探討Mismatch repair在細胞受到攜有pks island的大腸桿菌感染後引發的癌化現象中所扮演之角色

Study on potential roles of mismatch repair in tumor-promoting propensity of cells infected with E. coli pks+

廖偉翔(Wei-Hsiang Liao)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802578

摘要

大腸桿菌是微生物感染中最常見來源之一，同時也是人體腸內菌的一員。近來已發現某些大腸桿菌大有一大段基因名為pks+-island，帶有此基因的大腸桿菌稱為E. coli pks+。目前已知，E. coli pks+會造成細胞DNA斷裂，且細胞若存活下來會產生基因不穩定(genome instability)及癌化現象 (tumorigenesis)。而動物實驗也發現E. coli pks+加上腸道發炎的微環境會形成腫瘤。臨床上也發現大腸癌病患腸道中帶有E. coli pks+的比例較正常人高出2至3倍。大腸癌的發生是藉由一系列的基因突變而造成的，其中已知參與的基因有APS、K-Ras及p53。其中p53的突變是屬於最終一步的突變，並且會使腺癌(adenoma)轉變成惡性的癌瘤 (carcinoma)，由此可知p53的正常或突變對於大腸癌化扮演很重要的角色。錯配修復 (mismatch repair)的功能對於大腸癌進程也有相當的影響。研究顯示，部分大腸癌患者的細胞帶有錯配修復的缺失，且也發現錯配修復的缺失的大腸癌患者其腫瘤普遍具有侵略性且更為惡化。目前推論，若癌細胞帶有錯配修復的缺失，細胞易產生二次突變(secondary mutation)，倘若突變發生在抑癌基因(tumor suppressor gene)或原致癌基因(proto-oncogene)，則會使癌細胞更惡劣。在本篇研究，我們利用多株大腸癌細胞株配合著E. coli pks+以多個面向探討，包括: E. coli pks+造成的DNA 斷裂、E. coli pks+引發的抑制細胞生長、細胞受到E. coli pks+感染後DNA 修復的情形及E. coli pks+引發的癌化現象來探討。此外，我們利用HCT116 和HCT116 p53-/-細胞執行上述的實驗，來探討p53扮演的角色。另外，為了研究錯配修復機制，我們利用HCT116 + chr2 (錯配修復缺失的細胞)及HCT116 + chr3 (錯配修復正常的細胞) 執行上述的實驗，來探討錯配修復扮演的角色。目前實驗結果顯示，在HCT116 和HCT116 p53-/-細胞中，E. coli pks+抑制生長的能力及腫瘤生成能力皆差不多，我們推論p53可能沒有參與E. coli pks+引發的生物反應; 在HCT116 + chr2 及HCT116 + chr3細胞中，我們發現在彗星(comet)實驗中，細胞受到E. coli pks+感染後相較於HCT116 + chr3細胞，HCT116 + chr2產生較多的DNA斷裂，且產生的尾距 (tail moment)也較多。 而我們也發現E. coli pks+對於HCT116 + chr2產生的腫瘤生成能力比HCT116 + chr3還明顯。綜上所述，我們認為錯配修復對於E. coli pks+ 所引發的腫瘤生成能力扮演一定的角色，而在未來或許可以多加探討。

關鍵字

腸內菌 ； 大腸桿菌 ； 癌化 ； 大腸癌 ； 錯誤配對修飾

並列摘要

Escherichia coli is the most common cause of infections and a commensal of normal gut microbiota. In recent years, Scientists found that some E. coli carry a conserved genomic island named “ pks+ island”. This gene clusters allow for producing a putative genotoxin, Colibactin. According to previous researches, a short exposure of cultured mammalian epithelial cells to live E. coli pks+ would induce a transient DNA damage response, following by cell division with incomplete DNA repair. The exposed cells present a significant increase in anchorage-independent soft agar colony formation, indicating that the infection of E. coli pks+ may cuase genomic instability and mutagenic potential, thus favoring tumor progression. In my project, we try to elucidate the molecular pathways associated with the E. coli pks+ infected colon cells. p53 is a tumor suppressor gene, and plays an important role in colon cancer progression. In colon cacner progression, loss of p53 can be seen as the last step from adenoma to carcinoma. Recently, mismatch repair status is also key factor in cancer development. In colorectal cancer, loss of mismatch repair factor may induce secondary mutation. If secondary mutation is tumor suprressor gene or proto-oncogene, cancer may develop more tumorigenesis and invasion. In this thesis, we first build up the in vitro infection assay protocol. Next, we try to investigate the influence of E. coli pks+ on colon cancer cells from different aspect: Initial damage, recovery time, cell viability and tumorigenesis. We found that infection of E. coli pks+ increases the protein level of γH2AX and activation of phosphorylated ATM and Chk2, which indicated that E. coli pks+ may cause G2/M phase cell cycle arrest. In the other aspect, we attempt to repeat the results of anchorage-independent soft agar colony formation. According to our data, we found that E. coli pks+ would suppress cell proliferation in HCT116, HCT116 p53-/-, LoVo, SW480 and HT29 cell line. E. coli pks+ increases colony number in HCT116, HCT116 p53-/- and LoVo cell line but not in SW480 and HT29 cell line in anchorage-independent soft agar assay. To investigate more, we look back the genetic and mutation status of these colon cancer cell line. We found that mismatch repair (MMR) status may have some correlation with E. coli pks+ –increasing colony number. To confirm this hypothesis, we use HCT116 + chr2 (MMR-) and HCT116 + chr3 (MMR+) cell line as model, and perform the experiments described in previous. Western blot was used to check the MMR status and it showed that HCT116 + chr3 cells expressed MLH1 but HCT116 + chr2 cells did not express. In comet assay, we found that E. coli pks+ induced higher damage rate in HCT116 + chr2 cells (MMR-) compare to HCT116 + chr3 cells (MMR+) when cells were recovered 20 and 44 hours. Finally, combining with colony formation and soft agar assay, pks++E. coli may induce more colonies in HCT116 + chr2 cells and less colonies in HCT116 + chr3 cells. Together, mismatch repair may be involved in E. coli pks+-induced tumorigenicity and it shall be further investigated.

並列關鍵字

microbiota ； E. coli ； pks island ； tumorigenesis ； colon cancer ； mismatch repair

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