背景与目的：在全球范围内，肝细胞肝癌是最常见恶性肿瘤之一，探索有效的诊断和预后的生物标志物，对于提高肝癌诊治水平有着重要意义。MFSD2A作为转运蛋白参与了许多生理及病理过程，本研究进一步探索MFSD2A在 HCC发生发展过程中的作用。此外我们发现，MFSD2A与转录因子FOXA1表达呈显著负相关，我们探讨了FOXA1在HCC中的表达及其致癌作用。分析数据库下载的肝癌转录组数据，筛选Hub基因，探究肝细胞肝癌中E3泛素连接酶DTL的表达及预后价值。

方法：首先我们构建敲除MFSD2A HCC细胞系，通过克隆形成、流式检测细胞凋亡等功能实验验证MFSD2A对HCC细胞系恶性表型的影响。实验室前期工作中发现HCC组织中 MFSD2A和 FOXA1 mRNA表达呈负相关。从TCGA数据库和GEO数据库中下载HCC组织的转录组数据，用R软件进行分析。采用RT-PCR方法及Western Blot实验检测永生化肝细胞系及HCC细胞株中FOXA1的表达。构建过表达FOXA1的Huh7和L02细胞系，通过增殖、迁移实验验证过表达FOXA1对Huh7及L02细胞系恶性表型的影响，通过Western Blot实验检测，过表达FOXA1对Huh7细胞系E-cadherin、Vimentin等蛋白表达的影响。此外，我们还通过皮下注射肝癌细胞构建了NTG小鼠的移植瘤模型，观察FOXA1在小鼠体内的影响。从TCGA数据库获得424例包含完整临床信息的转录组数据，使用软件R中的survminer包寻找截断值，将数据按FOXA1的表达水平分为高表达组和低表达组，应用R软件中的Survival包进行预后分析。通过免疫组化实验检测49例肝癌及癌旁组织样本中FOXA1蛋白的表达水平，并分析FOXA1蛋白的表达与HCC临床病理参数及患者预后的关系。

从TCGA数据库中下载获得374例肝癌组织和50例癌旁组织转录组数据，用Wilcoxon秩和检验比较肝癌组织和正常组织中DTL的表达水平。用ROC曲线评价DTL的表达对HCC患者的诊断价值。采用Cox回归分析和Kaplan-Meier生存分析DTL表达在HCC中的预后价值。利用基于多变量Cox回归分析的列线图来预测DTL对预后的影响。

结果：

①成功构建MFSD2A敲除细胞系，实验结果表明，敲除MFSD2A后，Huh7细胞系的克隆增殖能力增强，细胞凋亡减少。

②通过GEO数据集共鉴定出1332个下调和939个上调的 DEGs。通过 PPI 网络中的模块分析，10个基因被确定为枢纽基因。通过分析TCGA数据库中下载的转录组数据，发现FOXA1在HCC组织中表达更高，FOXA1低表达组的HCC患者预后更好。RT-PCR及Western Blot实验结果显示FOXA1在肝癌细胞系中的表达均高于在永生化人肝细胞系中的表达，差异均具有统计学意义(P＜0.01)。增殖和划痕

实验表明，过表达FOXA1增加了Huh7及L02细胞系的增殖和迁移能力。Western Blot实验表明，FOXA1过表达能抑制E-cadherin的表达，增强Vimentin、C-myc的表达。皮下成瘤实验结果表明FOXA1过表达能在小鼠体内促进肝癌细胞的生长。免疫组化结果显示FOXA1蛋白在HCC组织中呈阳性高表达，预后分析显示FOXA1低表达组患者的疾病特异性生存时间和无进展间期存时间更长。

③分析TCGA数据库及GEO数据库中的转录组数据，发现DTL在肝癌组织的表达显著高于正常组织（P<0.001），并且与AFP水平、病理分级、病理分期、T分期等有关。ROC曲线显示DTL表达对HCC有显著的诊断能力（AUC = 0.96）。生存分析结果显示，DTL高表达患者的总生存期、无进展间期较差。通过构建列线图发现，DTL表达的C指数为0.663（95% CI为0.54~0.61）。GSEA结果发现，DTL可能通过影响细胞周期、染色体维持、DNA复制等通路影响肝癌的发生发展。

结论：MFSD2A在肝细胞肝癌的发生发展中发挥抑制作用。FOXA1蛋白在HCC细胞系及HCC患者组织显著高表达，并且其在HCC组织中的高表达提示肝癌患者的不良预后，表明其在促进肝细胞肝癌中发挥重要作用。DTL高表达预示了肝细胞肝癌患者的不良预后，提示其可作为肝细胞癌诊断和预后的潜在生物标志物。

Background and objctive：Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. It is of great importance to explore effective biomarkers for diagnosis and prognosis of HCC. MFSD2A is involved in many physiological and pathological processes as a transporter. This study further explored the role of MFSD2A in the development and progression of HCC. In addition, we found that MFSD2A was significantly negative correlated with the expression of transcription factor FOXA1, and we explored the expression of FOXA1 in HCC and its carcinogenic role. Liver cancer transcriptome data downloaded from database were analyzed to screen Hub genes, and explore the expression and prognostic value of E3 ubiquitin ligase DTL in HCC.

