| Abstract: | 最近研究指出，致癌病毒感染宿主細
胞，會經由使宿主細胞抑癌基因甲基化，
而導致細胞的腫瘤化，但其作用機轉仍不
清楚。已知高危險型HPV 所轉錄產生的致
癌蛋白E6 可經由降解p53 抑癌蛋白而促
使細胞的腫瘤化，亦有研究指出p53 抑癌
蛋白可抑制DNMT 轉錄起始區的轉錄活
性進而抑制DNMT 蛋白的表現，因此推測
HPV 可能藉由E6 蛋白使p53 蛋白去活化
而無法抑制DNMT 的轉錄，進而促使抑癌
基因p16 的甲基化。但在子宮頸癌的研究
發現，HPV 16/18 E7 會與HDAC 交互作
用，而使HDAC 維持DNA 雙股結構之能
力失效，使E2F 啟動有關細胞增生之基因
轉錄活化。這顯示HPV 16/18 E6 及E7 致
癌蛋白在肺癌細胞及子宮頸癌細胞株的甲
基化調控機制以及致癌機轉有所不同。除
p53 外，DNMT3b 轉錄起始區的基因多型
性亦影響其基因的轉錄調控，且HPV16 致
癌蛋白E6 是否會直接調控DNMT3b 的表
現則仍不清楚，因此本計畫擬構築不同基
因型之DNMT3b 轉錄起始區轉染至肺腺
癌細胞株A549 ， 利用report assay 及
Chromatin immunopreciption assay (CHIP)
方式分析釐清HPV E6 致癌蛋白是經由抑制p53 而活化DNMT3b，或是可直接經由
與DNMT3b 轉錄起始區結合而活化
DNMT 的表現，並釐清HPV 對不同基因
型之DNMT3b 的轉錄活性是否有影響，此
外亦利用MS-PCR 及同步定量反轉錄聚合
酶連鎖反應(real-time RT-PCR)分別偵測
p16基因的甲基化及其mRNA 表現，以證
明HPV 確實經由影響DNMT3b 造成p16
基因甲基化影響p16 抑癌基因表現而使細
胞腫瘤化。
Frequent promoter hypermthylation of
tumor suppressor genes (TSGs), such as p16,
RASSF1A, and ER, was frequently observed
in oncogenic virus-induced human cancers.
However, the underline mechanism is still
unclear. TSGs transcription was regulated by
the balance of acetylation and deactylation of
histone through histone acetylase (HAT) and
histone deacetylase (HDAC), respectively. In
cervical cancer, HPV 16/18 E7 can be
interacted with HDAC to open DNA strand
for E2F binding and then upregulates cellproliferation-associated gene expressions.
Our preliminary data showed that HDAC
expression was increased in lung cancer cells
after transfected with HPV 16 E6, E7,
respectively, and the hypermethylation
frequency of p16 and RASSF1A was
concomitantly increased in HPV-transfected
lung cancer cells as compared with parental
cells. Moreover, the expression levels of
DNMT1 and DNMT3b in cervical cancers
were significantly lower than those of lung
tumors with HPV infection (unpublished
data). This result was consistent with fewer
p16 hypermethylation in cervical cancer
compared to lung cancer. Based on the above
mentions, HPV infections in lung cancer and
cervical cancer had a different role in p16
inactivation. Beside p53，DNMT3b promoter
polymorphism will also affect on p16
transcriptional regulation, and whether
HPV16 E6 oncoprotein can regulate
DNMT3b expression directly or not is still
unclear. Therefore, we propose to construct
different genomic types of DNMT3b
promoters and transfect them into lung
adenocarcinoma cells A549, by using report
assay and Chromatin immunopreciption
assay (CHIP) to illuminate whether HPV E6
oncoprotein is through the inhibition of p53
and further activation of DNMT3b, or
through binding with DNMT3b promoter
directly to activate DNMT expression, also
to clarify whether HPV will affect the
regulatory activity of different genomic types
of DNMT3b promoters or not. Meanwhile,
p16 hypermethylation and its mRNA
expression will be examined by MS-PCR
and real-time RT-PCR, respectively, to prove
that HPV is through DNMT3b upregulation
and then causes p16 hypermethylation and
tumorigenesis. |
