ＡＨＣＹ ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ???
專(zhuān)利匯可以提供ＡＨＣＹ ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ???專(zhuān)利檢索，專(zhuān)利查詢(xún)，專(zhuān)利分析的服務(wù)。并且S-????????????????? (adenosylhomocysteine hydrolase; AHCY)? S-???????????????? L-??????????????, ?, ????? ???????????????? ?????. ???????????? AHCY?????????????. ?????????? AHCY???? XTT ??, ??????? ?????? (transwell assay)??????????????????. ???? AHCY? ERK ?????????????????????????????????????????. ????? AHCY????? ???????? AHCY ????????????????????????, ?????? AHCY ?????????????????. ????, ?????? AHCY? ERK ???????????????????????????????.，下面是ＡＨＣＹ ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ???專(zhuān)利的具體信息內(nèi)容。
?? AHCY ?? ???? AHCY ???? mRNA? ????? ???? ???? ????????? ???? ?? ??? ?? ? ?? ??? ???? ???.
?? AHCY ?? ???? shRNA (short hairpin RNA)?? ???? ?? ??? ?? ? ?? ??? ???? ???.
?? AHCY ?? ???? siRNA (short interfering RNA)?? ???? ?? ??? ?? ? ?? ??? ???? ???.
?? AHCY ?? ???? AHCY ???? ???? ? AHCY ???? ???? ?? ??? ?? ? ?? ??? ???? ???.
ＡＨＣＹ ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ??? {Pharmaceutical composition for inhibiting proliferation and metastasis of breast cancer containing AHCY expression or activity inhibitor}
? ??? S-?????????? ??????? (S-adenosylhomocysteine hydrolase; AHCY) ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ???? ?? ???.
S-?????????? ??????? (S-adenosylhomocysteine hydrolase; AHCY)? S-???? ????? (S-Adenosyl methionine; SAM) ????? ???? ????. ? ???? S-???? ?????? ??, ?? ? ?? ???? ???. AHCY? S-?????????? (SAH)? ????? S-???? ????? ??? ??? ???????? ????. ???, AHCY? ???????? (transmethylation)? ???? ???? 3 . ??? ??? ???? ??? ??? ???? ???? ??? ?? ??? ???? ?????? 4 -6 . ?? ?? AHCY? ??? ? ??? ?? ?? (transmethylation reactions)? ???? ??? ???? ?? ??? ? ? ?? ???? ??? ?? ? ?? 7 ,8 . ?? ???, AHCY ???? DZNep? ?? AHCY? ????? ? ??? ?????? ????? ???? 9 . ???? ??? AHCY? ??? ????? ?? ??? ????? ? ????? ???? ???? ???.
???? ??? ????? ?? ???? ???? ? ? ???? 10 , ??? ???? ?? ? ??? ??? ?? ? ???? 11 . ?? ?? ? ???? ??? ???? ?? ????? ?? ? ??. ?? ??? ?? ?? ????? (benign epithelial atypia)?? ??? ?? ??? (atypical ductal hyperplasia; ADH) ?? ??????? (lobular carcinoma in situ; LCIS)??? ??? ? ??. DCIS (ductal carcinoma in situ)? ????? ??? ?? ???? ???? ?? IDC (invasive ductal carcinoma)? ? ? ?? 1 2 ,13 . ??? ???? ?? ??? ?? ? ?? ??, ?? ????? ?? ??? ?? ??? ??? ?? Ⅰ??? Ⅳ???? ????. ??? ?? ??? ???? ? ??? ?? ???? ?? ??? ??? ??? ?? 14 . ??? ? ??? ?? ??? ????.
MAPK (Mitogen-activated protein kinase) ??? MAP3K, MEK ? ERK? ???? ?? 15 ,16 . ERK1/2? ??? ??? ?????? ??, ??, ?? ?? ? ?? ??? ???? 17 -19 . MEK-ERK ???? ?? ??? ???? ?????? ????? BRaf (MAP3K)? ? 31%? ? ??? ?? ?? ??? (basal triple negative breast cancer)?? ?????, BRaf ????? ???? ????? ?? ???? ??? 20 ,21 . BRaf-MEK-ERK ??? ?? ? ??? ????? ???? 21 ,22 . ????? ???? ????? ??? (ER)-?? ????? MEK/ERK ????? ???? ??? ??? 23 . ERK1/2? ???? ???? ??? ???? D1 ????? ??? ?????? ?? ???? ? ??? ?? (progenitor cell)? ??-?? (self-renewal)? ???? 24 . ERK1/2? ?? ??? ?? ? ??? ?? ?? ???? ??? ???? 25 . ??? ???? ERK1/2? ?? ???? ??? ??? ??? ??? ??? ??? ??? ?? 26 .
