肝細胞癌是最常見的原發性肝臟惡性腫瘤，是全球癌症相關致亡率的主要原因。我們實驗室先前的研究中發現Nei endonuclease VIII-like 3 (NEIL3)在肝癌組織的表現高於非肝癌組織，且NEIL3過表達會促進腫瘤增殖、侵襲及遷移。NEIL3是一種DNA醣苷酶(DNA glycosylase)，參與鹼基切除修復(Base Excision Repair, BER)途徑中，辨認並去除受損的DNA鹼基，同時具有DNA醣苷酶以及AP裂解酶的活性。值得注意的是，NEIL3野生型和NEIL3酵素突變型都能增加遷移和侵襲的能力，說明NEIL3可能是透過非鹼基切除修復機制來促進肝癌的演進。首先，本研究第一個目標是想自己製造NEIL3抗體，因市面上的NEIL3抗體辨認其蛋白的特異性不夠好。我們利用鎳離子柱及陽離子交換層析法將兩段NEIL3特異性蛋白純化後，送至LEADGENE生物技術公司進行兔多株克隆抗體的製造。之後我們檢測了這兩支NEIL3抗體的敏感性和特異性，發現只有在NEIL3過表達時，NEIL3抗體才能辨識到其蛋白。另外，我們嘗試提取核蛋白來檢測NEIL3抗體，結果顯示NEIL3抗體似乎就可以檢測到內源性的NEIL3。接著，本研究的第二個目標是透過酵母菌雙雜交系統(yeast two-hybrid system, Y2H)來篩選會和NEIL3結合之蛋白質。當Bait蛋白與Prey蛋白愛酵母菌中有相互作用時，酵母菌則可以在篩選培養基(-TULH)中生長。我們將人類睪丸library cDNA送入含有neil3/pGBKT7的AH109酵母菌，來篩選和NEIL3有結合的蛋白質，接下來，我們利用DNA測序對這些蛋白進行鑑定，發現SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily B, member 1 (SMARCB1)可能和NEIL3有結合，SMARCB1是SWI/SNF三磷酸腺苷依賴染色質重塑複合體中的核心次單元，參與基因表達的調控，且具有腫瘤抑制因子的作用。此外，我們也以免疫共同沉澱法證實了在哺乳動物細胞中NEIL3和SMARCB1的結合關係，更進一步發現NEIL3的N端是和SMARCB1結合的區域。另外，我們想研究NEIL3與SMARCB1結合後之相關功能作用。我們過去的研究顯示neil3可能在肝癌中是屬於proto-oncogene (原癌基因)，因此我們推測在腫瘤中NEIL3可能會抑制SMARCB1的功能。根據結果顯示，在neil3過表達及neil3敲落的細胞中，smarcb1的mRNA表現與對照組相比並無差異。此外，我們也想了解NEIL3和SMARCB1的結合是跟整個SWI/SNF複合物結合還是只跟其中的亞複合物進行結合的，以及NEIL3是否會影響SWI/SNF複合物之間的結合。免疫共同沉澱的結果表明，NEIL3可能是和SMARCB1、SMARCA4等次單元進行結合，且不影響SWI/SNF複合物次單元之間的結合。然而NEIL3如何影響SMARCB1的功能機制，在未來會進行更進一步的探討。

Hepatocellular carcinoma (HCC) is the most common primary liver tumor and is the third-leading cause of cancer-related death in the world. Preliminary data in our lab revealed that Nei endonuclease VIII-like 3 (NEIL3) was highly expressed in human HCC tumorous compared with non-tumorous tissues and overexpression of NEIL3 promoted tumor proliferation, invasion and migration. NEIL3 is an essential base excision repair (BER) for oxidative DNA damage and contains DNA glycosylase and apurinic/apyrimidinic lyase activities. Notably, cell invasion and migration abilities were increased in the neil3 knockdown cell that overexpressed with wild-type neil3 and loss-of-function neil3 mutants, indicating that NEIL3 facilitated HCC progression through a BER-independent mechanism. In this study, we first aimed to generate in-house NEIL3 antibodies because the commercial NEIL3 antibodies were not specific enough for our experiments. We purified two partial NEIL3 proteins by nickel column and cation exchange chromatography to near homogeneity. These purified proteins were sent to the LEADGENE biotech company for production of rabbit polyclonal antibodies. Then we tested the sensitivities and specificities of these two NEIL3 antibodies and found that only when NEIL3 was overexpressed, the NEIL3 antibodies could recognize its protein. In addition, we extracted the nuclear proteins to test the NEIL3 antibodies. The results showed that the endogenous NEIL3 seemed to be detected by NEIL3 antibodies. The second aim was to screen proteins that are associated with NEIL3 by using the yeast two-hybrid (Y2H) screening assay. When the Y2H bait NEIL3 interacts with the prey protein in the yeast, the yeast can grow in the -TULH selective plate. We transformed the human testis Y2H library DNA into AH109 which contained neil3/pGBKT7 to screen proteins that interacted with NEIL3. Next, we identified these proteins by DNA sequencing and found SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily B, member 1 (SMARCB1) was