背景和目的：

目前我国胆囊癌的发病率和死亡率正处于不断上升的阶段，其致命性源于其恶性程度较高，治疗上缺乏早期有效的预测指标、治疗手段和预后分析模型。近年来吉西他滨、顺铂和度伐利尤单抗的联合治疗以及靶向治疗药物的临床使用已经有效改善患者的预后，但其复发率和病人远期生存率依旧令人担忧，目前我国胆囊癌患者的5年生存率仅为24.7%。最近的研究发现三基序蛋白在信号传导、细胞周期停滞和细胞凋亡等细胞生长过程中扮演重要角色，然而，关于三基序蛋白27（TRIM27）在胆囊癌发展中的具体作用尚不明确。本项研究旨在利用生物信息学分析和体内外实验，探讨TRIM27在胆囊癌中的表达情况、生物学作用以及可能的下游调控机制。

方法：

1.利用生物信息学数据库的胆囊癌数据集探究胆囊癌临床病理特征及影响预后的相关性：选取6对本中心符合上述临床病理特点的胆囊癌标本验证TRIM27的表达情况。

2.体内、体外实验探究 TRIM27 对胆囊癌肿瘤表型的效应：明确胆囊癌细胞株（GBC-SD和NOZ）TRIM27的表达水平，构建TRIM27敲降和过表达的细胞株，CCK-8实验和克隆形成实验检测细胞的增殖能力，Transwell穿孔实验检测细胞的迁移和侵袭能力，细胞周期变化和细胞凋亡情况可用流式细胞学检测；利用稳定敲降和过表达的细胞株建立小鼠皮下成瘤模型，对比敲降和过表达及各自对照组的成瘤体积及重量明确TRIM27的致癌作用，瘤体中TRIM27的表达量通过免疫组化验证。

3.探究TRIM27可能的下游调控机制：利用免疫荧光标识定位TRIM27在胆囊癌细胞中的表达，对敲降TRIM27的胆囊癌细胞株（GBC-shTRIM27）行Illumina转录本测序，结合基因功能富集分析（GO、KEGG）寻找下游可能靶点；TRIM27过表达以及敲降后下游相关基因的表达水平改变情况使用RT-qPCR、Western Blot检测，通过Western Blot检测相关通路蛋白水平变化。使用 ChIP-seq、STRING蛋白互助网络分析技术验证胆囊癌细胞中TRIM27与下游差异基因可能的结合方式。Co-IP检测TRIM27与潜在靶点的蛋白是否存在关系；最后对下游基因行挽救实验验证通路调控。

结果：

1.是否行手术治疗和淋巴结受累是影响总生存期的最具统计学意义的危险因素；且转移淋巴结达到三个时提示胆囊癌病人更差的预后结果；通过选取我院符合上述特点的胆囊癌病人的组织标本行转录组测序，发现TRIM27基因呈现高表达。

2.细胞功能实验显示GBC-SD敲降TRIM27后细胞的增殖、迁移和侵袭能力受到抑制，更多细胞停留于S期，凋亡细胞数量增加；而NOZ过表达TRIM27后细胞的增殖、迁移和侵袭得以增强，更多细胞停留于G2/M期，凋亡细胞数量减少。动物成瘤实验则显示TRIM27过表达后小鼠皮下成瘤速度更快，体积更大，重量更大；TRIM27敲降后小鼠皮下成瘤速度更慢，体积更小，重量更小。

3.基因功能富集分析显示差异基因多富集于“PI3K-AKT通路”、“焦点粘附”、“细胞外基质受体相互作用”等过程；RT-qPCR 和 Western Blot 结果显示TRIM27敲降和过表达后，PI3K/Akt通路与上皮间质-转化过程的相关蛋白水平发生相应变化，表现为在shTRIM27组PTEN表达水平升高，其下游靶点Akt水平未见明显改变，而p-Akt水平较前下降，提示PI3K-Akt通路受到抑制，另可见E-cadherin蛋白表达上调，N-cadherin、Vimentin蛋白等表达下调。提示上皮-间质转化过程收到抑制。免疫荧光标识结果提示TRIM27主要表达于细胞核中， 但ChIP-seq 结果提示TRIM27并不直接与PTEN结合，STRING蛋白互助网络分析提示可能通过与PTEN蛋白相互作用，进而调控PI3K/Akt通路与影响上皮间质-转化，Co-IP验证了以上发现。而在 TRIM27 敲降细胞GBC-SD中敲低PTEN 后，发现 PI3K/Akt通路与上皮间质-转化过程及细胞增殖和迁移的能力可被逆转。

结论：

本研究揭示了TRIM27 通过与PTEN相互作用，进一步影响Akt底物的磷酸化，上调PI3K/Akt通路，促进上皮-间质转化的过程，进而参与胆囊癌肿瘤细胞的增殖、迁移、侵袭及凋亡。我们首次提出TRIM27作为胆囊癌中高表达的肿瘤标志基因在促进肿瘤发展中发挥的作用，并证实了TRIM27-PTEN/Akt信号通路在癌症发展过程中的潜在作用。

Background

Currently, the incidence and mortality rates of gallbladder cancer in China are on the rise. Its lethality stems from its high malignancy, but a comprehensive model for early effective prediction, treatment and prognostic analysis is under investigation. In recent years, the combined treatment of gemcitabine, cisplatin, and durvalumab, as well as the clinical use of targeted therapy drugs, have effectively improved the prognosis of patients. However, the recurrence rate and long-term survival rate of patients remain a concern. At present, the 5-year survival rate of gallbladder cancer patients in China is only 24.7%. Recent studies have found that tripartite motif (TRIM) proteins play important roles in cell growth processes such as signal transduction, cell cycle arrest, and apoptosis. However, the specific role of tripartite motif protein 27 (TRIM27) in the development of gallbladder cancer remains unclear. This study aims to explore the expression, biological function, and possible downstream regulatory mechanisms of TRIM27 in gallbladder cancer using bioinformatics analysis and in vitro and in vivo experiments.

