臺灣博碩士論文加值系統

長片段非編碼核醣核酸(lncRNA)是一群長度大於200個核苷酸非蛋白質編碼轉錄體，許多研究資料顯示長片段非編碼核醣核酸在癌症發展中扮演重要角色其中包括細胞生長、細胞週期、凋亡與轉移，Metformin已經被證實在許多癌症中擁有預防與治療癌症的潛力，在之前的研究中皆專注於尋找受metformin調控的蛋白質編碼基因和微小核醣核酸，而這些受調控的基因主要參與癌症生長，但是在胃癌中metformin如何透過調控長片段非編碼核醣核酸表現造成細胞生長的抑制目前並不清楚。因此本研究希望了解metformin在胃癌中的角色並且嘗試找尋其下游的調控基因，我們的結果顯示metformin處理可以誘導細胞週期停滯在G2/M，並且顯著性抑制胃癌細胞的生長，為了近一步了解詳細的調控機制，我們使用微陣列晶片分析胃癌細胞處理 metformin、控制組以及兩對胃癌病人的正常與腫瘤組織中的轉錄體，結果發現處理metformin後可以誘導4,160個基因表現上升以及3,383個基因表現下降，將這些受metformin調控的基因進一步作訊息傳遞路徑分析發現，這些metformin調控的基因顯著的聚集在調控細胞生長與細胞週期控制的路徑上，而這些metformin調控的基因中有61個蛋白質基因與20個長片段非編碼核醣核酸基因在胃癌檢體中呈現低表現,但當metformin處理之後會提升其表現量，相反的其中有61個蛋白質基因與7個長片段非編碼核醣核酸基因在胃癌檢體中呈現異常高表現,但處理metformin可以抑制其表現量，其中我們選定Loc100506691作深入探討，利用real-time PCR分析發現，胃癌細胞處理metformin後loc100506691表現量明顯降低，且與劑量多寡或時間長短呈現正相關。在臨床胃癌病人腫瘤比起正常組織中的Loc100506691呈現顯著高表現 (P<0.001)，利用siRNA方式剔除降低Loc100506691表現能夠有效的抑制胃癌細胞生長、轉移能力與細胞週期停滯。我們的結果揭示metformin抑制胃癌細胞生長的機制可能某些部份是因Loc100506691表現量受到抑制後而誘導細胞週期停滯在G2/M所引起。

The long-non coding RNAs (lncRNAs) are a group of non-protein coding transcripts, which are more than 200 nucleotides in length. Accumulating evidences showed that lncRNAs play critical role in cancer progression, including cell growth, cell cycle, apoptosis and metastasis. Metformin shows the potential as a preventive and therapeutic drug for human cancers. Previous studies indicated that metformin suppressed cancer cerll growth by regulating protein coding genes and microRNAs (miRNA). However, the role of lncRNA involved in metformin-induced inhibition of cell growth and its biological function in gastric cancer remains largely unknown. In this study, we like to identify metformin-associated lncRNAs in gastric cancer cell. Our results showed that metformin could significantly suppress gastric cancer cell growth via inducing cell cycle arrest at G2/M phase. In order to study detail mechanism, we performed the transcriptome profiles of HR cell with/without metformin treatment, adjacent normal tissues and cancer tissues form two gastric cancer patients using microarray approach. Our data revealed that 4,160 genes upregulated and 3,383 genes downregulated in HR cell with metformin treatment. Using pathway enrichment analysis revealed that these metformin-associated genes significantly involved in cell cycle-relative pathway.

Among these metformin-regulating genes, these are 61 protein-coding genes and 20 lncRNAs were downregulated in gastric cancer compared to adjacent normal tissues, but upregulated in HR after metformin treatment. Furthermore, 61 protein-coding genes and 7 lncRNAs upregulated in gastric cancer, but downregulated in HR cell by metformin treatment. Among them, the oncogenic lncRNA, Loc10050669, was selected to further examine in our study. The expression levels of Loc10050669 were decreased in dose- and time course-dependent manner after gastric cancer cells treatment with metformin. Moreover, Loc100506691 were significant overexpression in gastric cancer compared to adjacent normal tissues (P<0.001). Knockdown of Loc10050669 expression could significantly suppress gastric cancer cell growth and induce cell cycle arrest at G2/M phase. Furthermore, Loc100506691 knockdown also significantly suppressed cell invasion ability in gastric cancer cells. Our findings concluded that anti-proliferative effects of metformin in gastric cancer may result partly from induction of cell cycle arrest at G2/M by suppressing loc100506691 expression.

Acknowledgement ………………………………………………………ⅰ
Chinese Abstract ………………………………………………………. ⅱ
English Abstract ………………………………………………………. ⅲ
Table of Contents ……………………………………………………… ⅳ
List of Tables …………………………………………………………... ⅴ
List of Figures …………………………………………………………. ⅵ
I. Introduction ………………………………………………………. 01
II. Specific Aims ……………………………………………………… 07
III. Results ……………………………………………………………... 08
3-1 To investigate the effects of gastric cancer with metformin treatment …………………………………………………………… 08
3-1-1 Metformin inhibited gastric cancer cell proliferation …….. 08
3-1-2 Metformin affects DNA damage and induces cell cycle arrest at G2/M-phase in gastric cancer cell ……………………………………… 08
3-1-3 Metformin inhibited gastric cancer cell invasion …………. 09
3-1-4 Generate the RNA transcriptome profiles of HR cell with metformin treatment ……………………………………………………. 09
3-2 Identification of metformin-associated lncRNA candidates in gastric cancer …………………………………………… 11
3-2-1 Identify oncogene and tumor suppressor gene in gastric cancer …………………………………………………………………… 11
3-2-2 Identify metformin-regulating oncogene and tumor suppressor gene in gastric cancer ………………………………… 12
3-2-3 LncRNA expression were regulated by metformin ………….. 12
3-2-4 Examine the expression levels of Loc100506691 in gastric cancer ……………………………………………………………………. 13
3-3 The biological role of Loc100506691 in gastric cancer …………. 14
3-3-1 Identify full length of Loc100506691 sequence ……………... 14
3-3-2 Knockdown of Loc100506691 suppress gastric cancer cell proliferation ……………………………………………………... 14
3-3-3 Knockdown of Loc100506691 inhibit invasion ability on gastric cancer cell ………………………………………………………… 15
IV. Discussion ………………………………………………………….. 16
V. Methods …………………………………………………….......... 20
5-1 Chemicals ……………………………………………………… 20
5-2 Cell culture …………………………………………………….. 20
5-3 WST-1 Assay ………………………………………………….. 20
5-4 Image flow cytometry assay …………………………………… 21
5-5 Western blotting ……………………………………………….. 21
5-6 Invasion assay …………………………………………………. 22
5-7 Colony-forming assay …………………………………………. 22
5-8 Microarray ……………………………………………………... 23
5-9 Pathway enrichment analysis …………………………….. 23
5-10 RNA isolation ………………………………………………… 24
5-11 Reverse transcription-PCR …………………………………… 24
5-12 Gene expression by quantitative PCR ……….. 24
5-13 For full-length, RNA ligase-mediated rapid amplification of 5´ and 3´ cDNA ends (RLM-RACE) ………………………………………….. 25
5-14 Loc100506691 cDNA Sequences ……………………………. 25
5-14-1 Isoform-1 ………………………………………………… 25
5-14-2 Isoform-2 ………………………………………………… 26
5-14-3 Isoform-3 ………………………………………………… 26
5-15 Polymerase chain reaction, PCR ………………... 27
5-16 RNA Interference …………………………………………….. 28
VI. References …………………………………………………………. 29

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