Methods: The MFSD2A knockout HCC cell lines were constructed, and the effects of MFSD2A on malignant phenotypes of HCC cell lines were verified by functional experiments such as colony formation assay and flow cytometry.

In our preliminary work, we found that the mRNA expression of MFSD2A and FOXA1 was negatively correlated in HCC tissues.Transcriptome data of HCC tissues were downloaded from TCGA database and GEO database, and analyzed by R software. FOXA1 expression in immortalized liver cell lines and HCC cell lines was detected by RT-PCR and Western Blot. FOXA1 overexpression Huh7 and L02 cell lines were constructed, and the effects of FOXA1 overexpression on the malignant phenotype of Huh7 and L02 cell lines were verified by proliferation and migration experiments. We used the western Blot assay to detect the effects of FOXA1 overexpression on the expression of E-cadherin, Vimentin and other proteins in Huh7 cell lines. In addition, xenograft tumor model of NTG mice were established by subcutaneous injection of HCC cells to observe the effect of FOXA1 in mice. The transcriptome data of 424 patients with complete clinical information were obtained from the TCGA public database. Survminer package in R software was used to search for the cut-off value, and the data were divided into the low expression group and the high expression group according to FOXA1 expression level. The prognosis analysis was performed by Survival package in R software. The expression of FOXA1 protein in 49 HCC tissues and paracancer tissue samples was detected by immunohistochemical assay, and the relationship between FOXA1 protein expression and clinicopathological indicators and prognosis of HCC patients was analyzed.

Transcriptome data of 374 HCC tissues and 50 paracancer tissues were downloaded from TCGA database. We used Wilcoxon rank sum test to compare the expression level of DTL in HCC tissues and paracancer tissues. The diagnostic value of DTL expression in HCC patients was evaluated by ROC curve. The prognostic value of DTL expression in HCC was analyzed by Cox regression analysis and Kaplan-Meier survival analysis. The effect of DTL on prognosis was predicted by using multivariate Cox regression analysis.

Results:

①The MFSD2A knockout cell line was successfully constructed. The results showed that after MFSD2A was knocked out, the cloning and proliferation ability of Huh7 cell line was enhanced, and the apoptosis of Huh7 cell line was reduced.

②1332 down-regulated and 939 up-regulated DEGs were identified by GEO data set. Through module analysis in PPI network, 10 genes were identified as hub genes. Analysis of transcriptome data in TCGA database showed that expression of FOXA1 was higher in Liver cancer tissues, and HCC patients with low FOXA1 expression had better prognosis. The results of qpcr and Western Blot showed that the expression of FOXA1 in HCC cell lines was higher than that in immortalized human liver cell lines, with statistical significance (P < 0.01). Proliferation and scratch experiments showed that the overexpression of FOXA1 increased the proliferation and migration of Huh7 and L02 cell lines. Western Blot showed that the overexpression of FOXA1 inhibited the expression of E-cadherin and enhanced the expression of Vimentin and C-myc. The results of subcutaneous tumor-forming experiment showed that FOXA1 overexpression could promote the growth of hepatocellular carcinoma cells in mice. Immunohistochemical results showed that FOXA1 protein was highly expressed in Liver cancer tissues, and prognostic analysis showed that HCC patients with low FOXA1 expression had longer disease-specific survival time and progression-free survival time.

③Transcriptome data from TCGA database and GEO database were analyzed. The expression of DTL in liver cancer tissues was significantly higher than that in normal tissues (P<0.001). Analysis of the correlation between DTL expression and clinicopathological features showed that DTL expression was related to AFP level, histologic grade, pathological stage and T stage. ROC curve showed that DTL expression had significant diagnostic ability for HCC (AUC=0.96). The results of survival analysis showed that HCC patients with high DTL expression had poor overall survival without progression interval. The C-index of DTL expression was 0.663(95%CI of 0.54 to 0.61)

 by constructing nomograms. The results of GSEA showed that DTL may affect the occurrence and development of liver cancer by affecting DNA replication, cell cycle and other pathways.

Conclusion: MFSD2A plays an inhibitory role in the occurrence and advance of hepatocellular carcinoma.FOXA1 is significantly overexpressed in HCC cell lines and tissues of HCC patients, and its overexpression in HCC tissues is significantly correlated with poor prognosis of HCC patients, suggesting that FOXA1 plays a very important role in promoting hepatocellular carcinoma. High expression of DTL predicts poor prognosis of HCC, suggesting that DTL may be a potential biomarker for prognosis and diagnosis of hepatocellular carcinoma.