||The functional role of hsa-miR-181b in colorectal cancer
||Insitute of Bioinformatics and Biosignal Transduction
大腸直腸癌在台灣的致死率是第二高的，而其在全世界的死亡率每年也有高達五十萬人之多。關於大腸直腸癌的研究，在臨床上對於病患的預後與治療仍然是相當薄弱。近年來有許多研究指出，許多微小核糖核酸(micro-RNAs)都被發現有參與在大腸直腸癌的發育過程中。在過去文獻中提到微小核糖核酸-181b (miR-181b)在大腸直腸癌中都有較高的表現量，顯示miR-181b可能可以提供一個好的治療指標。在本篇研究中，我們分析了大腸直腸癌病患檢體。萃取檢體中的所有的核糖核酸，利用及時定量聚合酶連鎖反應分析微小核糖核酸及一般的訊息核糖核酸基因。我們確認了miR-181b在腫瘤組織中的表現量都有明顯高於在正常組織中的表現。因此我們進一步利用TargetScan的資料庫找到了微小核糖核酸-181b可能會結合的目標基因，組織金屬蛋白酶抑制物-3 (TIMP-3)。令我們興奮的是，在大腸直腸癌組織與細胞株中，miR-181b與TIMP-3的表現量呈現完全相反的現象。接著我們利用reporter assay發現miR-181b會藉由結合到TIMP-3的3’-UTR而降低TIMP-3的表現量。另外我們在細胞增生以及凋亡實驗的功能性分析中發現了在化療藥物 (5-FU)的處理下，miR-181b對於癌細胞的增生過程似乎會透過調控TIMP-3而在其中扮演重要的角色。首先我們發現擁有比較高miR-181b 表現量的SW620細胞株，對化療藥物 (5-FU)有較高的抗性。更重要的是，我們也發現TIMP-3 會降低原本受miR-181b 誘導增加的細胞增生能力。因此我們推測大腸直腸癌細胞中，可能會透過miR-181b 去降低TIMP-3 的表現而造成細胞對化療藥物變得更具抗藥性。最後我們提出一個理論，假使大腸直腸癌細胞帶有較高表現量的miR-181b和較低表現量的TIMP-3，則此細胞可能偏向屬於抗藥性的細胞株；相反則是對化療藥物較有效。因此我們認為，在大腸直腸癌的預後上，miR-181b與TIMP-3 可以做為治療性的指標。
Colorectal cancer is the second leading cause of death among cancer in Taiwan. It has been estimated that colorectal cancer is responsible for 500,000 deaths worldwide. Progress in diagnosis and treatment for patients with advanced disease or systemic metastasis is still very poor. Several recent reports suggest that microRNAs involved in the development of colorectal cancer. In our studies, the recurrent and non-recurrent colorectal cancer tissues, sixteen and thirty pairs of tumor and normal samples respectively, were analyzed. Total RNAs were isolated and the microRNA and gene expression level was quantified by real time PCR analysis. We found that the expression level of miR-181b in tumor tissues was higher than in normal tissues. Besides, we identified a miR-181b candidate target gene TIMP-3, a tissue metalloproteinase inhibitor, via the TargetScan website. Interestingly, the mRNA levels of miR-181b and TIMP-3 had an opposite behavior in CRC tissues and cell lines. Therefore, we verified the TIMP-3 is a direct target of miR-181b by reporter assay, and miR-181b can reduce the luciferase activity of TIMP-3 through binding to the seed regions on TIMP3 3’-UTR. In addition, the functional role of miR-181b in CRC cell lines were verified by proliferation and apoptosis assays. These data suggest that miR-181b plays a critical role on the regulation of cell proliferation and apoptosis under 5-Fluorouracil (5-FU) treatment. First we found the SW620, with higher miR-181b expression level, was more resistant to 5-FU treatment. And the data suggested that TIMP-3 can induce cell apoptosis and miR-181b can induce proliferation. Most importantly, TIMP-3 could reduce the miR-181b-upregulated proliferation. So we conjectured that the colorectal cancer cell line could be more resistant to 5-FU treatment through TIMP-3 down regulation by miR-181b.Finally, we categorize a rule that the CRC cell lines expressed higher miR-181b level and lower TIMP3 level may be prone to be a drug-resistant cell line, just like the SW620. If the condition is opposite to the former, the cells may be prone to be sensitive to drug treatment. Besides, we think these two factors of miR-181b and TIMP-3 could be good prognostic indicators in the clinical.
Chinese abstract I
English abstract III
Figure index X
I. The overview of colorectal cancer 1
i. The survival rate of every stages of colorectal cancer 2
ii. The chemotherapeutics of colorectal cancer 2
iii. The mechanism of chemotherapeutic drug (5-FU)-induced cell death 3
II. Introduction of microRNAs 4
i. MicroRNAs and discovery 4
ii. MicroRNA biosynthesis and functions 4
iii. The functional roles of miRNAs in colorectal cancer 5
iv. The miR-181family 6
v. MicroRNA-181b in colorectal cancers 6
III. Tissue inhibitors of metalloproteinases-3 (TIMP-3) 7
i. The overview of Tissue inhibitor of metalloproteinase-3 (TIMP-3) 7
ii. Physical functions of TIMP-3 in colorectal cancer 8
IV. Research aim 9
Materials and Methods
I. Materials 11
II. Primers list 16
A. Cell culture and 5-FU treatment 17
B. Reporter plasmid constructs 17
C. Transient transfection 18
i. Plasmid transfection with PolyJetTM in vitro transfection reagent 18
ii. SiRNA transfection with GenMuteTM siRNA transfection reagent 18
iii. Plasmid transfection with TranIT○R-2020 Transfection Reagent 19
D. RNA extraction 19
E. Preparation protein cell lysates 20
F. Reverse transcription (RT) 20
G. Western blot assay 21
H. Quantitative real-time PCR (qRT-PCR) 21
I. Reporter assay 22
J. Proliferation assay 22
K. Flowcytometry assay 23
I. The expression levels of hsa-miR-181b in tumor tissues were higher than in normal tissues 24
II. The expression levels of miR-181b are correlating with different stages of colorectal cancer cell line 24
i. SW620 cells with higher expression level of miR-181b displays more resistant to 5-FU treatment than SW480 25
ii. The malignant cell lines, SW620, has higher miR-181b level than in SW480 cell lines 25
III. The expression of miR-181b promotes the cell proliferation 26
IV. TIMP-3 3’-UTR contains two possible miR-181b target regions 27
V. The mRNA expression levels of TIMP-3 and miR-181b were opposite in the CRC cell lines 27
i. The RNA expression level of TIMP-3 and miR-181b in CRC samples and cell lines 27
ii. The endogenous TIMP-3 mRNA level was reduced in miR-181b-overexpressed cell lines 28
VI. TIMP-3 RNA levels could be reduced by miR-181b through binding on the seed regions of TIMP-3 3’UTR 28
i. The reporter constructs illustration 28
ii. Reporter assay to confirm the direct binding of miR-181b on TIMP-3 29
VII. To compare the expression fold change between tumor and normal samples of TIMP-3 and miR-181b in human colorectal cancer tissues 29
VIII. TIMP-3 was down-regulated the miR-181b-overexpressed proliferation 30
IX. Apoptosis level of SW620 was induced after TIMP-3 induction and reduced by miR-181b in SW480 30
X. Hypothesis model of this study 31
I. The drug resistant phenomenom and colorectal cancer 32
II. The functional roles of miR-181b in human cancers 33
III. The correlation between tissue inhibitor of metalloproteinase-3 (TIMP-3) and 5-FU drug resistance 34
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