????? ?? ??? ?? ?? ?? ???? ??? ?? 3-4? ??? ???? ???? ????, ?????, ?????? ? ?? ???? ?? ???? ?? ???? 27 . ? ????? ?? ?? (duct extension)? ??? ?? ?? ?? (stromal fat pad)? ???? ??? ??? (terminal end bud; TEB) ??? ???? 2 8 . ??? ?? ??? ?? ??, ?? ? ?? (involution)? ??? ??? ???. ?? ??? ?? (stroma) ?? ????? ???, ?? ?? ? ??? (side branching)? ???? 29 . ???? ?? ??? ?? ??? ? ???? ??? ?? (branched ductal tree)? ?????? ????? ???? 30 . ??? ??? ????? ??? ??? ??, ?????? ? ??? ??? ??? ? ??, ?????? ??? ??? ??? ?? ? ?? 30 .
Bartel, RL & Borchardt, RT Effects of adenosine dialdehyde on S-adenosylhomocysteine hydrolase and S-adenosylmethionine-dependent transmethylations in mouse L929 cells. Molecular pharmacology 25, 418-424 (1984). Paik, WK, Paik, DC & Kim, S. Historical review: the field of protein methylation. Trends in biochemical sciences 32, 146-152, doi:10.1016/j.tibs.2007.01.006 (2007). Gibson, KD, Wilson, JD & Udenfriend, S. The enzymatic conversion of phospholipid ethanolamine to phospholipid choline in rat liver. The Journal of biological chemistry 236, 673-679 (1961). Yideng, J. et al. Homocysteine-mediated expression of SAHH, DNMTs, MBD2, and DNA hypomethylation potential pathogenic mechanism in VSMCs. DNA and cell biology 26, 603-611, doi:10.1089/dna.2007.0584 (2007). Shu, S. et al. S-adenosylhomocysteine hydrolase is localized at the front of chemotaxing cells, suggesting a role for transmethylation during migration. Proceedings of the National Academy of Sciences of the United States of America 103, 19788-19793, doi:10.1073/pnas.0609385103 (2006). Radomski, N. et al. Interaction of S-adenosylhomocysteine hydrolase of Xenopus laevis with mRNA(guanine-7-)methyltransferase: implication on its nuclear compartmentalisation and on cap methylation of hnRNA. Biochimica et biophysica acta 1590, 93-102 (2002). Villanueva, JA & Halsted, CH Hepatic transmethylation reactions in micropigs with alcoholic liver disease. Hepatology 39, 1303-1310, doi:10.1002/hep.20168 (2004). Song, Z. et al. Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: possible involvement of mitochondrial S-adenosylmethionine transport. Biochemical pharmacology 74, 521-531, doi:10.1016/j.bcp.2007.05.005 (2007). Hayden, A., Johnson, PW, Packham, G. & Crabb, SJ S-adenosylhomocysteine hydrolase inhibition by 3-deazaneplanocin A analogues induces anti-cancer effects in breast cancer cell lines and synergy with both histone deacetylase and HER2 inhibition. Breast cancer research and treatment 127, 109-119, doi:10.1007/s10549-010-0982-0 (2011). Redig, AJ & McAllister, SS Breast cancer as a systemic disease: a view of metastasis. Journal of internal medicine 274, 113-126, doi:10.1111/joim.12084 (2013). Muthuswami, M. et al. Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition. PloS one 8, e77553, doi:10.1371/journal.pone.0077553 (2013). Claus, EB, Stowe, M. & Carter, D. Breast carcinoma in situ: risk factors and screening patterns. Journal of the National Cancer Institute 93, 1811-1817 (2001). Bombonati, A. & Sgroi, DC The molecular pathology of breast cancer progression. The Journal of pathology 223, 307-317, doi:10.1002/path.2808 (2011). Chatterjee, SJ & McCaffrey, L. Emerging role of cell polarity proteins in breast cancer progression and metastasis. Breast Cancer (Dove Med Press) 6, 15-27, doi:10.2147/BCTT.S43764 (2014). Dhanasekaran, DN & Johnson, GL MAPKs: function, regulation, role in cancer and therapeutic targeting. Oncogene 26, 3097-3099, doi:10.1038/sj.onc.1210395 (2007). Lawrence, MC et al. The roles of MAPKs in disease. Cell research 18, 436-442, doi:10.1038/cr.2008.37 (2008). Roskoski, R., Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacological research : the official journal of the Italian Pharmacological Society 66, 105-143, doi:10.1016/j.phrs.2012.04.005 (2012). Yoon, S. & Seger, R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 24, 21-44, doi:10.1080/02699050500284218 (2006). Carlson, SM et al. Large-scale discovery of ERK2 substrates identifies ERK-mediated transcriptional regulation by ETV3. Science signaling 4, rs11, doi:10.1126/scisignal.2002010 (2011). Weinstein, JN et al. The Cancer Genome Atlas Pan-Cancer analysis project. Nature genetics 45, 1113-1120, doi:10.1038/ng.2764 (2013). Comprehensive molecular portraits of human breast tumours. Nature 490, 61-70, doi:10.1038/nature11412 (2012). Duncan, JS et al. Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell 149, 307-321, doi:10.1016/j.cell.2012.02.053 (2012). McGlynn, LM et al. Ras/Raf-1/MAPK pathway mediates response to tamoxifen but not chemotherapy in breast cancer patients. Clinical cancer research : an official journal of the American Association for Cancer Research 15, 1487-1495, doi:10.1158/1078-0432.CCR-07-4967 (2009). Jeselsohn, R. et al. Cyclin D1 kinase activity is required for the self-renewal of mammary stem and progenitor cells that are targets of MMTV-ErbB2 tumorigenesis. Cancer cell 17, 65-76, doi:10.1016/j.ccr.2009.11.024 (2010). Whyte, J., Bergin, O., Bianchi, A., McNally, S. & Martin, F. Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development. Breast cancer research : BCR 11, 209, doi:10.1186/bcr2361 (2009). Mueller, H. et al. Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients. International journal of cancer. Journal international du cancer 89, 384-388 (2000). Humphreys, RC et al. Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development 122, 4013-4022 (1996). Wiseman, BS & Werb, Z. Stromal effects on mammary gland development and breast cancer. Science 296, 1046-1049, doi:10.1126/science.1067431 (2002). Hynes, NE & Watson, CJ Mammary gland growth factors: roles in normal development and in cancer. Cold Spring Harbor perspectives in biology 2, a003186, doi:10.1101/cshperspect.a003186 (2010). Villegas, E. et al. Plk2 regulates mitotic spindle orientation and mammary gland development. Development 141, 1562-1571, doi:10.1242/dev.108258 (2014). Kim, JH et al. Adenosine dialdehyde suppresses MMP-9-mediated invasion of cancer cells by blocking the Ras/Raf-1/ERK/AP-1 signaling pathway. Biochemical pharmacology 86, 1285-1300, doi:10.1016/j.bcp.2013.08.022 (2013). Tan, J. et al. Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development. Oncogene, doi:10.1038/onc.2013.434 (2013). Parsa, S. et al. Terminal end bud maintenance in mammary gland is dependent upon FGFR2b signaling. Developmental biology 317, 121-131, doi:10.1016/j.ydbio.2008.02.014 (2008).
? ??? ? ? ???? ??? ?? ? ?? ??? ???? ???? ????? ?? ???? ??.
? ??? S-?????????? ??????? (adenosylhomocysteine hydrolase; AHCY) ?? ?? ?? ???? ???? ??? ?? ? ?? ??? ???? ???? ?? ???.
??, ? ??? ?? AHCY ?? ???? AHCY ???? mRNA? ????? ???? ???? ???????, shRNA (short hairpin RNA) ? siRNA (short interfering RNA)? ??? ? ? ??? ?? ??? ?? ???? ?? ??? ?? ? ?? ??? ???? ???? ?? ???.
? ??? ??? ?????? ??? ??? ?? ??? AHCY? ???? ?? siRNA? ???? ?? ????.
??, ? ??? ?? AHCY ?? ???? AHCY ???? ???? ? AHCY ???? ???? ?? ??? ?? ? ?? ??? ???? ???? ?? ???.
