Background & Aims: By whole genome sequencing, we identified for the first time that solute carrier family 12 member 5 (SLC12A5) gene located on chromosome 20q13.12 was amplified in colorectal cancer (CRC). We aimed to determine the amplification status of SLC12A5 and its clinical implication in CRC, and characterize the functional mechanisms of SLC12A5 in colorectal carcinogenesis. / Materials and Methods: Protein expression level of SLC12A5 was evaluated by immunohistochemistry. SLC12A5 amplification was verified by fluorescence in situ hybridization (FISH). The correlations between SLC12A5 expression and clinicopathologic parameters as well as the prognosis impact of SLC12A5 were analyzed in 195 CRC patients. The biological function of SLC12A5 in CRC cell lines were determined by cell viability, colony formation, invasion, migration, flow cytometry and in vivo tumorigenicity assays. Standard tail vein metastatic assay was performed to examine the effect of SLC12A5 in lung metastasis in nude mice. Western blot, luciferase reporter assays and human tumor metastasis PCR array were performed to evaluate SLC12A5 downstream effectors and related pathways. / Results: RT-PCR showed SLC12A5 was readily expressed in 7 of 9 CRC cell lines, but was absent in normal colorectal tissues. The mean protein expression level of SLC12A5 was significantly higher in primary CRCs as compared to their adjacent normal tissues. Amplification of SLC12A5 was detected in 40.8% (78/191) of primary CRCs by FISH, which was positively correlated with its protein overexpression (P < 0.001). Overexpression of SLC12A5 was positively associated with a more advanced TNM stage (P < 0.05). Multivariate Cox regression analysis showed that SLC12A5 overexpression was an independent predictor of poorer survival of CRC patients (P = 0.018). We further tested the biological function of SLC12A5 in human colon cancer cells. Ectopic expression of SLC12A5 in colon cancer cells SW480 and SW1116 increased proliferation and colony formation. Silencing SLC12A5 expression in HCT116 by siRNA had the opposite effects in vitro, and knockdown of SLC12A5 by shRNA significantly inhibited xenograft tumor growth in nude mice. We further revealed that SLC12A5 inhibited apoptosis of colon cancer cells by mediating apoptosis-inducing factor (AIF) and endonuclease G (EndoG) -dependent apoptotic signaling pathway. Moreover, gain-and loss-of-function experiments showed that SLC12A5 enhanced cell invasion and migration in vitro. Knockdown of SLC12A5 by shRNA significantly inhibited lung metastasis in nude mice. SLC12A5 promoted tumor metastasis through regulating key elements of the matrix architecture, such as matrix metallopeptidase and fibronectin. / Conclusion: We have identified a novel amplification gene SLC12A5 which is overexpressed in CRC. SLC12A5 may be an independent prognostic marker for CRC and may play a pivotal oncogenic role in colorectal carcinogenesis by inhibiting apoptosis and promoting metastasis. / 背景和目的：通過對結直腸癌進行全基因組測序，我們首次發現位於染色體20q13.12的SLC12A5基因在結直腸中擴增。本研究旨在探索SLC12A5在結直腸癌中的擴增情況和臨床意義，并進一步研究SLC12A5在結直腸癌發生發展中的作用機制。 / 材料和方法：採用免疫组化方法檢測SLC12A5的蛋白表达水平。應用熒光原位雜交方法驗證SLC12A5基因的擴增情況。在195例結直腸癌患者中对SLC12A5表达與临床病理關係及其對預後的影響其进行分析。通过檢測細胞活力、細胞集落形成實驗、侵襲實驗、遷移實驗、流式細胞術和體內成瘤實驗以研究SLC12A5在結直腸癌中的生物学功能。進而通過免疫印跡、熒光素酶報告實驗和人腫瘤轉移的PCR陣列，探索SLC12A5調控的基因和相关途径。 / 结果：我們採用RT-PCR方法檢測SLC12A5在9株結直腸癌細胞株的表達情況，SLC12A5在7株結直腸癌細胞株中穩定表達，但是在正常大腸組織中表達沉默。SLC12A5在結直腸中的平均蛋白表達水平顯著高於其鄰近的正常組織。通過熒光原位雜交方法，在40.8% （78/ 191）的結直腸癌中檢測到SLC12A5的擴增，該基因的擴增與其蛋白高表達水平呈正相關關係。SLC12A5高表達水平跟晚期TNM分期密切相關（P <0.05）。多因素Cox回歸分析表明，SLC12A5高表達是結直腸癌患者較差的生存的獨立預測因子（P = 0.018）。我們進一步在人結腸癌細胞株中檢測SLC12A5的生物功能。在結腸癌細胞SW480和SW1116中過度表達SLC12A5促進細胞增殖和集落形成。siRNA敲低HCT116 細胞SLC12A5的表達在體外實驗中有相反的效果。此外，shRNA敲低SLC12A5的表達顯著抑制裸鼠移植瘤的生長。我們進一步發現，SLC12A5通過介導凋亡誘導因子（AIF）和核酸內切酶G（EndoG）-依賴的細胞凋亡信號轉導通路抑制結腸癌細胞的凋亡。此外，功能獲得性和功能缺失性的體外實驗表明，SLC12A5促進腫瘤細胞的侵襲和遷移。尾靜脈注射實驗表明shRNA敲低SLC12A5的表達顯著抑制裸鼠肺轉移。SLC12A5通過調節基質結構的關鍵因子，如基質金屬蛋白酶和纖維連接蛋白，促進腫瘤轉移。 / 结论：我們發現了一個新的擴增基因SLC12A5，該基因在結直腸癌中高表達。SLC12A5是結直腸癌的一個獨立的預後標誌物。SLC12A5通過抑制細胞凋亡和促進腫瘤轉移，在結直腸癌的發生發展中起了舉足輕重的致癌作用。 / Xu, Lixia. / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 107-120). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1291470 |
| Date | January 2014 |
| Contributors | Xu, Lixia (author.), Chan, Francis Ka-leung (thesis advisor.), Yu, Jun , 1963- (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Medical Sciences. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvii, 120 leaves) : illustrations, computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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