THRAP3藉由減少BCAR3表現量來抑制大腸直腸癌轉移

Abstract

大腸直腸癌是全世界常見的惡性腫瘤之一，而癌細胞的侵襲及轉移是造成患者死亡的主要原因，因此，對於大腸直腸癌轉移探究其機制是重要的研究方向。為了全面性的探討轉移機制，我們使用全基因體shRNA靜默(knockdown)技術試圖篩選出與大腸直腸癌轉移相關的基因。在實驗室先前的研究中，藉由細胞侵入實驗篩選出高侵襲性的HCT-116細胞，並利用次世代定序確認shRNA序列所對應的基因。經由實驗發現利用特異shRNA抑制THRAP3表現可提高細胞侵襲能力。此外，初步利用大腸直腸癌病患檢體進行驗證發現，病患具THRAP3表現量低者，其整體存活率顯著劣於表現量高之病患，亦發現THRAP3低表現量與淋巴或遠端轉移及癌症分期有高度相關性。迄今為止，THRAP3在腫瘤轉移過程中所扮演的角色仍然未知。為了進一步證實THRAP3在大腸直腸癌轉移中的作用，我們發現THRAP3靜默後，除了明顯促進HCT-116細胞的侵襲能力外，透過傷口癒合試驗發現細胞的移動能力也會顯著增加。另外，藉由外送THRAP3的表現質體於THRAP3被靜默的細胞中，可以顯著恢復對細胞侵襲能力的抑制作用。同時，我們在不同大腸直腸癌細胞株HCT-15和DLD-1中亦得到驗證，靜默THRAP3會提高其細胞侵襲能力。綜合上述，結果顯示THRAP3會抑制大腸直腸癌細胞的轉移。為了針對THRAP3做更深入地探討，透過次世代定序進行全轉錄組定序比較細胞在THRAP3靜默後所造成的基因表現量變化，從中挑選出表現量上升差異較大的基因，並進一步經由實驗驗證後，發現靜默BCAR3表現對細胞侵襲及移動能力有明顯抑制作用，可能作為受THRAP3調控進而影響大腸直腸癌轉移的候選基因，故我們將著重探討其之間的關係。根據全轉錄組定序的數據顯示THRAP3靜默會增加BCAR3 mRNA表現量，同時利用西方墨點法確認BCAR3蛋白表現量也有顯著上升。此外，由實驗結果發現在靜默THRAP3的細胞中抑制BCAR3表達後，可以減緩原由THRAP3靜默時導致細胞侵襲能力增加的現象，暗示THRAP3可能藉由調控BCAR3影響細胞侵襲能力。為了進一步釐清THRAP3調控BCAR3的機轉，我們探討了BCAR3 mRNA穩定度、pre-mRNA剪切效率或啟動子活性是否受到THRAP3調控，然而目前為止在這些實驗中並未沒有足夠的數據證明THRAP3是如何調控BCAR3。綜合我們實驗結果顯示，靜默THRAP3會增加BCAR3表現量而促進細胞侵襲能力，但是還需要更多研究才能明瞭其調控機轉。

Colorectal cancer (CRC) is one of the most common malignancies in the world, and metastasis is one main cause of cancer-related death. Therefore, it is an important issue to explore the mechanism of CRC metastasis. For a comprehensive perspective of metastatic CRC, we used a genome-wide shRNA library to discover metastasis-associated genes in CRC. In our previous study, the shRNA lentivirus-infected HCT-116 cells were subjected to invasiveness selection by a transwell invasion assay. The fragments corresponding to the shRNA of invaded HCT-116 cells were sequenced by Next Generation Sequencing (NGS). It was found that knockdown of THRAP3 increases invasion ability of HCT-116 cells. Additionally, the validation of the CRC patient cohort demonstrated that the low expression of THRAP3 is significantly associated with shorter overall survival, lymph node invasion, distant organ metastasis and tumor staging. However, the role of THRAP3 in cancer metastasis is still unknown. To further confirm the role of THRAP3 in CRC metastasis, the results showed that THRAP3 knockdown not only significantly promotes HCT-116 cell invasion, but also increases migration by wound healing assay. In addition, restoration of THRAP3 could recover the inhibition of cell invasion ability. Knockdown of THRAP3 increases the cell invasion ability was verified in two additional colorectal cancer cell lines, HCT-15 and DLD-1. Taken together, the results reveal that THRAP3 can inhibit CRC metastasis. To have insights into the molecular mechanism of THRAP3-involved in CRC metastasis, we performed RNA sequencing analysis. The top ranking fold-change genes whose expression up-regulated by loss of THRAP3 were selected and further verification revealed that BCAR3 knockdown dramatically decreases cell invasion and migration. BCAR3 may be a candidate gene that is regulated by THRAP3 to affect CRC metastasis, therefore, we next focused on the relationship between these two genes. According to RNA sequencing analysis data, both mRNA and protein expressions of BCAR3 were increased while THRAP3 knockdown. On the other hand, knockdown of BCAR3 abrogates the increase of invasion which is caused by THRAP3 knockdown, suggesting that THRAP3 modulates metastatic ability of cells through BCAR3. To further clarify the mechanism, we explored whether BCAR3 mRNA stability, pre-mRNA splicing efficiency, or promoter activity is regulated by THRAP3. However, there has not been enough data to prove how THRAP3 upregulates BCAR3. In summary, our results show that THRAP3 knockdown can increase BCAR3 expression and promote cell invasion, but further studies are necessary to understand its regulatory mechanism.