? ??? AHCY ???? ?? ?? ?? ???? ?????? ???? ??? ?? ? ?? ??? ????, ??? ? ??? ??? ?? ????? 0.0001 ?? 50 ??%? ????.
? ??? ???? ?? AHCY ???? ?? ?? ?? ???? ??? ?? ?? ??? ??? ???? ????? 1? ?? ??? ? ??. ? ??? ???? ?? ??? ?? ?? ???? ??? ???? ???? ??? ??? ??? ??? ? ??.
? ??? ???, AHCY? ?? ???? ?? ???? mRNA? ?? ???? ??????????? ? ??.
???? ??????????? ?? ??? ??? ?? ???? ???-??? ??? ???? ?? ????? ???? ??. ???? ???????? ?? DNA ?? RNA ??? ?? ????(?? ??)? ?? ??? DNA ?? ??(?? DNA ????)??. ???? ??????????? ??? ???? ??, ?? ?? ?????? ???? ??? ??? ???? DNA ?? RNA ??? ?? ???? ???? ??? ?? ?? ?????. ???? ??????????? ?? ?? ? ??? ?? ????? ????? ???? ??(Hogrefe, 1999). ??????????? ???? ??? ?? ???? ???? ?? ?? ?? ???? ??????????? ? ?? ??? ????? ??? ?? ????. ??? ??? ???? ??????????? ???? ?? ???? DNA, ???? ?? ???? RNA, DNA/RNA ?????, DNA ? RNA ???? ? ??, ? ?? ??(backbone) ??? ?? ??????????? ????. ??????????? ???? ????? ????? ??? ?? ???? ????? ?? ? ??? ??? ????? ??? ??? ?? ????. ?? ??? ? ?? ??? ??? ?? ?????????? ??? ??, ? ??? ?? ?? ??? ??? ???? ?? ???? ?? ???.
??, AHCY? ?? ???? ?? ???? siRNA(Small Interfering RNA)? ? ??. siRNA? ????? AHCY ???(mRNA)? ???? ? ??? 19~23?? ??? ????? ? ??.
siRNA? ?? ?? ? ?? ??? ?? ???? ??, ?? ??? ??? ???????? ?? ? ?? ? ??? ?? ???? ???? ?? ??? ??? ???? ??? ??? ?? ?? ?? ??? ?? ???? ???(Bertrand et al., 2002). siRNA? ?? ? siRNA? ??? ?? ?????/??? ????? ??? ??. ?? ??, ?? ?? ?2004/106567 ? 2004/0086884? ????? ??, ?????? ??, ??? ?? ???, ????? ?? ???, ?? ???? ???? ? ????? ?????? ??? ?? ?????? ??? ?? ?? ?????/??? ???? ??.
siRNA? ????? ?? ???? ???? ??????????? ?? ?? ? ?? ??? ????? ?? ??? ?? ? ?? ??? ???? ??????????? ???? ???? ??(Thompson, 2002). siRNA? ??? ??? ?? ??? ??? ??? ??? ???? ? ???? ???? ??. ???? ?? ?? ??? ??? ??? ???? ??? ???? ?? ???? ?????????? ? siRNA? ???? ???? ? ??(Andreas Henschel, Frank Buchholz1 and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32(Web Server Issue):W113-W120 ??). ?? ???? ???? ?????????? ?? siRNA? ?? ?? ?????/??? ??? ?? ??(???, ??? ????, ??? ????, ??-??? ???? ?? ?? ??)? ? ???? ??.
??? ?? ? ?? ??? ?? ???? ? ??? ???? ?????????? ?? siRNA? ?????? ???? ??? ????? ??? ???? ??? ??? ? ??. ?????? ???? ??? ?? ?? ?? ??? ?, ???, ?? ?? ???, ????, ????, ????? ??? ??? ??? ????. ??? ???? ?? ? ?? ??? ?? ??, ?? ?????? ??? ??? ????? ???? ? ??.
?? AHCY ???? ???? ???? ?????????? ?? siRNA ??? ?? ???? ??? ???? ???? ?? ??? ????. ??? ?? ?? ?? ???? ?? ???? ???? ??? ??? ?? ?? ????.
? ??? ?? AHCY ?????? ??? ???? ?? ???? ???? ??? ?? ? ??? ???? ????. ? ??? ???? ???? ?? ?? ???? ??? ???? ??? ??? ???? ????, ?? ???? ???? ???? ??? ??? ??? ? ??. AHCY ??? ????? PCT ?????? WO/2010/027935? ???? ?? ??? ?? ? ? ??.
