登革病毒屬於黃病毒屬，主要由埃及斑蚊及白線斑蚊帶原傳播，並盛行於熱帶及亞熱帶地區。感染登革病毒的患者通常為無症狀或輕症，只有少數患者會發展為重症，如登革出血熱及登革休克症候群。肝細胞癌約佔所有原發性肝癌病例之90%，並且為目前世界上死亡率第三的癌症，其治療方法至今仍是需要被解決的難題。CD133+或 CD34+的癌細胞已被發現具有癌症幹細胞特徵，而癌症幹細胞的存在被認為是肝細胞癌難以被有效治療的原因之一。目前，病毒已被指出可能會透過與人類幹細胞的相互作用引起疾病及影響細胞分化，或產生適應性方面的優勢。在先前的研究中，人類幹細胞已被證明能夠被登革病毒感染，並且一部分被感染的幹細胞能夠在一定時期內持續釋放出具感染力之登革病毒。然而，登革病毒感染肝癌幹細胞後會對其造成的影響尚未明瞭。因此，我們想研究登革病毒感染肝癌幹細胞後，是否會造成其細胞族群發生變化。首先，我們從HepG2細胞中分選出CD133+、CD133-CD34+ 以及CD133-CD34- 細胞，然後用登革病毒進行感染。我們觀察到，在感染期間， CD133+ 細胞在分選出的CD133-CD34+ 細胞和CD133-CD34- 細胞中有增加的趨勢，但在分選出的CD133+ 細胞並沒有此現象。並且，細胞增殖實驗指出，登革病毒感染分選細胞後，CD133+細胞數量增加不是因為病毒感染而促使細胞增殖速度所導致，顯示新增的這些CD133+細胞可能是源於CD133-細胞。基於細胞受感染後所產生之病毒效價的峰值，以及細胞表現NS1蛋白的比例，我們認為相較於CD133- 細胞，CD133+ 細胞更容易被登革病毒感染。此外，登革病毒感染可以顯著維持分選出的 CD133+ 細胞中的CD133+ 細胞族群比例，同時增加分選出的CD133- 細胞中的 CD133+ 細胞族群表現。在球體形成試驗中也觀察到登革病毒的感染提升了分選出的CD133+和CD133- HepG2細胞的球體形成能力。接著，在蛋白質組分析當中，我們發現在受感染細胞中，病毒之非結構蛋白-1 (NS1)與人類α-2-HS-醣蛋白（一種 TGF-β 拮抗劑）具有交互作用。據報導，乙型轉化生長因子 (TGF-β) 會藉由降低肝細胞癌中 CD133 甲基化的程度，以增加CD133的基因表達。我們透過實驗證實登革病毒感染細胞後不僅提升了 TGF-β 的分泌，也降低了CD133啟動子-1甲基化的比例。綜合以上結果，登革病毒感染確實會增加CD133- HepG2細胞中的CD133+ 細胞群，且這些新增的CD133+ 細胞可能來自於CD133- 細胞的轉變。而這樣的轉變可能是由登革病毒的NS1蛋白通過 AHSG/TGF-β 途徑減少CD133啟動子-1的甲基化，以增加CD133基因表現所導致。

Dengue virus (DENV) belongs to the genus Flavivirus and is transmitted by the principal mosquito vector Aedes aegypti. Symptoms in people infected by DENV include asymptomatic, mild symptoms, and a few affected subjects may develop into severe dengue. Hepatocellular carcinoma (HCC) represents approximately 90% of all cases of primary liver cancer. Cancer cells with CD133+ or CD34+ have cancer stem cells (CSCs) characteristics. The presence of CSC has been considered a reason for the difficult treatment for HCC. Viruses have been indicated may directly interact with human stem cells for causing aberration in dictated differentiation programs and benefit for viral fitness. Human stem cells have been shown to be infectable by DENV, and some DENV-infected stem cells sustain the infection and are able to release infectious DENV for a certain period. However, we still do not understand the effects of DENV infection on CSC in HCC. Our aim is therefore to investigate whether the population of CSCs has been altered upon DENV infection. To achieve the aim, we utilized HepG2 cells, a cell line that has been established from HCC. The approach was to sort out CD133+, CD133-CD34+, and CD133-CD34- cells from HepG2 cells, followed by infection with DENV. Results showed that the CD133+ cells were increased in sorted CD133-CD34+ cells as well as in sorted CD133-CD34- cells, but not in sorted CD133+ cells upon DENV infection. Proliferation studies in the CD133-CD34+ and CD133-CD34- sorted cells suggested that an increase of CD133+ cells did not result from the proliferation of these cells upon DENV infection, indicating that the increase of CD133+ cells was likely derived from CD133- cells during DENV infection. In addition, we observed that CD133+ cells were highly permissive to DENV infections based upon the peak of viral titers and the expression of NS1 protein, and that the infection could significantly maintain the CD133+ populations in sorted CD133+ cells while increasing the CD133+ populations in sorted CD133- cells. The functional assay revealed that the infection of DENV improved the spheroid forming ability of sorted CD133+ and CD133- cells from HepG2 cells. Proteomic analysis of NS1 pull-down proteins in DENV infected cells suggested that NS1 interacted with alpha-2-HS-glycoprotein (AHSG), a TGF-β antagonist. TGF-β has been reported can increase CD133 expression in HCC. Upon further investigations, we found that DENV infection increased not only the concentration of TGF-β but also the unmethylated CD133 promoter-1 in sorted CD133- cells. In conclusion, DENV infection increased the CD133+ cell population in CD133- HepG2 cells, suggesting that the origin of increased CD133+ cells is likely from the CD133- cells. Furthermore, DENV NS1 protein might be the viral factor that caused the alteration in transcription. Our working model suggested that NS1 might reduce the methylation of CD133 promoter-1 through the AHSG/TGF-β pathway to increase the CD133+ cell population.

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