轉錄因子SPZ1透過BRAF促進肝癌中的腫瘤生長

Abstract

肝癌（Hepatocellular carcinoma, HCC）是一種常見的惡性腫瘤，也是世界上最常見的癌症死亡原因，其預後差且復發率高。然而，目前對於肝腫瘤形成的詳細機制尚不清楚。轉錄因子Spermatogenic leucine zipper 1（SPZ1）是一種致癌基因，在精子發生過程中扮演重要角色，包括細胞生長和分化。先前的研究已發現SPZ1受到上游RAS-MAPK訊息傳遞路徑調控，從而導致癌細胞增生和腫瘤形成。然而，對於SPZ1如何調控下游基因的機制尚不清楚。BRAF為一種致癌基因，透過MAPK訊息傳遞路徑調控癌細胞生長、增生和存活。因此，我們提出SPZ1可能透過BRAF來調控腫瘤生長的假設。我們首先檢測197位肝癌患者的腫瘤與非腫瘤檢體中SPZ1及BRAF表現，結果發現在腫瘤組織中SPZ1與BRAF均有高度表現。另外，我們比較了SPZ1與BRAF表現在肝癌患者中臨床病理特徵的相關性，並進行存活分析，發現高度表現SPZ1與BRAF對於病人的預後較差。此外，我們觀察到SPZ1與BRAF mRNA表現呈中度正相關。為了確認SPZ1是否為BRAF的目標基因，我們利用shRNA在Huh7和SK-Hep1中抑制了SPZ1的表現。結果顯示，當抑制SPZ1表現時，BRAF蛋白質表現也隨之減少。接著，我們將不同片段的BRAF promoter接合上含有Luciferase gene的pGL4質體，並與含有SPZ1的質體共轉染到肝癌細胞株中。透過冷光素酶檢測法，我們發現SPZ1可能與BRAF promoter 的-200到-61位置處結合。最後，我們進一步研究了過度表達SPZ1和SPZ1/shBRAF對肝癌細胞生長的影響。結果顯示，SPZ1可能透過BRAF來調控細胞生長。綜上所述，我們的研究結果顯示，在肝癌發展中，SPZ1扮演著重要的角色，可能透過BRAF來促進細胞增生。未來的研究將繼續探索SPZ1/BRAF訊息傳遞路徑在肝癌細胞發展中的功能。Hepatocellular carcinoma (HCC) is a common malignant tumor and a leading cause of cancer-related mortality worldwide, characterized by poor prognosis and high recurrence rates. However, the precise mechanisms underlying HCC development remain elusive. The transcription factor Spermatogenic leucine zipper 1 (SPZ1), recognized as an oncogene, plays a crucial role in spermatogenesis, encompassing cellular growth and differentiation processes. Previous studies have highlighted SPZ1 regulation by the upstream RAS-MAPK signaling pathway, culminating in cellular proliferation and tumorigenesis. Nevertheless, the downstream gene modulation mechanisms of SPZ1 remain obscure. BRAF, an oncogene, orchestrates cell growth, proliferation, and survival through the MAPK signaling pathway. Thus, we hypothesize that SPZ1 may regulate tumor growth via BRAF. We initially examined the expression of SPZ1 and BRAF in tumor and non-tumor specimens from 197 liver cancer patients. The results revealed a significant upregulation of both SPZ1 and BRAF in the tumor tissues. Furthermore, we examined the correlation between SPZ1 and BRAF expression and clinical-pathological characteristics in HCC patients, conducting survival analyses that unveiled an unfavorable prognosis associated with heightened SPZ1 and BRAF expression. Additionally, we observed a moderate positive correlation between SPZ1 and BRAF mRNA expression. To substantiate SPZ1 as a putative BRAF target gene, SPZ1 expression was silenced using shRNA in Huh7 and SK-Hep1 cells. The results demonstrated that BRAF protein expression decreased concomitant with SPZ1 suppression. Employing luciferase reporter constructs harboring distinct segments of the BRAF promoter, we co-transfected them with a plasmid containing SPZ1 into HCC cell lines. Luminescence assays indicated potential SPZ1 binding to the -200 to -61 region of the BRAF promoter. Finally, we delved into the impact of SPZ1 overexpression and SPZ1/shBRAF on HCC cell growth. Outcomes suggested that SPZ1 may modulate cell growth via BRAF. To conclude, our study elucidates the pivotal role of SPZ1 in HCC progression, potentially fostering cellular proliferation through the SPZ1-BRAF pathway. Subsequent research will further explore the functional relevance of the SPZ1-BRAF signaling pathway in HCC cell development.