| 摘要: | 肝癌是世界排名第六名的常見癌症，在台灣則是男女性癌症死因的第一及第二名，對台灣人民的健康造成極大的威脅。而肝癌所造成的高死亡人數的主因，通常是病患太晚才發覺罹患癌症，此時癌細胞已經轉移或更惡性化，因此可以早期診斷出肝癌的方法已經成為重要的議題之一。
從許多文獻中得知微型核醣核酸(microRNA)可以參與很多生物體內的生理活動，包含了細胞增生、分化以及細胞凋亡等。近年來的研究也顯示微型核醣核酸可同時扮演抑癌基因(tumor suppressor)或者是致癌基因(oncogene)，所以當微型核醣核酸的表現量不正常，就可能造成癌症的發展與進程。先前本實驗室利用微型核醣核酸微陣列篩選方式，對不同惡性化肝癌細胞株Hep-3B, Hep-J5, Hep-G2, Hep-G2215, Sk-hep-1, 及Mahlavu進行篩選，找到了有明顯表現量差異的微型核醣核酸，從資料庫比對找出與肝癌細胞移行及藥物敏感性相關的hsa-miR-125b。為証實此段微型核醣核酸與肝癌細胞之移行及藥物敏感性有關，本實驗挑選了hsa-miR-125b表現量低，且移行能力也較其他肝癌細胞株差的Hep-G2進行後續實驗。利用lentivirus感染的方式，送入帶有hsa-miR-125b及篩選基因的質體，感染Hep-G2後挑出大量表現的stable clone。從細胞型態上可以觀察到，相較於野生型的Hep-G2細胞，大量表現miR-125b的Hep-G2細胞在型態上變得有較多觸角，也具有較強的移行能力，緊接著利用了二維蛋白電泳找出可能會被 hsa-miR-125b表現量所調控的蛋白，雖然從二維蛋白電泳比較中找出了與細胞移行相關的蛋白Profilin-1，然而從基因序列分析卻找不到hsa-miR-125b的結合位置。除此之外，本研究也利用mRNA維陣列分析，找出可能會被hsa-miR-125b所抑制的標的-Adenomatous polyposis coli (APC)，在蛋白質電泳的實驗也證實相較於野生型的Hep-G2而言，具有hsa-miR-125b的細胞株中APC表現確實較低。由於APC蛋白會影響β-Catenin的穩定性，因此具較低表現量的APC細胞會有較高量的β-Catenin表現，並確認其下游與細胞移行相關的路徑，如N-cadherin及Vimentin也因此表現量有所不同。除此之外，從對藥物的敏感性來看，hsa-miR-125b的表現會造成肝癌細胞株對doxorubicin等抗癌藥物敏感性變差。所以本研究認為 hsa-miR-125b 在肝癌細胞株 Hep-G2 中所扮演的角色為致癌基因，會與抑癌基因 APC結合並抑制 APC的表現量，進而影響其下游的路徑已達到使癌細胞更惡性化的情形。
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide. Over 600,000 cases of HCC are diagnosed each year all over the world, which about two-third of cases happen in Asia. HCC is one of the health threats in Taiwan, which are top one cancer mortality in men and the second most common in women. There are 7,000 people die from HCC every year in Taiwan. The main reason of high mortality rate is that the early symptoms of HCC are not so clear until the later stages of disease, therefore earlier diagnosis of HCC earlier has become the important issues.
According to previous studies, microRNAs were showed to participate lots of physiological activities which including cell proliferation, differentiation, and apoptosis. Recently, studies also showed that microRNA could act as oncogene or tumor suppressor gene. According to our preliminary data, several microRNAs were significant differentially expressed in different malignant liver cancer cell lines such as Hep-3B, Hep-J5, Hep-G2, Hep-G2215, SK-hep-1 and Mahlavu. Further analysis showed hsa-microRNA-125b (hsa-miR-125b) was one of over expression in the malignant HCC cell lines. In order to explore the correlation between hsa-mir-125b and malignant of HCC, we use hepatoma cancer cell line Hep-G2 detected lower expression of hsa-miR-125b, sending lentivirus, with miR-125b plasmid and selection gene, infected to evaluate the expression of hsa-mir-125b expression in Hep-G2 and singled out the performance of a large number of stable clone. Compare to the wild-type Hep-G2, cell morphology of hsa-mir-125 over-expression cells changed from flatted round to spindle-like shape companion. Further study showed that the migration ability of hsa-mir-125b over-expression Hep-G2 was stronger than wild-type and control . Then use two-dimensional electrophoresis to identify protein that may be regulation by hsa-mir-125, although identified the protein profilin-1 associated with cell migration, however profilin-1 has no binding site of hsa-mir-125b from the sequence analysis. Except that, hsa-mir-125b over-expressed Hep-G2 was more resistant to drug treatment. In addition, we also use mRNA-dimensional array analysis to identify the mRNA Adenomatous polyposis coli (APC) and has the 3’UTR binding site of hsa-mir-125b, in protein electrophoresis experiments and real-time PCR also confirmed that APC would be suppressed by hsa-mir-125b. Since the APC protein can affect the stability of β-Catenin, so with a lower expression of APC have the higher amount of β-Catenin and confirmed its downstream pathway factors associated with cell migration, such as N-cadherin and Vimentin. In addition to the sensitivity of the drug, the expression of miR-125b would decrease the drug sensitivity of doxorubicin . Therefore, this study suggests that miR-125b is a oncogene in Hep-G2 and down-regulated the expression of tumor suppressor gene APC, affecting downstream of cancer cells. |