a potential interactive protein with NEIL3. SMARCB1 is the core subunit of the SWI/SNF ATP-dependent chromatin remodeling complex, involved in regulating gene expression and acts as a tumor suppressor. Moreover, we also confirmed NEIL3-SMARCB1 interaction in mammalian cells by co-immunoprecipitation, and further found that the N-terminal domain of NEIL3 is the interaction region that directly binds with SMARCB1. Next, we wanted to investigate the effects of NEIL3-SMARCB1 interaction on the functions of these two proteins. Previous studies in our lab suggested that neil3 might be a proto-oncogene in HCC, so we hypothesized that NEIL3 might suppress the function of SMARCB1 in tumor. The results showed that the mRNA expression of smarcb1 in neil3 overexpression or knockdown cells had no difference with the control cells. Furthermore, we examined the co-immunoprecipitation of NEIL3 and SMARCB1 to detect the other subunits of SWI/SNF complex. The results indicated that NEIL3 might be associated with SMARCB1 and SMARCA4, and did not affect the binding between the subunits of the SWI/SNF complex. However, the further mechanism how NEIL3 affects the function of SMARCB1 will be investigated in the future.

Albelazi, M.S., P. R. Martin, S. Mohammed, L. Mutti, J. L. Parsons and R. H. Elder (2019). "The Biochemical Role of the Human NEIL1 and NEIL3 DNA Glycosylases on Model DNA Replication Forks." Genes (Basel) 10(4).
Ansari, D. and R. Andersson (2012). "Radiofrequency ablation or percutaneous ethanol injection for the treatment of liver tumors." World J Gastroenterol 18(10): 1003-1008.
Anzola, M. (2004). "Hepatocellular carcinoma: role of hepatitis B and hepatitis C viruses proteins in hepatocarcinogenesis." J Viral Hepat 11(5): 383-393.
Balogh, J., D. Victor, 3rd, E. H. Asham, S. G. Burroughs, M. Boktour, A. Saharia, X. Li, R. M. Ghobrial and H. P. Monsour, Jr. (2016). "Hepatocellular carcinoma: a review." J Hepatocell Carcinoma 3: 41-53.
Broustas, C. G. and H. B. Lieberman (2014). "DNA damage response genes and the development of cancer metastasis." Radiat Res 181(2): 111-130.
Brown, K. S. (2006). "Chemotherapy and other systemic therapies for hepatocellular carcinoma and liver metastases."Semin Intervent Radiol 23(1): 99-108.
Bruix, J. and M.Sherman (2011). "Management of hepatocellular carcinoma: an update." Hepatology 53(3): 1020-1022.
Carr, B. I., V. Kondragunta, S. C. Buch and R. A. Branch (2010). "Therapeutic equivalence in survival for hepatic arterial chemoembolization and yttrium 90 microsphere treatments in unresectable hepatocellular carcinoma: a two-cohort study." Cancer 116(5): 1305-1314.
Choo, Q. L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C. Dong, C. Gallegos, D. Coit, R. Medina-Selby, P. J. Barr and et al. (1991). "Genetic organization and diversity of the hepatitis C virus." Proc Natl Acad Sci U S A 88(6): 2451-2455.
Ciccia, A. and S. J. Elledge (2010). "The DNA damage response: making it safe to play with knives." Mol Cell 40(2): 179-204.
Dellian, M., B. P. Witwer, H. A. Salehi, F. Yuan and R. K. Jain (1996). "Quantitation and physiological characterization of angiogenic vessels in mice: effect of basic fibroblast growth factor, vascular endothelial growth factor/vascular permeability factor, and host microenviroment." Am J Pathol 149(1): 59-71.
