探讨环指蛋白，跨膜2（RNFT2）在肝细胞癌（肝癌）中表达及其对细胞增殖、侵袭和患者预后的影响。

利用阿拉巴马大学伯明翰分校癌症数据分析门户（UALCAN）获取RNFT2在肝癌和正常肝组织及肝癌不同肿瘤分期和病理分级中表达；R2基因组学分析和可视化平台分析RNFT2在肝癌和正常肝组织中的表达差异；肝细胞和组织中的基因表达谱（GepLiver）分析RNFT2表达与肝癌患者生存期的相关性。临床标本来源于2021年1月至2021年12月西安交通大学第一附属医院肝癌患者手术切除的24对肝癌组织及癌旁组织样本。患者男19例，女5例；年龄42~71岁，中位年龄52岁。利用Western blot检测RNFT2蛋白在肝癌和癌旁组织以及正常肝细胞和肝癌细胞中的表达。采用小干扰RNA（siRNA）技术下调HCCLM3和Huh7细胞中RNFT2表达，5-乙炔基-2'-脱氧尿苷（EdU）及Transwell实验分析敲低RNFT2对肝癌细胞增殖、迁移和侵袭能力的影响。采用单因素方差分析、LSD-t或t检验来分析各组之间的差异。生存分析采用Kaplan-Meier法和Log-rank检验。

UALCAN和R2平台分析TCGA和GEO数据库中肝癌组织及正常肝组织的转录组测序数据，结果显示RNFT2 mRNA在肝癌组织中表达明显高于正常肝组织（t=19.343，7.133，6.934；P<0.001）。24对肝癌与对应癌旁组织进行Western blot检测，结果显示肝癌组织RNFT2蛋白相对表达量为2.1（1.1，3.3），亦明显高于癌旁组织的1.1（0.5，1.7） （Z=-2.536；P<0.05）。UALCAN平台分析TCGA数据库发现RNFT2 mRNA与肿瘤分期及病理分级有关（F=5.082，8.008；P<0.05），Ⅱ和Ⅲ期的肝癌组织中RNFT2 mRNA表达明显高于Ⅰ期的肝癌组织（LSD-t=0.161，0.162；P<0.05），且RNFT2 mRNA在高病理分级（3级和4级）的肝癌组织中表达明显高于低病理分级（1级和2级）的肝癌组织（LSD-t=0.196，0.142和0.384，0.360；P<0.05）。GepLiver平台分析TCGA数据库中肝癌患者生存数据分析结果显示RNFT2 mRNA高表达提示肝癌患者总体生存期、疾病特异性生存期、无病生存期和无进展生存期明显缩短（HR=1.86，1.73，1.60，1.82；P<0.05）。RNFT2在肝癌细胞系中表达明显高于正常肝细胞（P<0.05），敲低RNFT2导致HCCLM3和Huh7细胞增殖、迁移和侵袭能力明显减弱（P<0.05）。

RNFT2在肝癌组织中表达上调，其高表达与患者预后不良有关。敲低RNFT2明显抑制肝癌细胞的增殖、迁移和侵袭能力。

To investigate the expression level of ring finger protein, transmembrane 2 (RNFT2) in hepatocellular carcinoma (HCC) and its effect on cell proliferation, invasion and prognosis.

The expression levels of RNFT2 in HCC, normal liver and HCC tissues with different tumor stages and pathological grades were obtained by using UALCAN. R2 genomics analysis and visualization platform were used to analyze the difference of RNFT2 expression level between HCC and normal liver tissues. The correlation between the expression of RNFT2 and survival of HCC patients was analyzed by gene expression profile (GepLiver) in liver cells and tissues. Clinical samples were collected from 24 pairs of HCC and adjacent tissues of HCC patients undergoing surgical resection at the First Affiliated Hospital of Xi’an Jiaotong University from January to December 2021. Among them, 19 patients were male and 5 female, aged from 42 to 71 years, with a median age of 52 years. The expression levels of RNFT2 protein in the HCC and adjacent tissues, and normal liver cells and HCC cells were detected by Western blot. The expression levels of RNFT2 in HCCLM3 and Huh7 cells were down-regulated using small interfering RNA (siRNA). The effect of RNFT2 knockdown on the proliferation, migration and invasion of HCC cells was analyzed by EdU-based assay and Transwell chamber test. One-way ANOVA, LSD-t test and t-test were used to analyze the differences among different groups. Survival analysis was performed by Kaplan-Meier method and Log-rank test.

The transcriptome sequencing data of HCC and normal liver tissues in TCGA and GEO databases were analyzed by UALCAN and R2 platforms. The results showed that the expression level of RNFT2 mRNA in HCC tissues was significantly higher than that in normal liver tissues (t=19.343, 7.133, 6.934; all P<0.001). Western blot of 24 pairs of HCC and matched adjacent tissues showed that the relative expression of RNFT2 protein in HCC was 2.1 (1.1, 3.3), significantly higher than 1.1 in adjacent tissues (0.5, 1.7) (Z=-2.536; P<0.05). Analysis of TCGA database by UALCAN platform found that RNFT2 mRNA was associated with tumor staging and pathological grading (F=5.082,8.008; both P<0.05). The expression of RNFT2 mRNA in stage Ⅱ and Ⅲ HCC tissues was significantly higher than that in stage Ⅰ HCC tissues (LSD-t=0.161, 0.162; both P<0.05), and the expression of RNFT2 mRNA in high pathological grade (grade Ⅲ and grade Ⅳ) was significantly higher than that in low pathological grade (grade Ⅰ and grade Ⅱ) (LSD-t=0.196, 0.142, 0.384, 0.360; all P<0.05). GepLiver platform analysis of the survival data of HCC patients in TCGA database showed that high expression of RNFT2 mRNA prompted that the overall survival, disease-specific survival, disease-free survival and progression-free survival of HCC patients were significantly shortened (HR=1.86, 1.73, 1.60, 1.82; all P<0.05). The expression of RNFT2 in HCC cell line was significantly higher than that in normal liver cells (P<0.05). RNFT2 knockdown significantly weakened the proliferation, migration and invasion of HCCLM3 and Huh7 cells (all P<0.05).

The expression level of RNFT2 is up-regulated in HCC tissues, which is associated with poor prognosis of HCC patients. RNFT2 knockdown significantly inhibits the proliferation, migration and invasion of HCC cells.