。研究显示,一个miRNA可以调控数以百计的不同的mRNA,而一个mRNA也可以被多种miRNA所调控。近年来的研究发现,miRNA通过调控多个细胞信号传导通路中的不同基因,可以影响细胞的增殖,分化及凋亡,进而参与肿瘤的多种病理生理过程,包括肿瘤的发生、发展。最近的研究发现miR-299-3p在多种肿瘤中发挥一定作用。Wang等通过体内和体外研究发现,miR-299-3p 通过下调 VEGFA(Vascular Endothelial Growth Factor A,血管内皮生长因子A)的表达,抑制人类结肠癌细胞的增殖和侵袭。在肝细胞肝癌中,miR-299-3p通过作用于Sirtuin5抑制肿瘤的生长、侵袭。Yu等报道了,miR-299-3p作用于TCF4(transcription factor 4)抑制宫颈癌的增殖和侵袭。Zhao等报道,抑制miR-299-3p的表达将会降低卵巢癌细胞SKOV3的侵袭、迁移、增殖,促进其凋亡。进一步研究证实,miR-299-3p的作用是通过调控OCT-4(organic cation/camitine transporter4)的表达得以实现。从上述文献中可以看到,miR-299-3p在结肠癌、肝细胞肝癌、宫颈癌中扮演抑癌基因角色,而在卵巢癌中扮演的却是促癌基因角色。到目前为止,miR-299-3p在甲状腺乳头状癌中的作用还不清楚。因此,本研究的目的是探索miR-299-3p在甲状腺乳头状癌中的表达情况以及发挥的作用。研究目的在我国,2015年甲状腺癌的发病率达到女性恶性肿瘤发病率的第四位,占女性恶性肿瘤的8.49%。甲状腺乳头状癌不但给患者带来了手术治疗的身体的创伤,同时也因对疾病复发恐惧,带来了沉重的心理负担。因此,进行相关的基础研究,阐明甲状腺乳头状癌的发生、发展的分子机制具有重要的价值。近年来的研究发现,miRNA调控了多个细胞信号传导通路中的不同基因,可以影响细胞的增殖,分化及凋亡,进而参与肿瘤的多种病理生理过程,包括肿瘤的发生、发展。其中miR-299-3p在结肠癌、肝细胞肝癌、宫颈癌中扮演抑癌基因角色,而在卵巢癌中扮演的却是促癌基因角色。到目前为止,miR-299-3p在甲状腺乳头状癌中的作用还不清楚。因此,本研究的目的是探索miR-299-3p在甲状腺乳头状癌中的表达情况以及发挥的作用。本研究将从以下几个方面进行研究:1.检测miR-299-3p在甲状腺乳头状癌组织及细胞中的表达情况;2.研究miR-299-3p在甲状腺乳头状癌细胞增殖、周期分布及凋亡中的功能。3.MiR-299-3p过表达对小鼠体内移植瘤生长的影响。4.MiR-299-3p靶基因的预测及验证。实验方法:1.采用实时荧光定量PCR的方法检测30对甲状腺乳头状癌及配对的癌旁组织中miR-299-3p的表达情况;相同方法检测甲状腺乳头状癌细胞系BCPAP和TPC-1,人类甲状腺滤泡上皮正常细胞Nthy-ori3-1及甲状腺未分化癌细胞系8505C等四组细胞系中miR-299-3p的表达情况,用统计学方法分析其差异。2.我们分别用 mimics 上调 miR-299-3p 表达,inhibitor 抑制 miR-299-3p 的表达,用CCK-8法和EdU细胞增殖实验来观察miR-299-3P表达的上调和下调对甲状腺乳头状癌细胞增殖的影响,用细胞周期实验、细胞凋亡实验分别来观察miR-299-3p表达的上调和下调对细胞周期分布及凋亡的影响。由于之前的结果显示BCPAP的miR-299-3p表达相对较高,而TCP-1的miR-299-3p表达相对较低,我们用miR-299-3p mimics及其阴性对照转染TCP-1细胞,用miR-299-3p inhibitor及其阴性对照转染BCPAP细胞。3.我们利用小鼠原位移植瘤模型,瘤内注射上调miR-299-3p表达的agomir,观察移植瘤的生长情况,并绘制移植瘤的生长曲线。采用实时荧光定量PCR的方法比较未干预组、ago-miR-299-3p组和ago-miR-NC组移植瘤miR-299-3p的表达情况;并比较未干预组、ago-miR-299-3p组和ago-miR-NC组的移植瘤所对应的生长曲线。4.我们首先利用RNA22,TargetScan,miRWalk及MiRanda四个公共数据库进行生物信息分析,预测miR-299-3p的靶基因。并利用双荧光素酶报告系统、实时荧光定量PCR及Western blot等实验来验证靶基因。分析甲状腺乳头状癌中SHOC2表达与miR-299-3p表达的关系。实验结果1、MiR-299-3p在组织及细胞系中的表达采用实时荧光定量PCR的方法检测30对甲状腺乳头状癌及其配对癌旁组织,结果显示甲状腺乳头状癌中的miR-299-3p表达显著低于其配对的癌旁组织,差异具有统计学意义。细胞系检测结果显示在甲状腺乳头状癌细胞系TCP-1、BCPAP及未分化癌细胞系8505C中miR-299-3p的表达显著低于人类甲状腺滤泡上皮正常细胞系Nthy-ori3-1,差异具有统计学意义。2、甲状腺乳头状癌细胞miR-299-3p表达的上调和抑制实时荧光定量PCR结果显示,转染miR-299-3p mimics的TCP-1细胞miR-299-3p表达显著高于未转染细胞和转染阴性对照的细胞;而转染miR-299-3p inhibitor后的BCPAP细胞miR-299-3p的表达显著低于未转染细胞和转染阴性对照的细胞。我们在不同的甲状腺乳头状癌细胞系中分别成功的上调和下调了 miR-299-3p的表达,这为我们后续的研究提供了条件。3、MiR-299-3p表达对甲状腺乳头状癌细胞增殖的影响细胞增殖曲线的比较可以看到:TPC-1转染miR-299-3p mimics后,其增殖曲线明显在转染阴性对照组的下方,增殖速度明显低于对照组。与之对应,BCPAP转染miR-299-3p inhibitor后,其增殖曲线明显在转染阴性对照组的上方,增殖速度明显高于对照组。EdU细胞增殖实验的结果显示,TPC-1转染miR-299-3p mimics后,EdU 阳性率显著低于空白对照组。而BCPAP转染miR-299-3p inhibitor后,EdU 阳性率显著高于空白对照组。4、MiR-299-3p表达对甲状腺乳头状癌细胞周期分布的影响TPC-1在上调miR-299-3p后,与阴性对照组相比较,G1/0期细胞比例显著上升,而S期细胞比例显著下降,这一结果证明,miR-299-3p的过表达可以抑制细胞周期从G1/0期向S期转换。而在BCPAP细胞中,抑制miR-299-3p表达后,BCPAP细胞G1/0期细胞比例显著下降,而S期细胞比例显著上升,这证实miR-299-3p的下调可以促进细胞周期从G1/0期向S期转换。5、MiR-299-3p表达对甲状腺乳头状癌细胞凋亡的影响MiR-299-3p上调的TPC-1细胞凋亡率明显高于其对照组;miR-299-3p抑制的BCPAP细胞凋亡率明显低于其对照组。这些结果显示,miR-299-3p的表达可以促进甲状腺乳头状癌细胞凋亡。6、移植瘤miR-299-3p表达的检测及miR-299-3p过表达对移植瘤的生长曲线的影响小鼠移植瘤体注射ago-miR-299-3p后,实时荧光定量PCR结果显示ago-miR-299-3p组移植瘤miR-299-3p表达显著高于未处理组和ago-miR-NC组。