【作者】 刘平；

【导师】 李红武；

【作者基本信息】 安徽医科大学 ， 耳鼻咽喉科学， 2023， 硕士

【摘要】 研究背景哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路参与了哺乳动物的多种代谢过程并发挥关键的作用。mTOR信号失调会呈现出不同的病理状况,如糖尿病、癌症、阿尔茨海默病等。mTOR信号经常在各种人类癌症中失调,如乳腺癌、前列腺癌、肾癌等。mTOR信号的上调可以通过促进生长因子受体信号、血管生成、糖代谢以及抑制自噬等机制促进肿瘤的生长和进展。同时,有研究报道,PI3K/Akt/mTOR通路在80%以上的头颈鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)中被过度激活,因此mTOR为HNSCC的临床治疗提供了一个极具潜力的关键分子靶点。mTOR有两个功能不同的复合物,mTOR复合物1(mammalian target of rapamycin complex 1,mTORC1)和mTOR复合物2(mammalian target of rapamycin complex 1,mTORC2)。异常激活的mTORC1在肿瘤血管生成中起重要作用,但其确切机制尚不清楚。MicroRNAs(miRNAs)是一类在真核生物中高度保守的非编码RNAs。miRNAs大多具有发夹结构,是大约由18-24个核苷酸组成的单链小RNA。miRNAs一般存在3种形式,原始的Pri-miRNA、pri-miRNA经过加工后形成的Pre-miRNA以及pre-miRNA经过Dicer酶酶切后形成的成熟miRNA。在功能上,miRNAs通过与靶基因互补结合从而沉默靶基因的表达。然而,miRNAs在mTORC1活化的肿瘤中的作用鲜有报道,并且非编码RNA是否参与mTORC1介导的血管生成尚不清楚。研究目的在本研究中,我们探讨了mTORC1活化介导的micro-RNA-130b-3p(miR-130b-3p)的表达变化及其在TSC肿瘤以及头颈肿瘤发生发展中的作用和分子机制。研究方法TSC2和TSC1缺失的小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)(Tsc2-/-和Tsc1-/-MEFs)及对照细胞(Tsc2+/+和Tsc1+/+MEFs)被广泛用于研究TSC相关疾病和mTOR信号通路。我们首先通过实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测了mTORC1活化的细胞和对照组细胞中miR-130b-3p的表达水平。然后利用微管形成试验、鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)实验研究了miR-130b-3p对血管生成的影响。接着我们通过生物信息学分析、Western blot、RNA免疫沉淀、免疫荧光、荧光素酶报告基因、染色质免疫沉淀(Chromatin immunoprecipitation,CHIP)、微管形成实验、CAM实验、HNSCC患者来源的异种移植(patient-derived xenograft,PDX)模型等方法,进一步研究了在MEFs以及人类肿瘤中信号转导和转录激活因子3(activator of transcription 3,STAT3)、miR-130b-3p和盲肌样蛋白1(muscleblind-like protein 1,MBNL1)之间的调节机制及其功能作用。研究结果我们首先证明了mTORC1活化上调miR-130b-3p的表达,且升高的miR-130b-3p在体外和体内都增强了mTORC1激活的细胞的血管生成和成瘤能力。然后我们发现mTORC1通过激活STAT3上调miR-130b-3p的表达,同时,STAT3可以通过直接结合miR-130b基因的启动子激活miR-130b-3p。随后我们确定MBNL1是miR-130b-3p的直接靶点,而在功能上MBNL1过表达后会抑制血管生成。接着,我们证实在MEFs中抑制miR-130b-3p后,敲低MBNL1可以挽救肿瘤生长受限以及血管生成减弱的现象。我们还发现在HNSCC中,miR-130b-3p表达显著上调,并与mTORC1信号转导呈正相关。并且在HNSCC PDX模型中,miR-130b-3p受到抑制后会影响血管生成和肿瘤生长。最后,我们发现MBNL1反馈抑制了mTORC1活化的细胞中STAT3的激活。研究结论STAT3/miR-130b-3p/MBNL1反馈环在mTORC1介导的血管生成和肿瘤进展中起着至关重要的作用。该通路可作为mTORC1相关肿瘤治疗干预的靶点。更多还原

