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研究生： 鄭亦宸
CHENG, YI-CHEN
論文名稱： IGF2BP3-LSS傳遞途徑調控膽固醇合成促進肝癌細胞生長的重要角色
Emerging role of IGF2BP3-LSS axis in cholesterol synthesis facilitates progression of Hepatocellular carcinoma
指導教授： 顏家瑞
Ten, Chia-Jui
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學暨分子生物學研究所
Department of Biochemistry and Molecular Biology
論文出版年： 2023
畢業學年度： 111
語文別： 英文
論文頁數： 70
中文關鍵詞： 肝細胞癌 、癌胚蛋白 、胰島素生長因子2結合蛋白3 、甲基轉移酶3 、羊毛甾醇合酶 、羊毛甾醇 、ELAV樣RNA結合蛋白1 、m6A修飾
外文關鍵詞： HCC, oncofetal protein, IGF2BP3, METTL3, LSS, lanosterol, ELAVL1, m6A modification
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    • 胰島素生長因子2結合蛋白3(IGF2BP3/IMP3)，是隸屬於IGF2BPs(IMPs)家族內的成員之一，作為RNA結合蛋白(RBP)存在，對mRNA進行轉錄後修飾來左右mRNA的後續反應，以此來在胎兒發育時發揮作用，且在一般成年人體內難以偵測到。而在近年來發現，其會作為癌胚蛋白在多種癌症中高度表達。在過往的研究中發現，IGF2BP3 在肝癌患者中占多數的肝細胞癌(HCC)中大量表現且與其生長情形相關聯，並被發現其與患者的總體生存率呈現負相關。為了研究透過膽固醇生合成造成HCC致病機制的IGF2BP3所調控的目標基因，我們透過整合分析甲基轉移酶3(METTL3) GSE資料和膽固醇生合成相關的蛋白，確認羊毛甾醇合酶(LSS)為IGF2BP3新的作用目標，透過抑制降低HCC細胞系中IGF2BP3的基因表達，觀察到LSS的mRNA與蛋白表現因此降低，此外，確認了抑制LSS的表達量會使HCC生長受到抑制。LSS是膽固醇生合成途徑的一份子，會生成反應中的第一個固醇類—羊毛甾醇，而此產物最終轉換成膽固醇，因此預期，抑制LSS會降低HCC細胞中的膽固醇含量，而在檢測中也呈現預期的趨勢。分子機制上，在METTL3基因敲落的HCC細胞中的LSS表達量顯著的降低；已知與IGF2BP3結合的穩定蛋白而為人熟知的ELAV樣RNA結合蛋白1 (ELAVL1)則明顯的促進了LSS的表現。臨床上，透過生物資訊資料庫得知，在肝癌患者身上可見METTL3-IGF2BP3-ELAVL1-LSS傳遞途徑。綜上所述，此研究發現了HCC中新的潛在調控機制—IGF2BP3結合ELAVL1而協同調控膽固醇生合成相關且具N6-甲基腺嘌呤(m6A) 修飾之LSS mRNA的表現促進HCC腫瘤生長，進而提供針對HCC治療的新穎治療標的。

    Insulin growth factor 2 binding protein 3 (IGF2BP3), one of IGF2BPs family member, acts as RNA binding protein (RBP) to stabilize mRNA by post-transcription modification. Normal expression of IGF2BP3 takes part in embryo development, but do not detect in adult tissue. Noticeably, IGF2BP3 belongs to an oncofetal protein, highly expressing in various types of cancer. Our previous study showed that IGF2BP3 promoted hepatocellular carcinoma (HCC) cell growth, was highly expressed in patients with HCC, and had bad overall survival rate. To discover the IGF2BP3-regulated target genes involving in the pathogenesis along cholesterol biosynthesis of HCC, we conduct and integrate the analysis of methyltransferase 3 (METTL3) Gene expression omnibus (GEO) database and protein involve in cholesterol biosynthesis to identify novel IGF2BP3’s target, lanosterol synthase (LSS). We confirm LSS downregulation in mRNA and protein level upon IGF2BP3 knockdown in HCC cells. Moreover, we found that the depletion of LSS markedly decreased HCC tumor growth. LSS takes part in cholesterol biosynthesis pathway in the synthesis of lanosterol, the first member of sterol produced and convert into cholesterol finally. As expected, knockdown of LSS reduced the content of cholesterol in HCC cells. Mechanistically, LSS expression is markedly decreased in METTL3-depleted HCC cells. Noticeably, ELAV like RNA binding protein 1 (ELAVL1), a well-known IGF2BP3-asscoiated RNA stabilizer, promotes LSS expression. Regarding clinical significance, METTL3-IGF2BP3-ELAVL1-LSS axes exist in liver cancer patients analyzed by online database. Taken together, these findings uncover a novel underlying mechanism that IGF2BP3 cooperates with ELAVL1 to facilitate HCC tumor growth by upregulation of cholesterol synthesis-related N6-methyladenine (m6A)-modified LSS mRNA expression, and further provide potential therapeutic targets for treatment of HCC.