证实了动物瘤体注射ago-miR-299-3p能够上调移植瘤miR-299-3p的表达。观察结果发现Ago-miR-299-3p组生长曲线及移植瘤重量显著低于未处理组和对照组。7、MiR-299-3p靶基因的筛选及验证(1)我们使用RNA22,TargetScan,miRWalk及MiRanda四个公共数据库进行生物信息分析,潜在靶基因有176个,筛选后选择SHOC2作为候选靶基因。(2)利用双荧光素酶报告系统证实SHOC2是miR-299-3p直接作用的靶基因为了验证SHOC2是否是miR-299-3p直接作用的靶基因我们首先构建了pGL3-SHOC2 3’UTR 和 pGL3-SHOC2 3’UTR-Mut 荧光报告质粒。TPC-1转染野生型荧光报告质粒pGL3-SHOC2 3’UTR并且同时转染miR-299-3p-mimics或者空白对照,转染miR-299-3p-mimics时荧光强度下降超过50%。而转染的突变的pGL3-SHOC2 3’UTR-Mut荧光报告质粒时,同时转染miR-299-3p-mimics或者空白对照相比较,荧光强度无显著差别。在BCPAP细胞中,我们进行了同样的实验,得到了相似的结果。这些结果说明miR-299-3p直接作用于SHOC2 3’UTR上的结合位点发挥作用,即SHOC2是miR-299-3p直接作用的靶基因。(3)在甲状腺乳头状癌中SHOC2表达与miR-299-3p表达的关系为了进一步研究甲状腺乳头状癌中miR-299-3p作用于SHOC2的机制,我们探索SHOC2表达与miR-299-3p表达的关系。①SHOC2在甲状腺乳头状癌组织与癌旁组织表达的差异。首先我们利用实时荧光定量PCR的方法,比较了 SHOC2在甲状腺乳头状癌组织与癌旁组织表达的差异。结果显示SHOC2在甲状腺乳头状癌组织中的表达显著高于癌旁组织的表达。这与miR-299-3p的情况恰恰相反。②甲状腺乳头状癌中SHOC2表达与miR-299-3p表达的相关性进而,我们利用30例甲状腺乳头状癌的手术标本提取的RNA探索了甲状腺乳头状癌中SHOC2表达与miR-299-3p表达的相关性。相关性分析结果显示,SHOC2的相对表达量与miR-299-3p相对表达量呈中度的负相关,相关系数-0.51(p=0.004)。③MiR-299-3p表达对SHOC2表达的影响为了进一步研究miR-299-3p表达对SHOC2表达的影响,我们在甲状腺乳头状癌细胞系中上调或下调miR-299-3p表达来观察SHOC2表达的变化。转染miR-299-3p-mimics 至 TPC-1 细胞,上调 miR-299-3p 后,western blot 结果显示SHOC2表达显著低于对照组;而在BCPAP细胞中加入miR-299-3p抑制剂下调miR-299-3p后,western blot结果显示SHOC2表达显著高于对照组。这些结果说明在甲状腺乳头状癌中miR-299-3p能够抑制SHOC2的表达。(4)Rescue assay为了进一步阐明miR-299-3p和SHOC2在甲状腺乳头状癌中的关系,我们进行Rescue assay实验。即研究抵消miR-299-3p-mimics引起SHOC2下调时,miR-299-3p上调所导致的细胞增殖、周期分布及凋亡变化是否也被恢复。我们比较同时转染miR-299-3p-mimics和SHOC2质粒与同时转染miR-299-3p-mimics和SHOC2阴性对照质粒时细胞增殖、周期分布及凋亡的差别。实时荧光定量PCR结果显示,同时转染mimics和SHOC2过表达质粒组的SHOC2表达显著高于mimics+阴性对照组。与miR-299-3p-mimics+SHOC2阴性对照质粒组相比较,同时转染mimics和SHOC2过表达质粒时,即SHOC2的表达rescue时,TPC-1细胞增殖显著增快;更多的细胞从G1/0期向S期转换;细胞凋亡也显著减少。这些结果说明miR-299-3p正是通过对SHOC2表达的负调控抑制了甲状腺乳头状癌细胞的增殖、周期转换,促进了细胞的凋亡。结论1.甲状腺乳头状癌中miR-299-3p的表达水平显著低于其配对的癌旁组织。甲状腺乳头状癌细胞系TCP-1及BCPAP中miR-299-3P的表达显著低于人类甲状腺滤泡上皮正常细胞系Nthy-ori3-1。2.MiR-229-3p的表达可以抑制甲状腺乳头状癌细胞的增殖。MiR-299-3p的表达使甲状腺乳头状癌细胞周期从G1/0期向S期的转换受阻。MiR-299-3p的表达可以促进甲状腺乳头状癌细胞凋亡。3.动物体内实验证明,过表达miR-299-3p可以抑制甲状腺乳头状癌细胞TPC-1移植瘤的生长。4.SHOC2是miR-299-3p直接作用的靶基因。MiR-299-3p通过对SHOC2表达的负调控抑制了甲状腺癌乳头状癌细胞的增殖、周期转换,促进了细胞的凋亡。意义1.我们的研究证实miR-299-3p通过靶向调节SHOC2的表达在甲状腺乳头状癌中发挥抑癌作用。2.我们的研究为阐明甲状腺乳头状癌进展的分子机制提供了新的见解,这可能为甲状腺乳头状癌发展中的生物标志物和治疗策略提供一些新的认识。更多还原

【Abstract】 BackgroundThyroid cancer(TC)is one of the most common malignancies in the world.Over the past decades,the incidence rate of thyroid cancer has increased significantly worldwide.In our country,the incidence rate of thyroid cancer reached fourth of the incidence rate of malignant tumors in 2015,accounting for 8.49%of the female malignant tumors.According to the origin of tumor cells,thyroid cancer is usually divided into four pathological types:papillary carcinoma,medullary carcinoma,follicular carcinoma and undifferentiated carcinoma.Among them,papillary carcinoma is the most common pathological type,accounting for 88%of all thyroid cancers.Some studies suggest that the increasing incidence rate of thyroid cancer has been mainly attributed to the increase of papillary carcinoma incidence rate in recent years.The prognosis of papillary thyroid carcinoma(PTC)is