【作者】 宋桂芹；

【导师】 耿佳；

【作者基本信息】 四川大学 ， 细胞生物学， 2020， 博士

【摘要】 食管癌(Esophageal Cancer,EC)是起源于食管粘膜上皮的常见消化道肿瘤,严重危害全球人类的健康。常见的组织病理学类型有食管鳞状细胞癌(Esophageal Squamous Cell Carcinoma,ESCC)和食管腺癌(Esophageal Adenocarcinoma,EAC)。我国是全球食管癌发病率和死亡率最高的国家之一,90%以上的食管癌病理类型是ESCC。多数患者临床确诊时已处于临床中晚期,约40～50%的患者发生远端转移,预后不良。因此,迫切需要深入阐明食管癌发生发展的机制,寻找潜在的临床治疗新靶点以及生物标记分子,同时提高早期筛查和诊断治疗水平,改善患者预后。SATB1(Special AT-rich sequence binding protein,SATB1)是一种组织特异性的核基质结合蛋白,该基因定位于3p23,编码763个氨基酸。研究表明SATB1除在细胞发育分化、X染色体失活等生物学过程中发挥重要调控作用外,还参与乳腺癌、胰腺癌、鼻咽癌、膀胱癌、前列腺癌等多种恶性肿瘤的发生发展,但食管鳞癌中的研究较少,具体的作用机制有待进一步研究。本研究首先利用实时荧光定量PCR、Western Blot检测了 20例食管鳞癌患者的癌组织和癌旁组织以及食管鳞癌细胞系中SATB1的表达水平,同时通过免疫组织化学技术检测144例食管鳞癌组织和癌旁组织中SATB1蛋白表达差异,分析了 SATB1的表达水平与患者临床病理参数之间的相关性。结果显示SATB1在鳞癌组织和细胞系中出现转录和翻译水平的相对高表达,免疫组织化学结果显示SATB1主要定位于细胞核,与肿瘤的分化程度密切相关。这些结果提示SATB1可能与食管鳞癌的发生发展有关;在此基础之上,进一步探究SATB1对食管鳞癌细胞生物学行为的影响。通过siRNA干扰技术对食管鳞癌细胞中的SATB1进行敲减,建立敲减细胞模型,通过MTT、流式细胞术和Transwell等体外细胞分子生物实验证实敲减SATB1后,TE-1细胞和Eca-109细胞的增殖和侵袭能力均下降、凋亡水平有所增加,这表明SATB1在体外能促进食管鳞癌细胞的增殖和侵袭,抑制凋亡。其次,为进一步探究SATB1基因影响食管鳞癌细胞生物学行为的机制,对敲减SATB1前后TE-1细胞进行高通量测序,分析其转录组水平的变化。生物信息学分析结果显示,差异表达基因共有433个,其中150个基因表达上调,283个基因表达下调,对差异表达基因进行GO和KEGG功能富集以及PPI蛋白网络分析等。结果提示FN1和PDGFR-β是SATB1发挥调节作用的关键节点基因;通过双荧光素酶实验证实SATB1对FN1和PDGFR-β有直接调控作用;接着通过相关实验证实FN1和PDGFR-β在体外促进食管鳞癌细胞的增殖和侵袭,而SATB1可正向调控FN1和PDGFR-β对食管鳞癌生物学行的影响。然后,进一步明确SATB1调控FN1和PDGFR-β参与食管鳞癌的发生发展机制。通过体外细胞水平实验证实SATB1通过调控FN1和PDGFR-β,激活AKT/mTOR信号通路,促进细胞的增殖、侵袭,通过影响Bax/Bcl2表达,抑制细胞凋亡。裸鼠移植瘤体内实验进一步验证SATB1参与食管鳞癌发展机制,结果与体外实验一致。食管鳞癌发生发展过程中有些肿瘤细胞从原发灶或转移灶脱落后,进入到血液中形成循环肿瘤细胞(Circulating tumor cells,CTCs),CTCs可用于辅助食管鳞癌的早期诊断、转移复发评估等方面,对阐明食管鳞癌的进展机制具有重要作用,但是由于CTCs在外周血中的含量极低,对其定量检测比较困难。本研究结合纳米孔传感技术和多级串联信号放大策略,建立了一种通用食管鳞癌CTCs定量检测方法。该方法利用适配体交联的DNA水凝胶作为载体对CTCs特异性结合,经过级联放大反应后,借助M2MspA蛋白质纳米孔对三螺旋DNA的特定易位事件进行测定,最终实现对CTCs的定量检测。结果显示,在电压的驱动下,三螺旋DNA通过M2MspA纳米孔时可产生一种独特的电流信号,其中台阶2是三螺旋DNA在M2MspA纳米孔收缩处解链所产生,台阶3是单链DNA通过或碰撞M2MspA纳米孔收缩处所产生,能实现CTCs的定量检测。本方法的检测下限是2个细胞,具有较高的灵敏度,具有应用于临床样本检测的潜力。综上所述,本研究深入探索了 SATB1参与食管鳞癌发生发展过程的机制,建立了一种食管鳞癌CTCs检测方法。首次证实SATB1通过正向调控FN1和PDGFR-β,进而激活AKT/mTOR信号通路,影响Bax/Bcl2,促进食管鳞癌的增殖和侵袭、抑制凋亡;建立的基于三螺旋DNA-适配体的新型食管鳞癌CTCs检测方法灵活,其灵敏性和特异性较高,为食管鳞癌的治疗提供了新的靶点和预后评估手段。更多还原

