食管癌是我国常见的恶性肿瘤之一。2018年中国癌症报告显示，食管癌发病率和死亡率在我国癌症中分居第六位和第四位。食管癌有两种病理类型：食管鳞癌与食管腺癌。在我国，90％以上的食管癌为食管鳞癌。尽管诊疗技术有所发展，但食管鳞癌发病隐匿，进展迅速，其五年生存率仅为10-30%。因此，揭示食管癌发生发展的分子机制，寻找潜在诊断标志物与治疗靶点，研发新的治疗药物是我国肿瘤研究领域的重要工作。

Ras-相关结构域家族蛋白6（RASSF6）是Ras相关结构域蛋白家族中的一员。RASSF6基因启动子区的高甲基化，导致其在结肠癌、乳腺癌及黑色素瘤等肿瘤中表达显著下调，可通过影响FAK、Hippo等信号通路调控肿瘤的生长与凋亡。本课题组食管鳞癌转录组测序数据显示，RASSF6在食管鳞癌中表达异常上调，这提示RASSF6在食管鳞癌中有着不同于其他癌种的作用机制。因此，我们以RASSF6为研究对象，深入探讨RASSF6的异常表达在食管鳞癌发生发展中的功能与机制。

在本研究中，我们首先在43对食管鳞癌组织中检测RASSF6的表达，结果显示RASSF6在食管鳞癌组织中的mRNA及蛋白表达水平显著高于癌旁组织。随后对84对食管鳞癌组织进行免疫组化染色，发现RASSF6在食管鳞癌组织中表达上调，而且它的异常高表达与食管鳞癌患者的不良预后显著相关。CoBRA实验表明，在食管鳞癌中RASSF6启动子区未发生显著的甲基化。

体外功能实验显示，过表达RASSF6可促进食管鳞癌细胞的增殖、克隆形成以及侵袭迁移能力，而敲降RASSF6可抑制食管鳞癌细胞的增殖、克隆形成、侵袭迁移能力，并导致细胞周期G1/S期的阻滞。动物实验表明，敲降RASSF6后，食管鳞癌细胞皮下成瘤以及肺转移能力显著减弱。

我们通过免疫共沉淀联合质谱技术发现，RASSF6可与抑癌基因TRIM16结合。进一步研究表明，RASSF6可通过结合TRIM16促进其泛素化过程，从而影响TRIM16蛋白的稳定性。机制研究发现，RASSF6可通过调控TRIM16影响Cyclin D1和Snail的表达，促进食管鳞癌细胞的增殖与侵袭迁移。同时，TRIM16可部分回复RASSF6对食管鳞癌恶性表型的影响。

综上所述，本课题首次发现RASSF6在食管鳞癌中异常高表达，RASSF6可通过结合TRIM16影响其稳定性促进食管鳞癌的发生发展，为食管鳞癌的诊治提供了新的思路。

近年来研究发现，在基因组发生转录的基因中，编码蛋白质的RNA仅占3%左右，其余的均以RNA的形式发挥功能，这提示非编码RNA在人体的生理过程中发挥重要作用。食管癌发病机制不清，缺乏有效的诊断标志物和治疗靶点。过去的研究多集中于蛋白在食管癌发生发展中的作用，非编码RNA在食管癌中的作用及分子机制尚不明确。

长链非编码RNA（long non coding RNA，LncRNA）是一类由RNA聚合酶Ⅱ 转录、长度大于200nt，无蛋白编码能力的非编码RNA。其种类繁多，具有丰富的二级结构，表达具有较强的组织特异性。LncRNA可以通过自身的折叠或结合染色质、RNA及蛋白等方式，在多个水平调控基因的表达，影响肿瘤的恶性增殖、转移、代谢和凋亡等。

本课题组前期对91例食管鳞癌及配对的癌旁组织进行转录组测序数据显示，LncRNA-581在食管鳞癌组织中表达上调；在独立于转录组测序样本的另外85例食管癌组织及癌旁配对组织进行qPCR，结果显示，LncRNA-581在食管鳞癌组织中高表达；UCSC数据库显示，LncRNA-581在食管癌中表达上调，物种之间的保守性较差，不具有蛋白编码能力。然而LncRNA-581在肿瘤中的功能与作用机制尚未见报道。

我们通过RACE及Northern Blot实验鉴定了LncRNA-581在食管鳞癌中的全长是668nt。核浆分提实验表明LncRNA-581定位于细胞浆。细胞功能实验显示，LncRNA-581促进食管鳞癌细胞的增殖、克隆形成以及侵袭迁移。构建LncRNA-581稳定低表达的食管癌细胞系，发现敲降LncRNA-581后，食管鳞癌细胞体内成瘤能力减弱。初步机制研究发现，LncRNA-581可以结合并影响防御素HNP-1的表达，进而影响单核细胞向M1型巨噬细胞的极化过程。