good.It is reported that the 20-year survival rate of papillary thyroid carcinoma is 99%.Although the postoperative survival rate of papillary thyroid carcinoma is very high,the long-term follow-up results show that the recurrence rate is as high as 25%.Papillary thyroid cancer not only brings the body trauma of surgical treatment to patients,but also brings heavy psychological burden because of the fear of recurrence.Therefore,it is of great value to study the molecular mechanism of the occurrence and development of papillary thyroid carcinoma.MicroRNA(miRNA)is a kind of noncoding single stranded small RNA molecules with 19-25 nucleotides encoded by endogenous genes.They can modulate post-transcriptional gene expression by translational repression or degradation.Research shows that a miRNA can regulate hundreds of different mRNAs,and a mRNA can also be regulated by multiple miRNAs.MiRNAs have been well studied by numerous researchers,and the physiological roles of miRNAs have been reported.The biological functions of miRNAs are various,including cell differential,proliferation,apoptosis,metastasis,invasion and senescence.Moreover,it has been reported that aberrant expression of miRNAs can lead to multiple different malignant tumors including GC.In recent studies,miR-299-3p has been found to play some roles in the occurrence and development of a variety of tumors.Wang et al.found that miR-299-3p inhibited the proliferation and invasion of human colon cancer cells by down regulating the expression of vascular endothelial growth factor A(VEGFA)in vivo and in vitro.In HCC(hepatocellular carcinoma),miR-299-3p inhibits tumor growth and invasion by acting on sirtuin 5.Yu et al reported that miR-299-3p could inhibit the proliferation and invasion of cervical cancer by acting on TCF4(transcription factor 4).Zhao et al reported that inhibiting the expression of miR-299-3p would reduce the invasion,migration,proliferation and promote the apoptosis of SKOV3 cells.Further study confirmed that the role of miR-299-3p was realized by regulating the expression of OCT-4(organic cation/carnitine transporter4).It can be seen from the above literature that miR-299-3p plays the role of tumor suppressor gene in colon cancer,liver cancer and cervical cancer,while it plays the role of oncogene in ovarian cancer.So far,the biological role of miR-299-3p in papillary thyroid carcinoma remains unknown.Therefore,the aim of our current research is to investigate the expression and role of miR-299-3p in papillary thyroid carcinoma.ObjectiveIn China,the incidence rate of thyroid cancer reached fourth of the incidence rate of female malignant tumors in 2015,accounting for 8.49%of the female malignant tumors.Papillary thyroid cancer not only brings the body trauma of surgical treatment to patients,but also brings heavy psychological burden because of the fear of recurrence.Therefore,it is of great value to study the molecular mechanism of the occurrence and development of papillary thyroid carcinoma.In recent years,it has been found that miRNA can affect cell