??? ? ??? ?? ???? ?? ???? ??? ?? ? ?? ?? ??? ????.
?? ???? ?? ???? ? ??? ?????? ??? AHCY ???? ?? ??? ? ??. ?? ???? ?? ???? ??? ???? ???? ???? ?? ?? ??? ?? ???? ??? ?? ??, ???? ?? ??? ? ??. ??, ???? ?? ??? ?? ???? ???? ???? ??? ??? ?? ?? ?? ???? ???? ??? ?? ??? ?? ???? ??? ?? ??.
? ??? ??? ?? ? ?? ??? ???? ??? ??? ?? ??? ?? ?? ??? ??? ?????? ???? ??? 1? ?? ???? ???? ???? ????? ???? ? ??. ? ??? ???? ?? ???? ??? ?? ?????? ???? ??? ?? ???? ?? ?? ?? ???? ????? ??? ?? ???, ??? ?? ???? ?? ???? ?? ??? ??? ? ??.
?? ? ??? ??? ?? ? ?? ??? ???? ?? ?? ? ??? ??? ?? ?? ?? ??? ???? ?? ??? ? ??. ? ??? ??? ?? ? ?? ??? ???? ????? ? ??? ??????(chemotherapeutic agent)? ??? ??? ? ??.
? ??? ??? ?? ? ?? ??? ???? ?? ?? ?? ??? ?????? ???? ?????? ???? ???? ??? ? ???, ??? ????? ???, ???, ???, ???, ???, ???, ???, ??? ?? ???? ?? ??.
?? ? ??? ???? ??? ??? ?? ??? ?? ?? ??? ??? ?????? ???? ??? 1? ?? ???? ???? ???? ????? ???? ? ??.
?? ???? ????? ???? ??? ???? ???? ???? ?? ? ??? ??? ????, ???, ???, ???, ?? ???, ??? ????, ????? ??, ?????? ??, ????, ??? ? ?? ?? ? 1 ?? ??? ???? ??? ? ???, ??? ?? ????, ???, ??? ? ?? ??? ???? ??? ? ??. ?? ???, ???, ?????, ??? ? ???? ????? ???? ???, ???, ??? ?? ?? ??? ??, ??, ??, ?? ?? ??? ???? ? ??. ? ??? ????? ??? ???? Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA? ???? ?? ??? ???? ? ??? ?? ?? ??? ?? ????? ???? ? ??.
? ??? ??? ?? ? ?? ??? ???? ?? ?? ??? ???, ??, ???, ??, ???, ??, ??, ?, ???, ??, ??? ?? ?? ??? ?? ? ?? ???? ???? ?? ?? ? ? ??.
? ??? ??? ?? ? ?? ??? ???? ?? ?, ?? ?, ?? ?, ?? ?, ?? ?, ?? ?, ?? ?, ??, ?? ?, ??, ??, ??, ??, ?? ? ?? ?? ??? ?? ???? ???? ??? ? ??.
? ??? ?? ??? ???, "???"? ??? ?? ? ??? ???? ??? ???? ???? ?? ????. ???, ? ??? ?? ??? "???"? ??? ??, ??? ???, ???? ??? ?? ?? ? ?? ??? ?? ? ??, ??? ?? ? ??? ??, ??, ?? ?? ??, ?? ? ??, ?? ??, ?? ?? ? ???? ???, ????, ?? ???? ??? ??? ??? ??? ?? ??? ? ??. ??? ??, ?? ??? ?? ???? ???? 1? 1? ?? ?? ???, siRNA? ?? 0.01ng/㎏~10㎎/㎏, ?? ???? mRNA? ?? ???? ??????????? ?? 0.01ng/㎏~10㎎/㎏, ???? ?? 0.1ng/㎏~10㎎/㎏, ?? ???? ?? ???? ??? ?? 0.1ng/kg~10㎎/㎏? ???? ???? ?? ?????.
? ??? ???, ?? AHCY ???? ??? ???? ? ?? ??? ??? ??, ?? ?? ??? ????? ? ??.
? ???? ????? ??? ??? ??? ??? ?? ??(Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) ? ???? ?? ?? (Frederick M. Ausubel et al., Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994))? ???? ?? ??? ?? ?? ?????.
? ??? ???? ????, ?? ??? ??, ?? ?? ? ???? ?? ???? ???? ?? ?? ???? ??? ? ??.