    中文摘要 i Abstract iii Acknowledgement v Abbreviation ix Content xi Introduction 1 Oncogenic role of Insulin-like growth factor 2 mRNA binding protein 3(IGF2BP3) in Hepatocellular carcinoma (HCC). 1 Cholesterol metabolism in HCC. 3 The role of Lanosterol synthase (LSS) in HCC. 6 Specific aim 8 Significance 9 Materials and Methods 10 Cell culture 10 Plasmid purification 10 RNA preparation 11 Reverse Transcription (RT-PCR) 12 Real-time polymerase chain reaction (qPCR) 12 Lentivirus production 13 Lentivirus titration 13 Lentivirus infection 14 Protein extraction 14 BCA protein assay 14 Western blot 15 Viability assay 15 Clonogenic assay 16 Cholesterol/Cholesterol ester assay 16 Statistical analysis 16 Results 18 LSS is identified as a novel target of METTL3 in HCC. 18 METTL3, IGF2BP3, and LSS are associated with poor prognosis in HCC. 20 LSS knockdown impairs cell proliferation, colony formation, and cholesterol biosynthesis in HCC cells. 20 METTL3 facilitates LSS expression, tumor growth, and cholesterol biosynthesis in HCC cells. 21 IGF2BP3 upregulates expression of LSS, promotes tumor growth, and increases cholesterol biosynthesis in HCC cells. 21 IGF2BP3-associated RNA stabilizer ELAVL1 positively regulates LSS level in HCC cell. 22 METTL3- LSS, IGF2BP3- LSS, and ELAVL1-LSS axis exist in HCC patients. 22 Conclusion and Discussion 24 References 27 Figures 36 Figure 1. Identification of novel IGF2BP3’s mRNA targets regulated by METTL3 involving in HCC cholesterol biosynthesis pathway using GEO database. 36 Figure 2. Clinical significance of IGF2BP3’s mRNA targets in HCC cholesterol biosynthesis pathway. 37 Figure 3. Clinical value of METTL3, IGF2BP3, and LSS expression is determined 38 Figure 4. Clinical correlation of METTL3, IGF2BP3, and LSS expression is determined 39 Figure 5. Clinical correlation of METTL3, IGF2BP3, and LSS expression is determined 40 Figure 6. Overall survival curve of METTL3, IGF2BP3, and LSS in HCC patients. 41 Figure 7. Potential m6A sites on LSS mRNA are predicted by SRAMP. 42 Figure 8. LSS is efficiently knocked down in HCC cell lines using lentivirus-mediated transduction of shRNA. 43 Figure 9. LSS knockdown impairs colony formation in HCC cell lines. 44 Figure 10. LSS knockdown reduces cholesterol biosynthesis in HCC cell lines. 45 Figure 11. Depletion of METTL3 results in a decreased expression of LSS protein in HCC cell lines. 46 Figure 12. Depletion of METTL3 leads to a decreased level of LSS mRNA in HCC cell lines. 47 Figure 13. METTL3 promotes colony formation in HCC cell lines. 48 Figure 14. METTL3 positively regulates cholesterol biosynthesis in HCC cell lines. 49 Figure 15. Depletion of IGF2BP3 downregulates LSS protein expression in HCC cell lines. 50 Figure 16. Depletion of IGF2BP3 causes a decreased expression of LSS mRNA in HCC cell lines. 51 Figure 17. IGF2BP3 facilitates the cell growth in HCC cell lines. 52 Figure 18. IGF2BP3 promotes cholesterol biosynthesis in HCC cell lines. 53 Figure 19. IGF2BP3-associated ELAVL1 mRNA stabilizer promotes LSS expression and have higher correlation with LSS compared with PABPC1. 55 Figure 20. ELAVL1 promotes LSS expression in HCC cell lines. 57 Figure 21. ELAVL1 promotes colony formation in HCC cell lines. 58 Figure 22. ELAVL1 enhances cholesterol biosynthesis in HCC cell lines. 59 Figure 23. Clinical value of ELAVL1 in HCC. 61 Figure 24. Correlation analysis between either METTL3, IGF2BP3, or ELAVL1 and LSS in HCC. 63 Figure 25. Survival curve analysis of METTL3-LSS, IGF2BP3-LSS, and ELAVL1-LSS axis in HCC using TCGA public database. 64 Tables 65 Table. 1. List of IGF2BP3’s mRNA targets in HCC cholesterol biosynthesis pathway associated METTL3 using GSE102620. 65 Table 2. List of antibodies. 66 Table 3. List of lentiviral vector-based shRNAs. 67 Table 4. List of TaqMan assay. 68 Appendix 69 Appendix Figure 1. Incident rates and mortality rates of liver cancer. 70

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