proliferation,differentiation and apoptosis by regulating different genes in multiple cell signaling pathways,and then participate in a variety of pathophysiological processes of tumor,including the occurrence and development of tumor.MiR-299-3p plays the role of tumor suppressor gene in colon cancer,liver cancer and cervical cancer,while it plays the role of oncogene in ovarian cancer.So far,the biological role of miR-299-3p in papillary thyroid carcinoma remains unknown.Therefore,the aim of our current research is to investigate the expression and role of miR-299-3p in papillary thyroid carcinoma.This study will be carried out from the following aspects:1.To detect the expression of miR-299-3p in papillary thyroid carcinoma;2.To study the function of miR-299-3p in the proliferation,cycle distribution and apoptosis of thyroid papillary carcinoma cells.3.The effect of over expression of miR-299-3p on the growth of transplanted tumor in mice.4.Prediction and verification of miR-299-3p target gene.Methods1.The expression of miR-299-3p in 30 pairs of papillary thyroid carcinoma and matched paracancerous tissues were detected by real-time fluorescence quantitative PCR;the expression of miR-299-3p in four groups of cell lines,BCPAP and TPC-1(thyroid papillary carcinoma cell lines,),Nthy-ori3-1(normal human thyroid follicular epithelial cell line)and 8505C(undifferentiated thyroid carcinoma cell line),were detected by the same method;the difference was analyzed by statistical methods.2.We used mimics to upregulate the expression of miR-299-3p and inhibitor to inhibit the expression of miR-299-3p,CCK-8 assay and EdU cell proliferation experiment were used to observe the effect of up-regulation and down-regulation of miR-299-3p expression on the proliferation of thyroid papillary carcinoma cells,the effect of up-regulation and down-regulation of miR-299-3p expression on cell cycle distribution was observed by cell cycle experiment,the effect of up-regulation and down-regulation of miR-299-3p expression on apoptosis was observed by apoptosis assay.As the previous results showed that the expression of miR-299-3p in BCPAP was relatively high,while that of miR-299-3p in TCP-1 was relatively low,we used miR-299-3p mimics and its negative control to transfect TCP-1 cells,and miR-299-3p inhibitor and its negative control to transfect BCPAP cells.3.We used the mouse orthotopic tumor model,injected with agomir(the expression of miR-299-3p is up-regulated),observe the growth of transplanted tumor and draw the growth curve of transplanted tumor.The expression of miR-299-3p in the transplanted tumor of the non-intervention group,ago-miR-299-3p group and ago-miR-NC group were compared by Real time fluorescence quantitative PCR,the growth curves of the transplanted tumors were compared among the three groups.4.Firstly,we used four public databases:RNA22,TargetScan,miRWalk and MiRanda,to analyze biological information and predict the target gene of miR-299-3p.The target gene was verified by double Luciferase Report System,real-time fluorescence quantitative PCR and