利用Pooled RNAi Library尋找肝癌生長的特異基因

Abstract

肝癌為全球盛行癌症第五名，對於其治療或是基因檢測還沒有很好的指標，所以想要利用human pooled shRNA lentiviruses library以negative selection的方式尋找肝癌生長的特異基因。將human pooled shRNA lentiviruses library pool 2分別感染四種不同的癌症細胞株，原本包含了9984個shRNA，結果發現人類肝細胞癌細胞株Huh7、人類肺腺癌細胞株A549、人類子宮頸癌細胞株HeLa、人類胃癌細胞株SCM1共同沒有比對到的shRNA有423個，涵蓋307個基因，而只有人類肝細胞癌細胞株Huh7沒有比對到的shRNA則有334個，涵蓋303個基因。有研究顯示抑制色胺酸雙加氧酶(tryptophan 2,3-dioxygenase, TDO2)可使T細胞重新獲得對抗腫瘤的能力，所以想要探討是否可以藉由shRNA抑制TDO2基因表現來抑制肝腫瘤的生長。利用定量及時聚合酶連鎖反應，發現shRNA clone ID 874、33分別抑制人類肝癌細胞株Huh7、小鼠肝癌細胞株Hep55.1c和小鼠肝臟上皮細胞株BNL1.MEA的TDO2基因表現的效果最好。以西方墨點法偵測被shRNA clone ID 33慢病毒感染後的Hep55.1c細胞株、被scramble shRNA慢病毒感染後的Hep55.1c細胞株以及未被感染的Hep55.1c細胞株之色胺酸雙加氧酶表現量，因為陽性控制組沒有明顯的band，所以無法確定細胞色胺酸雙加氧酶表現量。以MTT試驗測定細胞增生，在第五天時，未被感染的Hep55.1c細胞株之粒線體活性比被shRNA clone ID 33慢病毒感染後的Hep55.1c細胞株和被scramble shRNA慢病毒感染後的Hep55.1c細胞株數值高，且有顯著差異，表示未被感染的Hep55.1c細胞株生長速度較快。皮下注射動物實驗方面，在C57BL/6小鼠打入被shRNA clone ID 33慢病毒感染後的細胞形成的腫瘤比被scramble shRNA慢病毒感染後的細胞形成的腫瘤小，且在第十五天有顯著差異，但在BALB/C小鼠則沒有顯著差異。肝臟注射動物實驗方面，不管是在C57BL/6或BALB/C小鼠，打入被shRNA clone ID 33慢病毒感染後的細胞形成的腫瘤大小與被scramble shRNA慢病毒感染後的細胞形成的腫瘤大小都沒有顯著差異。

Hepatocellular carcinoma is the fifth most prevalent cancer worldwide. So far, there are no good indicators for its treatment or early diagnosis. Therefore, we wanted to use human pooled shRNA lentiviruses library to identify genes that are specific to hepatocellular carcinoma growth by negative selection. Four different types of cancer cell lines were infected by human pooled shRNA lentiviruses library pool 2 which had 9984 shRNA. There were 423 shRNA including 307 genes which were not aligned in Huh7, A549, HeLa and SCM1 cells. Additionally, there were 334 shRNA including 303 genes which were not aligned only in Huh7 cell. Previous studies showed that inhibiting tryptophan 2,3-dioxygenase (TDO2) allowed T cell to regain the ability to attack tumor. Thus, we wanted to investigate whether using shRNA to inhibit TDO2 gene expression can repress the growth of hepatocellular carcinoma. By real time qPCR, we found that shRNA clone ID 874 and 33 inhibited the TDO2 gene expression levels of Huh7, Hep55.1c and BNL1.MEA cells most efficiently respectively. The protein levels of tryptophan 2,3-dioxygenase of scramble shRNA treated Hep55.1c, shRNA clone ID 33 treated Hep55.1c and non-treated Hep55.1c cells were detected by western blot. We couldn’t confirm the protein levels of tryptophan 2,3-dioxygenase of these cells because there wasn’t a distinct band in positive control. Using MTT assay to detect the proliferation rate of non-treated Hep55.1c, scramble shRNA treated Hep55.1c and shRNA clone ID 33 treated Hep55.1c cells. We found that the activity of mitochondria in non-treated Hep55.1c cells was significantly higher than scramble shRNA treated Hep55.1c and shRNA clone ID 33 treated Hep55.1c cells at day 5 indicating that the proliferation rate of non-treated Hep55.1c cells was faster. In subcutaneous injection, the size of tumors derived from shRNA clone ID 33 treated cells was significantly smaller than those from scramble shRNA treated cells at day 15 in C57BL/6 but not in BALB/C mice. However, in liver injection, there was no significant difference between the size of tumors derived from shRNA clone ID 33 treated cells and those from scramble shRNA treated cells in C57BL/6 or BALB/C mice.