| 摘要: | 腎細胞癌（ RCC）的發生和死亡率逐年增加。該病在早期大多數都無症狀，但
轉移的機率高。在 RCC 亞型中，透明腎細胞癌（ccRCC）最常見，死亡人數最
多。其中有超過 70％的 ccRCC 病例在 von Hippel-Lindau 抑癌基因（VHL）中攜
帶基因組或表觀遺傳缺陷。而我們實驗室先前就已經建立了一種包含腎臟小管
中 Vhlh（小鼠 VHL 基因座）基因條件失活的小鼠模型，這小鼠模型會導致腎臟
增生，炎性和纖維化病變。最初的微陣列分析確定了與野生型相比， HAVCR-1
在突變小鼠腎臟中呈現高表達。 HAVCR-1（也稱為 KIM-1 或 TIM-1）是 1 型膜
蛋白，已被公認是腎臟疾病的診斷標記。最近，一些研究還表明， KIM-1 在包
括 ccRCC 的人類癌症中異常表達，所以我們假設 KIM-1 可能有助於 ccRCC 的
發展。但是 KIM-1 和 VHL 之間的功能相關性仍然是未知的。在這裡，我們分別
在有或沒有 VHL抑制的近端腎小管上皮細胞系（HK-2）中產生了 KIM-1抑制，
並進行了RNA序列分析，比較對照VHL抑制和KIM-1的差異表達基因（DEG）。
我們發現組合式和 VHL-KIM-1 雙組合式有許多 DEG，並且使用 Ingenuity
Pathway Analysis（IPA）對 DEG 進行富集分析，在 VHL 抑制的細胞中，炎症，
增殖和間充質的轉化途徑有增加，而細胞死亡和凋亡途徑則呈現減少。熱圖聚
類分析揭示了 4 個不同的基因組，它們在不同的基因敲低背景下顯示出不同的
表達模式，表明 VHL 和 KIM-1 之間的功能關係可能不是簡單的線性途徑。有趣
的是通過傷口癒合實驗對細胞遷移進行了測試，我們發現 VHL 基因敲落細胞中
的 KIM-1 基因敲落可以抑制細胞遷移。我們的結果表明，與 VHL 單敲除相比，
HK-2中的KIM-1和VHL的雙重敲除降低了多種細胞因子誘導的炎症途徑表達，
並且影響了腫瘤的發展途徑。因此，這些結果在未來可以用來創造新 ccRCC 或
腎癌治療的標靶。;The occurrence and death rates of renal cell carcinoma (RCC) are increasing each year.
However, this disease is mostly asymptomatic at an early stage but highly metastatic.
Among the RCC subtypes, the clear-cell RCC (ccRCC) is the most prevalent and
accounts for the most deaths. Importantly, more than 70% of the ccRCC cases carry
either genomic or epigenetic defects in the von Hippel-Lindau tumor suppressor gene
(VHL). Our laboratory has previously established a mouse model containing
conditional inactivation of the Vhlh (the mouse VHL locus) gene in the kidney tubules,
which results in hyperplastic, inflammatory, and fibrotic lesions in the kidneys. Initial
microarray analysis identified high expression of HAVCR-1 in mutant mouse kidneys
compared to wild-type. HAVCR-1 (also known as KIM-1 or TIM-1) is a type 1
membrane protein and has been recognized as a diagnostic marker for kidney disease.
Recently, several studies have also shown that KIM-1 is expressed abnormally in
human cancers, including ccRCC. This leads us to hypothesize that KIM-1 may
contribute to the progression of ccRCC. However, the functional correlation between
KIM-1 and VHL are still unknown. Here, we generated KIM-1 knockdown in proximal
tubule epithelial cell line (HK-2) with or without VHL knockdown, and RNA sequencing analysis was performed to analyze the differentially expressed genes
(DEGs) comparing control, VHL knockdown, KIM-1 knockdown, and VHL-KIM-1
double knockdown. Many DEGs were found, and the enrichment analysis using the
Ingenuity Pathway Analysis (IPA) software on DEGs showed that in VHL knockdown
cells, inflammatory, proliferative, and mesenchymal transition pathways are increased,
while cell death and apoptosis pathways are reduced, consistent with the tumorigenic
property of these VHL-defective kidney cells. Heatmap clustering analysis revealed 4
distinct groups of genes that show different expression patterns in different gene
knockdown backgrounds, suggesting the functional relationship between VHL and
KIM-1 is probably not a simple linear pathway. Interestingly, cell migration was tested
by wound healing experiment, and we found that KIM-1 knockdown in VHL
knockdown cells can inhibit cell migration. Our results showed that double knockdown
of KIM-1 and VHL in HK-2, compared with VHL single knockdown, reduces the
expression of multiple cytokines-induced inflammation pathways, and influences
tumor progression pathways. Thereby, these results together point to a new ccRCC or
renal cancer treatment targets in general. |