Di Bisceglie, A. M. (2009). "Hepatitis B and hepatocellular carcinoma." Hepatology 49(5 Suppl): S56-60.
El-Serag, H. (2008). "The association between obesity and GERD: a review of the epidemiological evidence." Dig Dis Sci 53(9): 2307-2312.
El-Serag, H. B. and K. L. Rudolph (2007). "Hepatocellular carcinoma: epidemiology and molecular carcinogenesis." Gastroenterology 132(7): 2557-2576.
Feitelson, M. A. (1999). "Hepatitis B virus in hepatocarcinogenesis." Journal of Cellular Physiology 181(2): 188-202.
Ferlay, J., H. R. Shin, F. Bray, D. Forman, C. Mathers and D. M. Parkin (2010). "Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008." Int J Cancer 127(12): 2893-2917.
Forner, A., J. M. Llovet and J. Bruix (2012). "Hepatocellular carcinoma." Lancet 379(9822): 1245-1255.
Fortini, P., C. Ferretti and E. Dogliotti (2013). "The response to DNA damage during differentiation: pathways and consequences."Mutat Res 743-744:160-168.
Franken, N. A., H. M. Rodermond, J. Stap, J. Haveman and C. van Bree (2006). "Clonogenic assay of cells in vitro." Nat Protoc 1(5): 2315-2319.
Giglia-Mari, G., A. Zotter and W. Vermeulen (2011). "DNA damage response."Cold Spring Harb Perspect Biol 3(1): a000745.
Gupta, R. A., N. Shah, K. C. Wang, J. Kim, H. M. Horlings, D. J. Wong, M. C. Tsai, T. Hung, P. Argani, J. L. Rinn, Y. Wang, P. Brzoska, B. Kong, R. Li, R. B. West, M. J. van de Vijver, S. Sukumar and H. Y. Chang (2010). "Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis."Nature 464(7291): 1071-1076.
Helleday, T., E. Petermann, C. Lundin, B. Hodgson and R. A. Sharma (2008). "DNA repair pathways as targets for cancer therapy." Nat Rev Cancer 8(3): 193-204.
Hildrestrand, G. A., C. G. Neurauter, D. B. Diep, C. G. Castellanos, S. Krauss, M. Bjoras and L. Luna (2009). "Expression patterns of Neil3 during embryonic brain development and neoplasia." BMC Neurosci 10:45.
Hoeijmakers, J. H. (2001). "Genome maintenance mechanisms for preventing cancer." Nature 411(6835): 366-374.
Jemal, A., F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman (2011). "Global cancer statistics." CA Cancer J Clin 61(2): 69-90.
Kim, Y. J. and D. M. Wilson, 3rd (2012). "Overview of base excision repair biochemistry." Curr Mol Pharmacol 5(1): 3-13.
Kohashi, K. and Y. Oda (2017). "Oncogenic roles of SMARCB1/INI1 and its deficient tumors." Cancer Sci 108(4): 547–552.
Krokeide, S. Z., J. K. Laerdahl, M. Salah, L. Luna, F. H. Cederkvist, A. M. Fleming, C. J. Burrows, B. Dalhus and M. Bjoras (2013). "Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroimindiohydantoin and guanidinohydantoin." DNA repair (Amst) 12(12): 1159-1164.
Lindahl, T. (1993). "Instability and decay of the primary structure of DNA." Nature 362(6422): 709-715.
Liou, G. Y. and Storz (2010). " Reactive oxygen species in cancer." Free Radic Res 44(5): 479-496.
Liu, M., S. Doublie and S. S. Wallace (2013). "Neil3, the final frontier for the DNA glycosylases that recognize oxidative damage." Mutat Res 743-744: 4-11.
Liu, M., K. Imamura, A. M. Averill, S.S. Wallace and S. Doublie (2013). "Structural characterization of a mouse ortholog of human NEIL3 with a marked preference for single-stranded DNA." Structure 21(2): 247-256.
Liu, Y. and F. Wu (2010). "Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment." Environ Health Perspect 118(6): 818-824.
Llovet, J. M., A. Burroughs and J. Bruix (2003). "Hepatocellular carcinoma." Lancet 362(9399): 1907-1917.
