背景:晚期结直肠癌(CRC)通常预后差,死亡率高。明确CRC进展的分子机制对于开发新的诊断和治疗策略以改善CRC预后和降低死亡率是必要的。转录因子IIIA(GTF3A),RNA聚合酶III转录因子,是肿瘤发生的关键驱动因素,并加剧CRC细胞生长。
目的:确认GTF3A是否通过调节胱抑素A(Csta)基因的表达促进CRC进展,并研究GTF3A是否可以作为CRC患者的预后标志物和治疗靶点。
方法:包含90对CRC组织和邻近非肿瘤组织的人组织微阵列,和包含20对CRC组织的人体组织微阵列,邻近的非肿瘤组织,使用免疫组织化学检查转移组织的GTF3A表达。分析患者的生存率。短发夹GTF3As和CSTAs被设计并包装到病毒中,以阻断Gtf3a和Csta基因的表达,分别。进行体内肿瘤生长测定以确认GTF3A是否在体内促进CRC细胞增殖。采用电泳迁移率变化法和荧光原位杂交法检测GTF3A与Csta的相互作用,而荧光素酶活性测定用于评估Gtf3a和Csta基因的表达。RNA测序(RNA-Seq)和数据分析用于筛选GTF3A的靶基因。
结果:GTF3A在CRC组织和淋巴结转移组织中的表达高于癌旁正常组织。GTF3A与CRC预后相关,Gtf3a基因的敲除损害了CRC细胞的增殖,入侵,和体外和体内的运动性。此外,RNA-Seq分析显示GTF3A可能上调Csta的表达,而荧光素酶活性测定显示GTF3A与Csta基因启动子结合并增加Csta转录。此外,CSTA调节上皮-间质转化(EMT)标志物的表达。
结论:GTF3A通过与Csta启动子结合增加CSTA表达,增加的CSTA水平通过调节EMT促进CRC进展。GTF3A的抑制防止CRC进展。因此,GTF3A是CRC潜在的新型治疗靶标和生物标志物。
BACKGROUND: Advanced colorectal cancer (CRC) generally has poor outcomes and high mortality rates. Clarifying the molecular mechanisms underlying CRC progression is necessary to develop new diagnostic and therapeutic strategies to improve CRC outcome and decrease mortality. Transcriptional factor III A (GTF3A), an RNA polymerase III transcriptional factor, is a critical driver of tumorgenesis and aggravates CRC cell growth.
OBJECTIVE: To confirm whether GTF3A promotes CRC progression by regulating the expression of cystatin A (Csta) gene and investigate whether GTF3A can serve as a prognostic biomarker and therapeutic target for patients with CRC.
METHODS: Human tissue microarrays containing 90 pairs of CRC tissues and adjacent non-tumor tissues, and human tissue microarrays containing 20 pairs of CRC tissues, adjacent non-tumor tissues, and metastatic tissues were examined for GTF3A expression using immunohistochemistry. The survival rates of patients were analyzed. Short hairpin GTF3As and CSTAs were designed and packaged into the virus to block the expression of Gtf3a and Csta genes, respectively. In vivo tumor growth assays were performed to confirm whether GTF3A promotes CRC cell proliferation in vivo. Electrophoretic mobility shift assay and fluorescence in situ hybridization assay were used to detect the interaction of GTF3A with Csta, whereas luciferase activity assay was used to evaluate the expression of the Gtf3a and Csta genes. RNA-Sequencing (RNA-Seq) and data analyses were used to screen for target genes of GTF3A.
RESULTS: The expression of GTF3A was higher in CRC tissues and lymph node metastatic tissues than in the adjacent normal tissues. GTF3A was associated with CRC prognosis, and knockdown of the Gtf3a gene impaired CRC cell proliferation, invasion, and motility in vitro and in vivo. Moreover, RNA-Seq analysis revealed that GTF3A might upregulate the expression of Csta, whereas the luciferase activity assay showed that GTF3A bound to the promoter of Csta gene and increased Csta transcription. Furthermore, CSTA regulated the expression of epithelial-mesenchymal transition (EMT) markers.
CONCLUSIONS: GTF3A increases CSTA expression by binding to the Csta promoter, and increased CSTA level promotes CRC progression by regulating the EMT. Inhibition of GTF3A prevents CRC progression. Therefore, GTF3A is a potential novel therapeutic target and biomarker for CRC.