鼻咽癌 (nasopharyngeal carcinoma, NPC) 是一種鱗狀細胞癌，盛行於中國東南部。 Simons等於1974年首先指出遺傳因子可能會使個人具有罹患鼻咽癌的傾向，而他們亦指出與 HLA-A2 基因是有相關。Lu 等人(2005)指出鼻咽癌的易感受性基因是位在 D6S510 和 D6S211 兩個microsatellite markers之間，總共132kb長，並且包含了HLA-A這個區域。藉由 primer walking 與 direct sequencing 方式，我們發現一個candidate 基因是HLA complex group 4 pseudogene 6 又稱 HCGIV-06，在此基因上有許多基因多型性存在，並且透過生物資訊的預測結果，發現HCGIV-06不會形成任何蛋白質，但目前對於此基因之功能並沒有深入探討。利用北方點墨法偵測到在不同的細胞株中包含鼻咽癌細胞株有HCGIV-06 primary transcript之存在，而其大小約是1.2kb。因此本研究之目標為探討HCGIV-06是否生成non-coding RNA，以及其所調節的基因在鼻咽癌形成時所扮演的角色。本研究首先利用PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probe), PCR-SSP (polymerase chain reaction-sequence specific primer), real-time PCR等方法鑑定在HCGIV-06上的基因多型性。利用real-time PCR 方式鑑定HCGIV-06上的對偶基因SNP G16980A (OR = 2.24, p = 0.02) 以及利用PCR-SSOP方法鑑定SNP G17139A (rs : 2517779 ) (OR=3.0, p<0.05)。發現這兩個對偶基因皆與鼻咽癌有相關。同時也利用Softberry生物資訊軟體預測此基因能產生十個microRNA,其中只有二個預測的microRNA (miR-17206G, miR-17759G) 位在HCGIV-06上的對偶基因上。本研究利用multiple alignment的方法看到這二個microRNA在是有演化上面的保守情形。同時利用生物資訊軟體 (Softberry and RNAhybrid) 預測這二個microRNA有可能結合在HLA-C 3’UTR並且可能調節其postranscriptional level，而在不同的物種間HLA-C 3’UTR這段區域也具有序列保守性的情形。本研究中而利用Rnase protection assay偵測此二個microRNA結果， 只有miR-17759G可以被偵測的到，並且存在於鼻咽癌細胞株(HONE-1, NPCTW01, NPCTW03, NPCTW04)。同樣利用北方點墨法也可以在鼻咽癌細胞株(HONE-1, NPCTW01, NPCTW03, NPCTW04) 偵測到miR-17759G。我們進一步分析此microRNA是否能影響HLA-C在後轉錄階段的表現，我們建立了報導基因包含HLA-C 3’UTR和microRNA表現的載體，發現此microRNA可能調控包含HLA-C 3’UTR 報導基因的表現。此等結果可以更深入了解鼻咽癌形成的分子機轉。

Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that is highly prevalent in southern China. The first indication that there might be genetic factors predisposing individuals susceptible to NPC came from the study of Simons et al., (1974), which indicated that HLA-A2 was associated with NPC. Lu et al., (2005) further demonstrated that the NPC susceptibility gene was most likely located between the microsatellitte markers D6S510 and D6S211, within a 132 kb DNA segment containing the HLA-A locus. By primer walking and direct sequencing, we have identified a candidate gene HLA complex group 4 pseudogene 6 (also named HCGIV-06), which was categorized as pseudogene. Dozens of single nucleotide polymorphisms were recognized within the transcript of HCGIV-06. Bioinformatic prediction revealed that it did not have any protein-coding capacity. No known function has been ascribed to it. Northern blot analysis identified a 1.2 Kb transcript in several cell lines, including NPC cell lines. We hypothesized that HCGIV may be a non-coding RNA and associated with genetic susceptibility to NPC. The aims of my study was first to investigate whether the HCGIV-06 could encode non-coding RNA that may play a role in the development of NPC. Second, what are the targets and their significance in the pathogenetic mechanism of NPC. In this study, genotyping methods, including PCR-SSOP (polymerase chain reaction-sequence specific oligonucleotide probe), PCR-SSP (polymerase chain reaction-sequence specific primer), and real-time PCR were applied to identify several polymorphisms in HCGIV-06. SNP G16980A (OR = 2.24, p = 0.02) and G17139A (rs : 2517779 ) (OR=3.0, p<0.05) were found to associate with NPC. miRNA prediction software (Softberry) revealed that HCGIV-06 may encode ten potential microRNAs. Two contained the SNP (miR-17206G, miR-17759G) were selected for further analysis. We found that these two potential microRNAs are evolutionary conserved by multiple alignment. We also found that HLA-C 3’UTR are conserved across different species. Two target prediction programs (Softberry and RNAhybrid) predicted two potential microRNA binding site within the HLA-C 3’UTR. Among the two miRNAs analyzed by Rnase protection assay, only miR-17759G could be detected in human nasopharyngeal carcinoma cell lines (HONE-1, NPCTW01, NPCTW03, and NPCTW04). At the same time, microRNA Northern blot also detected miR-17759G in human nasopharyngeal carcinoma cell lines (HONE-1, NPCTW01, NPCTW03, and NPCTW04). We further constructed a reporter gene containing HLA-C 3’UTR and microRNA expression vector. We found that this microRNA may downregulate luciferase reporter gene containing HLA-C 3’UTR. These results will be of help in understanding the possible function of HCGIV-06.

Abelson, J.F., Kwan, K.Y., O'Roak, B.J., Baek, D.Y., Stillman, A.A., Morgan, T.M., Mathews, C.A., Pa