Marrero, J. A. (2013). "Multidisciplinary management of hepatocellular carcinoma: where are we today ? " Semin Liver Dis 33 Suppl: S3-10.
Mashtalir, N., A. R. D’Avino, B. C. Michel, J. Luo, J. Pan, J. E. Otto, H. J. Zullow, Z. M. McKenzie1, R. L. Kubiak, R. St. Pierre, A. M. Valencia, S. J. Poynter, S. H.Cassel, J. A. Ranish and C. Kadoch (2018). "Modular Organization and Assemblyof SWI/SNF Family Chromatin Remodeling Complexes." Cell 175(5): 1272–1288.
Massaad, M. J., J. Zhou, D. Tsuchimoto, J. Chou, H. Jabara, E. Janssen, S. Glauzy, B. G. Olson, H. Morbach, T. K. Ohsumi, K. Schmitz, M. Kyriacos, J. Kane, K. Torisu, Y. Nakabeppu, L. D. Notarangelo, E. Chouery, A. Megarbane, P. B. Kang, E. Al-Idrissi, H. Aldhekri, E. Meffre, M. Mizui, G. C. Tsokos, J. P. Manis, W. Al-Herz, S. S. Wallace and R. S. Geha (2016). "Deficiency of base excision repair enzyme NEIL3 drives increased predisposition to autoimmunity." J Clin Invest 126(11): 4219-4536.
McLean, M. and M. F. Dutton (1995). "Cellular interactions and metabolism of aflatoxin: an update." Pharmacol Ther 65(2): 163-192.
Mena, S., A. Ortega and J. M. Estrela (2009). "Oxidative stress in environmental-induced carcinogenesis." Mutat Res 674(1-2): 36-44.
Mruk, D. D. and C. Y. Cheng (2011). "Enhanced chemiluminescence (ECL) for routine immunoblotting: An inexpensive alternative to commercially available kits." Spermatogenesis 1(2): 121-122.
Neurauter, C. G., L. Luna and M. Bjoras (2012). "Release from quiescence stimulates the expression of human NEIL3 under the control of the Ras dependent ERK-MAP kinase pathway." DNA Repair (Amst) 11(4): 401-409.
Orman, E. S., G. Odena and R. Bataller (2013). "Alcoholic liver disease: pathogenesis, management, and novel targets for therapy." J Gastroenterol Hepatol 28 Suppl 1: 77-84.
Park, E. M., M. K. Shigenaga, P. Degan, T. S. Korn, J. W. Kitzler, C. M. Wehr, P. Kolachana and B. N. Ames (1992). "Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column." Proc Natl Acad Sci U S A 89(8): 3375-3379.
Poli, A. and F. Marangoni (2014). "Response to the letter by Testino et al. on moderate alcohol use and health." Nutr Metab Cardiovasc Dis 24(3): e13-14.
Pollicino, T., I. Cacciola, F. Saffioti and G. Raimondo (2014). "Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications." J Hepatol 61(2): 408-417.
Prakash, A., S. Doublie and S. S. Wallace (2012). "The Fpg/Nei family of DNA glycosylases: substrates, structures and search for damage." Prog Mol Biol Transl Sci 110: 71-91.
Puoti, M., S. Babudieri, G. Rezza, P. Viale, M. G. Antonini, I. Maida, S. Rossi, B. Zanini, V. Putzolu, L. Fenu, C. Baiguera, S. Sassu, G. Carosi and M. S. Mura (2004). "Use of pegylated interferons is associated with an increased incidence of infections during combination treatment of chronic hepatitis C: a side effect of pegylation ? " Antivir Ther 9(4): 627-630.
Reis, A. and O. Hermanson (2012)." The DNA glycosylases OGG1 and NEIL3 influence differentiation potential, proliferation, and senescence-associated signs in neural stem cells." Biochem Biophys Res Commun 423(4): 621-626.
Reuter, S., S. C. Gupta, M. M. Chaturvedi and B. B. Aggarwal (2010). "Oxidative stress, inflammation, and cancer: how are they linked ? " Free Radic Biol Med 49(11): 1603-1616.
Ryder, S. D. (2003). "Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. " Gut 52 Suppl 3:iii1-8.
