尋找染色體3p25.1-p26.3之大腸直腸癌相關的抑癌基因

Abstract

於實驗室先前研究，利用分布在第三號染色體的23個微衛星標記，分析112對大腸直腸癌檢體，定義3p25.1-p26.3為共同刪除區域I (common deletion region I, CDRI)。並發現CDRI發生刪除與，其癌症患者的年齡顯著較年輕，且與腫瘤發生遠端器官轉移和病人預後較差有顯著相關。因此，本論文研究目的乃於CDRI尋找和大腸直腸癌相關的抑癌基因。首先在CDRI選出6個異合子頻率較高的微衛星標記，其平均間距約2 Mb，分析179對大腸直腸癌檢體之微衛星標記的失異合性。結果顯示D3S2397有最高的刪除頻率，為27.8%。針對至少有一個微衛星標記發生失異合性的56個案例進行統計分析，則D3S2397刪除頻率高達83.3%。結合CDRI之D3S1297，定義出最小刪除區域(minimal deletion region, MDR)為D3S1297-D3S2397之區域(3p25.3-p26.1)。分析6個微衛星標記之失異合性的臨床相關性，其單變項分析結果為：(1) D3S2387之失異合性較發生於男性(p= 0.0462)。 (2) D3S2397之失異合性和病人手術年齡有顯著相關(p= 0.0462)，並與遠端器官轉移(p= 0.0069)和Dukes氏分期(代表癌症進展)(p= 0.0176)有相關。 (3) D3S2403之失異合性和年齡有顯著相關(p= 0.0473)。 (4) MDR發生刪除和遠端器官轉移(p= 0.0033)以及Dukes氏分期(p= 0.0139)顯著相關。存活分析結果：病患較差之預後分別與D3S2397之失異合性(p= 0.0397) 和MDR發生刪除(p= 0.0333)顯著相關。於是進一步在MDR尋找可能的抑癌基因，針對9個已知功能的基因，先用RT-PCR於12個大腸癌細胞株和10對腫瘤檢體和正常黏膜組織分析其表現量，而於腫瘤表現量下降的基因包括：CNTN4、IL5RA和LRRN1；接續以即時反轉錄聚合酶連鎖反應，針對52對大腸直腸癌檢體進行定量分析，結果顯示：CNTN4和IL5RA 分別於42.3% (22/52)和44.2% (23/52)的腫瘤案例表現量下降；而52例大腸直腸癌腫瘤，相較於其正常黏膜組織，CNTN4和IL5RA的表現量顯著下降 (p< 0.0001和p= 0.0001)，為CDRI的候選抑癌基因(candidate tumor suppressor gene)。綜合本論結果並結合文獻研究，決定優先建構CNTN4的表現載體，以利將來對其抑癌功能的鑑定。

In previous studies, we carried out deletion mapping of chromosome 3 in 112 cases of sporadic colorectal cancer (CRC) via loss of heterozygosity (LOH) study with 23 microsatellite markers spanning from 3pter to 3qter, and thus defined the common deletion region I (CDRI) from 3p25.1-p26.3. Furthermore, genetic loss of CDRI was significantly associated with younger age at onset, tumor metastasis, and poorer overall survival. The specific aim of the study is to explore candidate tumor suppressor genes (TSGs) associated with CRC in CDRI. We conducted LOH analysis in 179 sporadic CRC with 6 new microsatellite markers. The highest LOH frequency occurred at D3S2397 locus, which was involved in 27.8% of 179 informative cases and 83.3% of 56 tumors with LOH at one or more microsatellite loci. Combined with previous study, a minimal deletion region (MDR) was defined between D3S1297 and D3S2397 at 3p25.3-p26.1. In addition, the clinical relevance of these genetic losses was assessed via univariate analysis. The results showed that, (1) LOH frequency of D3S2387 was higher in male patients with CRC (p= 0.0462); (2) genetic loss of D3S2397 was significantly associated with younger age at onset (p= 0.0462), distant metastasis (p= 0.0069) and Dukes’ stage (p= 0.0176); (3) genetic loss of D3S2403 locus was significantly associated with younger age at onset (p= 0.0473); (4) genetic loss of MDR was significantly associated with distant metastasis (p= 0.0033) and Dukes’ stage (p= 0.0139). Furthermore, poor prognosis of patients with CRC was significantly associated with genetic loss of D3S2397 or MDR (p= 0.0397 and 0.033, respectively). Accordingly, we proposed that there might be CRC-associated TSGs in the MDR defined. We first screened gene expression of 9 genes with known function in MDR with 12 CRC cell lines and 10 pairs of CRC primary tissues by RT-PCR. The gene expression of CNTN4, IL5RA and LRRN1 were down-regulated in certain CRC cells and tumors. We further validated the down-regulation of 3 candidate genes in 52 pairs of CRC primary tissues by quantitative RT-PCR, and then revealed that CNTN4 and IL5RA RNA transcripts were obviously decreased in 42.3% (22/52) and 44.2% (23/52) of colorectal carcinomas as compared with their matched normal mucosa (p< 0.0001 and p= 0.0001, respectively) Combined with literature review, CNTN4 expression plasmid was constructed for further study to identify tumor suppressor functions of CNTN4 in colorectal tumorigenesis.