【Abstract】 BackgroundThe mammalian target rapamycin(mammalian target of rapamycin,mTOR)signaling pathway is involved in multiple metabolic processes in mammals and plays an important role.Dysregulation of mTOR signaling presents in different pathological conditions such as diabetes,cancer,and Alzheimer’s disease.mTOR signaling is often dysregulated in various human cancers,such as breast cancer,prostate cancer,and kidney cancer.Upregulation of mTOR signal can promote tumor growth and progression by promoting growth factor receptor signal,angiogenesis,glucose metabolism and inhibition of autophagy.Meanwhile,it has been reported that PI3K/Akt/mTOR pathway is overactivated in more than 80% of head and neck squamous cell carcinoma(head and neck squamous cell carcinoma,HNSCC),thus mTOR provides a potential key molecular target for clinical treatment of HNSCC.MTOR has two complexes with different functions,mTOR complex 1(mammalian target of rapamycin complex 1,mTORC1)and mTOR complex 2(mammalian target of rapamycin complex 2,mTORC2).Abnormally activated mTORC1 plays an important role in tumor angiogenesis,but the exact mechanism still unclear.MicroRNAs(miRNAs)are a class of non-coding RNAs that are highly conserved in eukaryotes.Most of the miRNAs have hairpin structure,which is a small single-stranded RNA composed of about 18-24 nucleotides.miRNAs generally exist in three forms,the original Pri-miRNA,the Pre-miRNA formed by pri-miRNA after processing,and the mature miRNA that is cleaved by Dicer enzymes.Functionally,miRNAs silence the expression of target genes by complementary binding with target genes.However,the role of miRNAs in mTORC1-activated tumors is rarely reported,and it is still unclear whether non-coding RNA is involved in mTORC1-mediated angiogenesis.ObjectivesIn this study,we explored the expression changes of micro-RNA-130b-3p(miR-130b-3p)mediated by mTORC1 activation and its role and molecular mechanism in the development and progression of TSC tumors and head and neck tumors.MethodsTSC2 and TSC1 deficient mouse embryonic fibroblasts(MEFs)(Tsc2-/-and Tsc1-/-MEFs)and control cells(Tsc2+/+ and Tsc1+/+ MEFs)are widely used to study TSCrelated diseases and the mTOR signaling pathway.We first detected the expression of miR-130b-3p in mTORC1-activated cells and control cells by quantitative real-time PCR(q RT-PCR).Then we explored the effect of miR-130b-3p on angiogenesis using tube formation assay and chick embryo chorioallantoic membrane(CAM)assay.Next,we used bioinformatics analysis,Western blot,RNA immunoprecipitation,immunofluorescence,luciferase reporter gene,chromatin immunoprecipitation(CHIP),tube formation assay,CAM assay,HNSCC patient-derived xenotransplantation(patientderived xenograft,PDX)model and other experiments,furtherly explored the regulatory mechanism and function of signal transducer and activator of transcription 3(activator of transcription 3,STAT3),miR-130b-3p and Muscleblind-like protein 1(muscleblind-like protein 1,MBNL1)in MEFs and human tumors.ResultsWe first proved that mTORC1 activation up-regulated the expression of miR-130b-3p,and the increased miR-130b-3p enhanced the angiogenesis and tumorigenesis of mTORC1-activated cells both in vitro and in vivo.Then we found that mTORC1 upregulated the expression of miR-130b-3p by activating STAT3,while STAT3 could activate miR-130b-3p by directly binding to the promoter of miR-130 b gene.Subsequently we determined that MBNL1 is the direct target of miR-130b-3p,and functionally overexpression of MBNL1 will inhibit angiogenesis.Then,we demonstrated MBNL1 depletion rescued the compromised angiogenesis and tumor growth caused by miR-130b-3p inhibition.We also found that the expression of miR-130b-3p was significantly up-regulated in HNSCC and positively correlated with mTORC1 signal transduction.And the inhibition of miR-130b-3p will affect angiogenesis and tumor growth in the HNSCC PDX model.Finally,we found that MBNL1 feedback inhibited the activation of STAT3 in mTORC1-activated cells.ConclusionThe STAT3/miR-130b-3p/MBNL1 feedback loop plays a vital role in mTORC1-mediated angiogenesis and tumor progression.This pathway could be targeted for therapeutic intervention of mTORC1-related cancers.更多还原