Sanyal, A. J., N. Chalasani, K. V. Kowdley, A. McCullough, A. M. Diehl, N. M. Bass, B. A. Neuschwander-Tetri, J. E. Lavine, J. Tonascia, A. Unalp, M. Van Natta, J. Clark, E. M. Brunt, D. E. Kleiner, J. H. Hoofnagle and P. R. Robuck (2010). "Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. " N Engl J Med 362(18): 1675-1685.
Schuster, M. A., E. A. McGlynn and R. H. Brook (2005). "How good is the quality of health care in the United States ? 1998. " Milbank Q 83(4): 843-895.
Shaaban, S., A. Negm, E. E. Ibrahim and A. A. Elrazak (2014). "Chemotherapeutic agents for the treatment of hepatocellular carcinoma: efficacy and mode of action." Oncol Rev 8(1): 246.
Sobczak, M., J. Pietrzak, T. Płoszaj and A. Robaszkiewicz (2020). "BRG1 Activates Proliferation and Transcription of Cell Cycle-Dependent Genes in Breast Cancer Cells." Cancers 12(2): 349.
Sontag, E.M., G. P. Lotz, G. Yang, C. J. Sontag, B. J. Cummings, C. G. Glabe, P. J. Muchowski and L. M. Thompson (2012). "Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules." J Huntingtons Dis 1(1): 119-132.
Takao, M., Y. Oohata, K. Kitadokoro, K. Kobayashi, S. Iwai, A. Yasui, S. Yonei and Q. M. Zhang (2009). " Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant. " Genes Cells 14(2): 261-270.
Thannickal, V. J. and B. L. Fanburg (2000). "Reactive oxygen species in cell signaling." Am J Physiol Lung Cell Mol Physiol 279(6): L1005-1028.
Tinkle, C. L. and Haas-Kogan (2012). "Hepatocellular carcinoma: natural history, current management, and emerging tools." Biologics 6: 207-219.
Torgovnick, A. and B. Schumacher (2015). "DNA repair mechanisms in caner development and therapy. " Front Genet 6: 157.
Tsikitis, M., Z. Zhang, W. Edelman, D. Zagzag, and G. V. Kalpana (2005). "Genetic ablation of Cyclin D1 abrogates genesis of rhabdoid tumors resulting from Ini1 loss." Proc Natl Acad Sci U S A 102(34): 12129–12134.
Versteege, I., S. Medjkane, D. Rouillard, and O. Delattre (2002). "A key role of the hSNF5/INI1 tumour suppressor in the control of the G1-S transition of the cell cycle." Oncogene 21: 6403-6412.
Wallace, J. L. (2013). "Mechanisms, prevention and clinical implications of nonsteroidal anti-inflammatory drug-enteropathy. " World J Gastroenterol 19(12): 1861-1876.
Wang, D., D. B. Xiang, X. Q. Yang, L.S. Chen, M. X. Li, Z. Y. Zhong and Y. S. Zhang (2009). "APE1 overexpression is associated with cisplatin resistance in non-small cell lung cancer and targeted inhibition of APE1 enhances the activity of cisplatin in A549 cells." Lung Cancer 66(3): 298-304.
Wang, X., R. S. Lee, B. H. Alver, J. R. Haswell, S. Wang, J. Mieczkowski, Y. Drier, S. M. Gillespie, T. C. Archer, J. N. Wu, E. P. Tzvetkov, E. C. Troisi, S. L. Pomeroy, J. A. Biegel, M. Y. Tolstorukov, B. E. Bernstein, P. J. Park and C. W. M Roberts (2017). "SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation." Nat Genet 49(2): 289-295.
Wilson, D. M., 3rd and M. M. Seidman (2010). "A novel link to base excision repair ? " Trends Biochem Sci 35(5): 247-252.
Zhang, H., H. Ma, Q. Wang, M. Chen, D. Weng, H. Wang, J. Zhou, Y. Li, J. Sun, Y. Chen, X. Liang, J. Zhao, K. Pan, H. Wang and J. Xia (2010). "Analysis of loss of heterozygosity on chromosome 4q in hepatocellular carcinoma using high-throughput SNP array." Oncol Rep 23(2): 445-455.
Zhang, Z. K., K. P. Davies, J. Allen, L. Zhu, R. G. Pestell, D. Zagzag, and G. V. Kalpana1 (2002). "Cell Cycle Arrest and Repression of Cyclin D1 Transcription by INI1/hSNF5." Mol Cell Biol 22(16): 5975–5988.