Western blot assay.And then we analyzed the relationship between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinoma.Results1.Expression of miR-299-3p in tissues and cellsThirty pairs of papillary thyroid carcinoma and their matched paracancerous tissues were detected by real-time fluorescence quantitative PCR,the results showed that the expression of miR-299-3p in papillary thyroid carcinoma was significantly lower than that in the paracancerous tissues.The results of cell line detection showed that the expression of miR-299-3p in thyroid cancer cell lines TCP-1,BCPAP and undifferentiated cancer cell line 8505C were significantly lower than that in human thyroid follicular epithelial normal cell line Nthy-ori3-1.The differences were statistically significant.2.Upregulation and inhibition of miR-299-3p expression in papillary thyroid carcinoma cellsThe results of real-time fluorescent quantitative PCR showed that the expression of miR-299-3p in TCP-1 cells transfected with miR-299-3p mimics was significantly higher than that in the non transfected cells and the negative control cells.However,the expression of miR-299-3p in BCPAP cells transfected with miR-299-3p inhibitor was significantly lower than that in the non transfected cells and negative control cells.We successfully up-regulated and down-regulated the expression of miR-299-3p in different cell lines of thyroid papillary carcinoma,which provided conditions for our follow-up study.3.Effect of miR-299-3p expression on proliferation of thyroid papillary carcinoma cellsThe comparison of cell proliferation curve showed that the proliferation curve of TPC-1 transfected with miR-299-3p mimics was significantly lower than that of the negative control group,and the proliferation rate was significantly lower than that of the control group.In contrast,after BCPAP transfected with miR-299-3p inhibitor,the proliferation curve of the experimental group was greatly above the negative control group,and the proliferation speed was significantly higher than that of the control group.The results of EdU cell proliferation assay showed that the positive rate of EdU in TPC-1 transfected with miR-299-3p mimics was significantly lower than that in the blank control group.But after BCPAP transfected with miR-299-3p inhibitor,its EdU positive rate was significantly higher than that of the blank control group.4.Effect of miR-299-3p expression on cell cycle distribution of thyroid papillary carcinomaAfter upregulating the expression of miR-299-3p in TPC-1,compared with the negative control group,the proportion of G1/0 phase cells increased greatly,while the proportion of s phase cells decreased significantly,the results showed that over expression of miR-299-3p could inhibit the cell cycle transition from G1/0 to S phase.However,in BCPAP cells,inhibition of miR-299-3p expression significantly reduced the G1/0 phase cell proportion of BCPAP,but significantly increased the S phase cell proportion.It is confirmed that the down-regulation of miR-299-3p can promote the cell cycle transition from G1/0 