? ??? ?? ? ??, ??? ???? ???? ??? ???? ???? ?? ????? ???? ???? ???. ??? ? ??? ???? ???? ????? ???? ?? ??? ?? ?? ??? ??? ??? ???, ?? ? ????? ? ??? ??? ????? ??, ? ??? ??? ?? ???? ??? ??? ?? ??? ??? ??? ???? ???? ?? ???? ???, ? ??? ???? ??? ?? ??? ???.
? ??? ??? AHCY? ????? ??? ??? ??? ??? ?????. ???, AHCY? ?? ?? ??? ?? ???? ???? ???? ?? ? ??? ???? ??? ??? ? ??.
? 1? ??? ?? ??? ????? AHCY ?? ??? ????. ? 1a? ?? ??? ????? ??? RT-PCR? ??? AHCY mRNA ??????. ? 1b? ??? ???? ??? ?? ??? ???? AHCY ??? ??????.
? 2? ?? ??? ??? ??? ?? AHCY ???? ??? ????. ? 2a? MCF7-ADR/shCon ? MCF7-ADR/shAhcy ??? ?? ???? 4? ?? ?????? 10% WST-1?? ???? ??? ????. ? 2b? ?? ??? ??? ????? AHCY ?? ??? ??? RT-PCR? ??? ???? ??? ???.
? 3? MCF7-ADR ???? AHCY? ???? ???? ??? ???? ????. ???? ????? ?? ??? MCF7-ADR/shCon ? MCF7-ADR/shAhcy ???? ??? ???? ?????.
? 4? AHCY ??? ?? ??? ??? ????? ????? ???? ????. ? 4a? MCF7-ADR ??? ???? ???? ??? AHCY ? ?? ??? ??????? ?? ??? ????. ??? ?? ??? ??? 10% ????? ?? ?????. ? 4b? ?? ?? ???? ?? MCF7-ADR ???? ?? ??? ?? AHCY? ??? ??? ????. ??? ????? ??? ? ?? ? ??? ?? ???? ????? 24?? ? ? 48?? ?? ?????.
? 5? AHCY +/- ????? ??? ??? (mammary ductal terminal end buds)? ?? ???? ????. ? 5a? ??? ? AHCY +/- ??? 6?? ? 8??? ? ??? ?? (Whole-mount) ??? (carmine) ??? ??? 4?? ?? ??? ???. ? 5b? 6?? 8? ?? ??? (Terminal end bud; TEB)? ??? ???. ?? ? ? ?? ??? ????? ??? ?? ??? ???.
????? ???? ???? ?? ? ??? ??? ? ? ??? ????. ???, ? ??? ??? ???? ??? ??? ???? ?? ??? ? ??? ??? ?????? ??? ??? ?? ??? ????.
?? ??? ?????
MCF7-ADR, MDA-MB-435s, MDA-MB-468, T47D? 10% ???? (Fetal bovine serum, qualified, Canada origin, Gibco)? ??? ???? ??? DMEM ?? (Welgene, Republic of Korea)?? 5% CO 2 , ?? ???? 37 ℃?? ?????. MCF7-ADR ??? 10% FBS-DMEM? ?? 6-? ?? ????? 2ⅹ10 5 cells/well ??? ???? ?? ?? ?????. ??? ? 50%? ????? ? ??? (MOI 1:5, Santa Cruz, sc-108080)? AHCY shRNA ?????? ?? (lentiviral particles) (MOI 1:5, Santa Cruz, sc-62972-V)? 10 ?/?? ???? (polybrene; Santa Cruz, sc-134220)? ???? 10% FBS-DMEM?? ??? ?????. 12?? ? ??? ???? ?? ???? 24?? ?? ?????, ? ? 10 ?/?? ????? (puromycin; Santa Cruz, sc-108071)? ?? shRNA? ???? ??? ??? ???? 2 ?/?? ?????? ???? 10% FBS-DMEM?? ?????. MDA-MB-468 ??? 60 ㎜ ????? 5ⅹ10 5 ??? ????, FuGENE ?????? (Promega)? ???? 5 ?? pCMV-AHCY? ????? ????????.