以上研究表明，LncRNA-581在食管鳞癌中的表达上调，可结合并影响HNP-1的表达，影响单核细胞向巨噬细胞极化，进而促进食管鳞癌的恶性进程。我们后续将会进一步探究LncRNA-581对食管鳞癌细胞免疫逃逸的影响。

esophageal cancer is one of the common malignant tumors in china. the 2018 china cancer report showed that the incidence and mortality of esophageal cancer ranked the sixth and fourth in china. there are two pathological types of esophageal cancer, esophageal squamous cell carcinoma (escc) and esophageal adenocarcinoma, the former is the main type in china. in spite of the development of diagnosis and treatment technology, the pathogenesis of escc is concealed, its progress is rapid, and the five-year survival rate is only 10-30%. therefore, it is of great significance to study the mechanism of escc and to screen the molecular markers and effective drug targets of esophageal cancer.

ras-associated domain family protein 6 (rassf6) is one member of the ras associated domain protein family. in colon cancer, breast cancer and melanoma, the expression of rassf6 was significantly reduced due to epigenetic modification. rassf6 could affecte the growth and apoptosis of tumor by regulating fak, hippo and other signaling pathways. however, our tranomics sequencing data in the early stage showed that the expression of rassf6 in escc was increased abnormally，which was different from that in other cancers. therefore, we study the function and mechanism of rassf6 in the development of escc.

 in this study, we first tested the expression of rassf6 in 43 pairs of escc tissue. the results showed that the mrna and protein expression levels of rassf6 in escc were significantly higher than that of adjacent tissues. the subsequent immunohistochemical staining of 84 esophageal squamous cell carcinoma tissues showed that the expression of rassf6 in escc was increased, and its abnormal expression was significantly correlated with the adverse prognosis of patients with escc. then we conducted cobra and found no methylation in the promoter region of rassf6 in escc tissues.

the vitro functional experiments showed that overexpression of rassf6 promoted the proliferation, colony formation and migration and invasion of escc cells, while knockdown of rassf6 inhibited the proliferation, colony formation, invasion and migration of escc cells, and inducing the g1/s phase arrest. animal experiments showed that the abilities of subcutaneous tumor formation and lung metastasis of escc were significantly attenuated after knockdown of rassf6.
     we found that rassf6 bound to the tumor suppressor gene trim16 by immunoprecipitation combined with mass spectrometry. further studies have shown that rassf6 could promote the ubiquitination process of trim16, thereby affecting the stability of trim16 protein. mechanism studies have found that rassf6 could affect the expression of cyclin d1 and snail by regulating trim16, and promoted the proliferation and migration and invasion of escc cells. at the same time, trim16 partially restored the effect of rassf6 on the malignant phenotype of escc.
    in summary, this study found that rassf6 was abnormally highly expressed in escc for the first time. furthermore, rassf6 could promote the development of escc by combining trim16. all of these provide a new idea for the diagnosis and treatment of escc.

esophageal cancer is one of the common malignant tumors in china. the 2018 china cancer report showed that the incidence and mortality of esophageal cancer were respectively rank sixth and fourth in china. there are two pathological types of esophageal cancer: esophageal squamous cell carcinoma and esophageal adenocarcinoma. most patients with esophageal cancer in china are esophageal squamous cell carcinoma. despite the development of diagnosis and treatment technology，the pathogenesis of esophageal squamous cell carcinoma is concealed and the progress is rapid，the five-year survival rate is still maintained at the lower level of the scale. therefore, it is of great clinical significance to study the mechanism of the occurrence and development and to screen the molecular markers and effective drug target of esophageal cancer.  

long-chain non-encoded rna (long non-coding rna, lncrna) is a class of non-encoding rna transcribed by rna polymerase ⅱ, longer than 200nt in length, and with little protein encoding ability. the kind of it is abundant, the sondary structure of it is complicated, and the expression is organizationally specific. recent studies have shown that lncrna can regulate gene expression at multiple levels through its own folding or binding dna, rna or protein, and affect the proliferation, metastasis, metabolism and apoptosis of tumor cells.

in the early work, 91 cases of esophageal squamous cell carcinoma and paired adjacent tissues were sequenced by tranion group. comparing the two groups of data, it was found that the expression of lncrna-581 in esophageal squamous cell carcinoma was upregulated, and there was no report about it. the noncode database shows that lncrna-581 has four tranion copies and only expresses in placental tissue. the ucsc database shows that the expression of lncrna-581 in esophageal cancer is increased without protein coding ability. the qpcr of 85 patients with esophageal carcinoma and adjacent tissues isolated from the tranion group sequencing samples showed that lncrna-581 was highly expressed in esophageal squamous cell carcinoma.

the results of race and northern blot showed that lncrna-581 in esophageal squamous cell carcinoma was 668nt in length. lncrna-581 was located in the cytoplasm. cell function experiments showed that lncrna-581 promoted the proliferation, cloning, and invasion and migration of esophageal squamous cell carcinoma. it was found that the tumor forming ability of esophageal squamous cell tumor was weakened after stably knocking down lncrna-581.preliminary mechanism studies have found that lncrna-581 can combine and influence the expression of hnp-1, which in turn affected the polarization process of m1 macrophages.

the above studies showed that lncrna-581 was upregulated in esophageal squamous cell carcinoma. it could combine and influence the expression of hnp-1, affecting the polarization of monocytes to macrophages, and then promoted the malignant process of esophageal squamous cell carcinoma. we will further explore the effects of lncrna-581 on the immune escape of esophageal squamous cell carcinoma.