to S phase.5.Effect of miR-299-3p expression on apoptosis of thyroid papillary carcinoma cellsThe apoptotic rate of TPC-1 cells with upregulated miR-299-3p was significantly higher than that of the control group;the apoptosis rate of BCPAP cells with inhibited miR-299-3p was significantly lower than that of the control group.These results showed that the expression of miR-299-3p could promote the apoptosis of thyroid papillary carcinoma cells.6.Detection of miR-299-3p expression in transplanted tumor and the effect of over expression of miR-299-3p on the growth curve of transplanted tumorAfter injection of ago-miR-299-3p into the transplanted tumor of mice,the results of real-time fluorescent quantitative PCR showed that the expression of miR-299-3p in the group of ago-miR-299-3p was significantly higher than that in the group of untreated and ago-miR-NC.It is confirmed that the expression of miR-299-3p in transplanted tumor can be up-regulated by the injection of ago-miR-299-3p.The results showed that the growth curve and weight of transplanted tumor in ago-miR-299-3p group were significantly lower than those in untreated group and negative control group.7.Screening and verification of miR-299-3p target gene(1)We used four public databases,RNA22,TargetScan,miRWalk and MiRanda,to analyze biological information.There were 176 potential target genes.After screening,SHOC2 was selected as the candidate target gene.(2)It was confirmed that SHOC2 was the target gene of miR-299-3p by double Luciferase Report SystemIn order to verify whether SHOC2 is a direct target gene of miR-299-3p,we first constructed pGL3-SHOC2 3’UTR and pGL3-SHOC2 3’UTR-Mut fluorescence reporting plasmids.TPC-1 was co-transfected with pGL3-SHOC2 3’UTR and miR-299-3p-mimics or blank control,when miR-299-3p-mimics was transfected,the fluorescence intensity decreased by more than 50%.However,when the mutant pGL3-SHOC2 3’UTR-Mut was co-transfected with miR-299-3p-mimics or blank control,there was no obvious difference in fluorescence intensity.In BCPAP cells,we did the same experiment and got similar results.These results indicate that miR-299-3p directly acts on the binding site of SHOC2 3’UTR,and SHOC2 was a direct downstream target of miR-299-3p in PTC.(3)The relationship between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinomaIn order to further study the mechanism of miR-299-3p acting on SHOC2 in papillary thyroid carcinoma,we explored the relationship between the expression of SHOC2 and miR-299-3p.① The difference of expression of SHOC2 between papillary thyroid carcinoma and paracancerous tissueFirst of all,we compared the expression of SHOC2 in papillary thyroid carcinoma and its matched paracarcinoma tissues by real-time fluorescence quantitative PCR.The results showed that the expression of SHOC2 in papillary thyroid carcinoma was significantly higher than that in matched paracancerous tissue.This is the opposite of miR-299-3p.