Methods

Explore the correlation between the clinicopathological features of gallbladder cancer and prognosis using the gallbladder cancer dataset from bioinformatics databases. We select 6 pairs of gallbladder cancer specimens from our center that meet the above-mentioned clinicopathological characteristics to verify the expression of TRIM27.

Investigate the effects of TRIM27 on the tumor phenotype of gallbladder cancer through in vitro and in vivo experiments. We firstly determine the expression levels of TRIM27 in gallbladder cancer cell lines (GBC - SD and NOZ), further constructing cell lines with TRIM27 knockdown and overexpression. Using the CCK - 8 assay, colony formation assay, and Transwell assay to detect the proliferation and migration abilities of cells. Cell cycle changes and apoptosis were assessed by flow cytometry. We finally construct mouse subcutaneous tumor models with TRIM27 overexpression and knockdown cell lines, compare the final tumor volumes and weights of the two groups to clarify the carcinogenic effect of TRIM27, and verify the expression level of TRIM27 in the tumor by immunohistochemistry.

Explore the possible downstream regulatory mechanisms of TRIM27: We use immunofluorescence labeling to locate the expression of TRIM27 in gallbladder cancer cells. By performing Illumina transcriptome sequencing on the gallbladder cancer cell line with TRIM27 knockdown (GBC - shTRIM27), we conduct gene function enrichment analysis (GO, KEGG) on the transcriptome data to find possible downstream targets. The changes in the expression levels of downstream related genes after TRIM27 overexpression and knockdown were assessed by RT - qPCR and Western Blot. The changes in the protein levels of related pathways  were also assessed by Western Blot. We use ChIP - seq and STRING protein interaction network analysis techniques to verify the possible binding mode of TRIM27 and downstream differential genes in gallbladder cancer cells. Co - IP was performed to detect the potential relationship between TRIM27 and target proteins. Finally, a rescue experiment on downstream genes was used to verify the potential pathway regulation.

Results

Surgical treatment  and lymph node involvement are the most statistically significant risk factors affecting overall survival in gallbladder cancer patients. When the number of metastatic lymph nodes reaches three, it indicates a worse prognosis for gallbladder cancer patients.By performing transcriptome sequencing on tissue specimens from gallbladder cancer patients in our hospital who met the above characteristics, we found that the TRIM27 gene was highly expressed.

In vitro experiments, after TRIM27 knockdown in GBC - SD cells, cell proliferation and migration abilities were inhibited, the cell cycle was arrested in the S phase, and cell apoptosis was promoted. After TRIM27 was overexpressed in NOZ cells, cell proliferation and migration abilities were enhanced, the number of cells in the G2/M phase increased, and the number of apoptotic cells decreased. In vivo experiments showed that after TRIM27 overexpression, the subcutaneous tumor formation in mice was faster, with a larger volume and weight; after TRIM27 knockdown, the subcutaneous tumor formation in mice was slower, with a smaller volume and weight.

By performing gene function enrichment analysis, it was predicted that the differential genes were concentrated to varying degrees in processes such as the "PI3K - AKT pathway", "focal adhesion", and "extracellular matrix - receptor interaction". The results of RT - qPCR and Western Blot showed that after TRIM27 knockdown and overexpression, the protein levels related to the PI3K/Akt pathway and the epithelial-mesenchymal transition process changed accordingly. In the shTRIM27 group, the expression level of PTEN increased, the level of its downstream target Akt did not change significantly, while the level of p-Akt decreased, indicating that the PI3K-Akt pathway was inhibited. In addition, the expression of E-cadherin protein was up-regulated, and the expressions of N-cadherin, Vimentin proteins were down-regulated, indicating that the EMT process was inhibited. The immunofluorescence labeling results indicated that TRIM27 was mainly expressed in the nucleus, but the ChIP - seq results suggested that TRIM27 did not directly bind to PTEN. The STRING protein interaction network analysis suggested that it might interact with the PTEN protein to regulate the PI3K/Akt pathway and affect the EMT. Co - IP verified the above findings. In the GBC - SD cells with TRIM27 knockdown, after knocking down PTEN, the PI3K/Akt pathway, the EMT process, and the cell proliferation and migration abilities could be reversed.

Conclusions

This study reveals that TRIM27 participates in the regulation of the proliferation, migration, invasion, cell cycle, and apoptosis of gallbladder cancer tumor cells by interacting with PTEN, up-regulating the PI3K/Akt pathway, and promoting the EMT process. We first propose the role of TRIM27, a oncogene highly expressed in gallbladder cancer, in promoting tumor development and confirm the potential role of the TRIM27 - PTEN/Akt signaling pathway in the cancer development process.