??? ??? PCR
NucleoSpin® RNA/Protein kit (Macherey-Nagel)? ???? ???? ???? ??? ?????? RNA? ?????. cDNA (5 ?)? M-MLV ????? (Promega, M170A), RNasin® ??????? ??? (Promega, N211A), 100 nM oligo-dT, 2.5 mM dNTP ???? ???? RNA? ????? ?????. 0.1 ?? cDNA? Rotor-Gene (Corbett research, RG-3000A) ??? HiFast SYBR Lo-Rox (Genepole, Q100240)? ????? ???? ??? ??? PCR? ????? ?????. ????? ??? ??: AHCY (???: 5'-AGC CCC AGG TGG ACC GCT A-3', ???: 5'-TCA CGA AGC TGG GGT GTC CC-3', POSTN (???: 5'-TGC CCT GGT TAT ATG AGA ATG G-3', ???: 5'-AGA ATA GCG CTG CGT TGT GGT-3', ?? 18s rRNA (???: 5'-GTC GGC GTC CCC CAA CTT CTT-3', ???: 5'-CGT GCA GCC CCG GAC ATC TA-3'. ????? Bioneer (South Korea)?? ?????, ?? 18s rRNA ? ???? ?????.
??? ??
NucleoSpin® RNA/Protein kit (Macherey-Nagel)? ???? ???? ???? ??? ?????? ???? ?????. BCA (Bicinchoninic acid) ?? (Sigma, B9643)? ???? ?? (Copper(Ⅱ) sulfate solution; Sigma, C2284)? ???? ??? ???? ?????. ???? SDS-PAGE (10% ??? ?, 5% ??? ?; H 2 O, 30% ??????? (Bio-Rad, #161-0156), 1.5 M pH 8.8 Tris, 10% SDS, 10% ??? ?????, TEMED (Sigma, T9281)) ??? ???? ??? ?? ???? (Atto, AE-6667-P)?? ???. ???? ?-AHCY ?? (Abcam, ab134966), ?-phospho-Akt ?? (Cell signaling, #4060), ?-Akt ?? (Cell signaling, #4691), ?-phospho-Erk1/2 ?? (Cell signaling, #4377), ?-Erk1/2 ?? (Cell signaling, #9102), ?-cyclinD1 ?? (Cell signaling, #2978), ?-CDK6 ?? (Cell signaling, #3136), ?-p21 waf1/cip1 ?? (Cell signaling, #2946), ?-p53 ?? (Cell signaling, #2527) ? ?-???? ?? (anti-cytoskeletal actin) ?? (Bethyl, A300-291A)? ???? ?????. ??? ?? ??? ?? ?-?? IgG, pAb (HRP conjugate, Enzo, ADI-SAB-300-J), ?? ?-??? IgG, pAb (HRP conjugate, Enzo, ADI-SAB-100), ?-???? HRP? ??? ?? (Cell signaling, #7075)??. ????? LAS3000 (Fujifilm)? ???? ?????.
???? ??
??? 12-? ?? ?????? 10% FBS-DMEM? ?? 1x10 4 cells/well ??? ?????. ??? ?? ?? ?? ?, 100 ?/well? ?????? WST-1 (Roche, 644807001)? ???, ??? ???? ? ?? ?? ?????. ? ?? 100 ?? ??? 96-? ?? ????? ??? ELISA (Bio-tek instruments, PowerwaveX340)? ???? 450 ??? ?????. ??? 1% FBS-DMEM?? ??? ??? 100 ?/well WST-1?? ? ?? ?? ???? 96-? ????? ?? 1??? 4??? ?? ?? ??? ???? ?????. ? ?? ???? ??? ???? ???? ????.
?? ?? ???
60 ㎜ ???? 10% FBS-DMEM? ?? ??? 1ⅹ10 5 cells/?? ??? ?? ?? ???? 37 ℃? 24?? ?? ?????. ?? 24?? ?? ??? 1% FBS-DMEM?? ?????. ???? ??? ??? ?? ?? ?? ????? ??? 10 ? ?? ??? ?? ?????. ?? 0??, 24?? ? 48 ?? ? ???? ??? ???? ???? 40??? ?????.
???? ?? ???