②Correlation between the expression of SHOC2 and miR-299-3p in papillary thyroid carcinomaFurthermore,we extracted RNA from 30 surgical specimens of thyroid papillary carcinoma and explored the relationship between the expression of SHOC2 and miR-299-3p in PTC.The results of correlation analysis showed that the relative expression of SHOC2 was negatively correlated with that of miR-299-3p,and the correlation coefficient was-0.51(P=0.004).③ The effect of miR-299-3p expression on the expression of SHOC2In order to further study the effect of miR-299-3p expression on the expression of SHOC2,we upregulated or downregulated miR-299-3p expression in thyroid papillary carcinoma cell line to observe the change of SHOC2 expression.After miR-299-3p-mimics was transfected into TPC-1 cells and miR-299-3p was upregulated,western blot showed that the expression of SHOC2 was significantly lower than that of the control group;However,after transfection of miR-299-3p inhibitor into BCPAP cells and down-regulation of miR-299-3p,western blot showed that the expression of SHOC2 was significantly higher than that in the control group.These results show that miR-299-3p can inhibit the expression of SHOC2 in papillary thyroid carcinoma.(4)Rescue assayTo further determine the role of miR-299-3p and SHOC2 in PTC,we recruited rescue assay.The results showed that when co-transfected with SHOC2 overexpression plasmid and miR-299-3p-mimics,the expression level of SHOC2 was significantly up-regulated.The up-regulated SHOC2 reversed the influence of miR-299-3p in cell proliferation.Similarly,SHOC2 overexpression promoted cell cycle progression and suppressed the cell cycle ar-rest effect of miR-299-3p.Up-regulated SHOC2 abolished the influence of miR-299-3p in cell apoptosis in PTC.In sum,we validated that miR-299-3p functioned as a tumor suppressor in PTC by targeting SHOC2.Conclusions1.The expression level of miR-299-3p in papillary thyroid carcinoma was significantly lower than that in matched paracancerous tissues.The expression of miR-299-3p in thyroid papillary carcinoma cell line TCP-1 and BCPAP was significantly lower than that in human thyroid follicular epithelial normal cell line Nthy-ori3-1.2.The expression of miR-229-3p can inhibit the proliferation of papillary thyroid carcinoma cells.The expression of miR-299-3p blocked the cell cycle transition from G0/0 to S phase.The expression of miR-299-3p can promote the apoptosis of thyroid papillary carcinoma cells.3.In vivo experiments showed that overexpression of miR-299-3p in TPC-1 could inhibit the growth of its corresponding transplanted tumor.4.SHOC2 is a direct target gene of miR-299-3p.MiR-299-3p can inhibit the proliferation and cycle transformation of papillary thyroid cancer cells and promote the apoptosis of the cells through the negative regulation of the expression of SHOC2.Significances1.Our study confirmed that miR-299-3p functioned as a tumor suppressor in thyroid papillary carcinoma by targeting the expression of SHOC2.2.Our research provided novel insights into the molecular mechanism underlying PTC progression,which might afford some new understanding in biomarkers and therapeutic strategies in PTC development.更多还原