【Abstract】 Esophageal cancer(EC)is a common gastrointestinal tumor originating from the mucosal epithelium of the esophagus and it has been a serious threat to human health worldwide.There are two major pathological types of esophageal cancer,Esophageal Squamous Cell Carcinoma(ESCC)and Esophageal Adenocarcinoma(EAC).China is one of the countries with the highest incidence and mortality rates of esophageal cancer in the world,and more than 90%of esophageal cancer pathology is ESCC,most of the patients are already in advanced clinical stage at the time of diagnosis,and about 40-50%of them develop distant metastasis and have poor prognosis.Therefore,there is an urgent need to elucidate the mechanism of esophageal cancer development,and to search for potential new clinical therapeutic targets and biomarker molecules,so as to improve early screening,diagnosis and treatment,and patient prognosis.SATB1(Special AT rich sequence binding protein,SATB1)is a tissuespecific nuclear matrix binding protein which is localized at 3p23 and encodes 763 amino acids.Studies have shown that SATB1 plays an important regulatory role in biological processes such as cell differentiation and X chromosome inactivation,and is also involved in the development of breast cancer,pancreatic cancer,nasopharyngeal carcinoma,bladder cancer,prostate cancer and other malignant tumors,however,it has been less studied in ESCC,and the detailed mechanism needs further study.First,we used real-time quantitative fluorescence PCR and Western Blot to detect the transcription and translation levels of SATB1 in clinical esophageal squamous cell carcinoma tissues,paraneoplastic tissues and squamous cell lines,and immunohistochemistry to detect the expression of SATB1 in pathological tissue specimens of ESCC patients with clinical pathological correlations between the parameters were analyzed.The results showed that SATB1 showed relatively high expression at transcriptional and translation levels in ESCC tissues and cell lines,and the immunohistochemical results showed that SATB1 was mainly localized in the nucleus closely related to the degree of tumor differentiation.These results suggest that SATB1 may be associated with the development of ESCC;then,we further investigated the effect of SATB1 on the biological behavior of ESCC cells.We found that the proliferation and invasion ability of TE-1 cells and Eca-109 cells decreased and apoptosis level increased after knockdown of SATB1,indicating that SATB1 could promote proliferation and invasion and inhibit apoptosis in ESCC cells in vitro by MTT,flow cytometry and Transwell esperiments.Secondly,TE-1 cells were subjected to high-throughput sequencing before and after knockdown of SATB1 to analyze the changes in their transcriptome levels.Bioinformatics analysis showed a total of 433 differentially expressed genes,of which 150 genes were up-regulated and 283 genes were downregulated,GO and KEGG correlation analyses were performed on the differentially expressed genes.The biofunctional network analysis showed that FN1 and PDGFRB were key genes for the regulatory role of S ATB1 in TE-1 cells;the positive regulation of FN1 and PDGFR-β by SATB1 was confirmed by dual luciferase assay;FN1 and PDGFR-β promoted the proliferation and invasion of ESCC cells in vitro.Subsequently,the mechanism by which SATB1 regulates FN1 and PDGFRβ involved in the development of ESCC was further clarified.In vitro cell level experiments confirmed that SATB1 activates AKT/mTOR signaling pathway to promote cell proliferation and invasion by regulating FN1 and PDGFR-β,and inhibits apoptosis by affecting Bax/Bcl2 expression.The results of the in vivo experiments in nude mouse transplantation tumors were consistent with the in vitro experiments.In addition,more effective adjunctive methods need to be established to improve the early diagnosis and efficacy assessment detection of esophageal cancer.Circulating tumor cells(CTCs)are tumor cells that enter the bloodstream after detaching from the primary lesion or metastatic focus of tumor.The potential for application,however,is limited by the extremely low content of CTCs in peripheral blood and the presence of heterogeneity,which limits the clinical application of most CTCs detection methods.Biological nanopore sensing technology has high sensitivity and versatility,and is expected to be applied in the detection of CTCs in esophageal cancer.In this study,we developed a universal method for the quantitative detection of ESCC CTCs by combining nanopore sensing technology and a multi-stage tandem signal amplification strategy.In this method,the aptamer cross-linked DNA hydrogels was used as carriers to specifically capture specific esophageal cancer cells,and after a cascade amplification reaction,the specific translocation events of triplehelix DNA was measured with the help of M2MspA protein nanopores to finally achieve the quantitative detection of CTCs.The results show that under the driving of voltage,a unique current signal can be generated when the triple-helix DNA passes through the M2MspA nanopore,in which step 2 is generated by the unstranding of triple-helix DNA at the contraction of M2MspA nanopore,and step 3 is generated by the passage or collision of single-stranded DNA at the contraction of M2MspA nanopore,which can realize the quantitative detection of CTCs.The method has a lower limit of detection of 2 cells,which is highly sensitive and has the potential to be applied to clinical samples.In summary,this study explored the mechanism of SATB1 involvement in esophageal squamous cell carcinogenesis and established a method to detect CTCs in esophageal cancer.It was proved for the first time that S ATB1 positively regulates FN1 and PDGFR-β,and then activates AKT/mTOR signaling pathway,affects Bax/Bcl2 to promotes esophageal squamous cell carcinoma proliferation and invasion,and inhibit apoptosis;the new triple-helix DNA-adaptor-based esophageal cancer CTCs detection method is flexible,sensitive and specific,which provides new targets and prognosis for esophageal cancer treatment.更多还原