?? ??? Biocoat Matrigel Invasion Chamber (BD Biosciences, 354480)? ???? ???? ??? ?? ?????. 24-? ?? ??? 500 ?? ??? ??? DMEM? ? ?? ?? ??, ?? ??? ??? ??????. ?? ??? DMEM? 1% FBS-DMEM ?? 5ⅹ10 4 cells/ml 500 ?? ?????, ?? ??? DMEM ?? ???????? 5 ?/? ????? (Sigma, F2006)? ??? 10% FBS-DMEM 750 ?? ?????. ????? 5% CO 2 ???? 37 ℃? 24?? ?? ?????. ???? ???? ???? ?? ???? ?????. ???? ?? ??? ??? ???? 100% ???? ????? ???? ???? (Sigma, C0775)?? ?????. ?? ??? ?? ??? ? ???? ??? ?? ?? ?????? ???? ?????. ??? ???? ????? 40? ??? ?????. ELISA (Bio-tek instruments, PowerwaveX340)? ???? ?? ??? ???? ??? ?? ??? 10% ????? ????.
?? ??? ?? ?? ( Whole - mount analysis of mammary glands )
6?? ? 8?? ??? ??? ??? ???? ???? ???? ?????. ??? Carnoy? ???? (60% ???, 30% ????? ? 10% ??? ????)?? ???? 24?? ????? 70% ???? ???? ?? ? ?? ?????. ? ???? ??? ?? (Carmine Alum) (Stemcell, 07070)?? 24?? ?? 4 ℃? ???? ??? ?? ???? ?? ? ?? ????? (70%?? 95% ? 100%??). ? ???? ????? ???? ? ?? Permount ® ?? (Fisher scientific, SP15-100)? ?? ???? ???? ?? ??? ??? ?????.
?? ??
?? ? ??? ??? ?? ? ? ?? ?????, ?? ± ????? ?????. ???? ???? P < 0.05 ???? Student's t tests? ???? ?????. ? ?? (??? ??? ???) ??? ? ??? ?? ??? ???? ?? ?????.
?? 1: ??? ???? AHCY ? ??
?? ??? ????? AHCY? ??? ?????? ??? ? ????? ??? qPCR? ??? ???? AHCY? ??? ?????. AHCY? MCF7-ADR ? MDA-MB-435s ???? ?? ???? ??, MDA-MB-468 ? T47D ????? ?? ??? ??? (? 1). AHCY? ???? MCF7-ADR? AHCY sh RNA (small hairpin RNA) ?????? ??? ???? AHCY (MCF7-ADR/shAhcy) ??? ?????. AHCY ?? ??? RNA? ????? ?? MCF7-ADR/shAhcy ???? ?? ????? (? 1). AHCY ?? ??? T47D ??? AHCY ???? ??? pCMV-AHCY ??? ????????. AHCY ??? ??? G418 (T47D/Ahcy)? ???? ?????. AHCY ??? T47D/Con??? T47D/Ahcy ???? ? ??? (? 1).
?? 2: ?? ??? ?? AHCY ? ??
?? ???? ??? AHCY? ??? ???? ??? ?????? WST-1? AHCY? ???? ???? ???? ?? ???? ?????. ? ??, MCF7-ADR/shCon ??? ???? ????? MCF7-ADR/shAhcy ???? ?? ???? ????? (? 2a). AHCY ???? ?? ??? ??? ?????? ??? ???? ??? AHCY shRNA ??-????? ??? MCF7-ADR ??? ???? BrdU ??? ?????. ? ??? 5% FBS-DMEM?? ??? ??? ??? AHCY? ??? ? ? 50% ????? (? 2b), ?? ? 2a? ?????.
?? ? ????? ??? qPCR ? ??? ???? ?? ??? ? ??? ??? ??? ??? ????? ? ?????? ?????. AHCY mRNA ??? MCF7-ADR/shCon ??? ???? MCF7-ADR/shAhcy ???? ? 60% ????? (? 2b). ? ??? ???, AHCY ???? ??? ???? ?? ???? ??? ??? ????.
?? 3: AHCY ? ERK ???? ??? ??? ????? ????.
AHCY ???? ???? ?? ??? ????? ???? ??? ? ????? AHCY ??? ??? ????? ????D1 (CyclinD1), ????E (CyclinE), CDK6, CDK2, p21 ? p53? ?? ???? ??????? ??? ?????. ????? G 0 /G 1 ??? ??? ????D1? CDK6? MCF7-ADR/shAhcy ???? ?????. ??, G 1 /S ??? ???? ????E? CDK2? MCF7-ADR/shAhcy ???? ??? ??? (? 3a). ? ??? AHCY ???? G 1 /S ??? ?? G 0 /G 1 ??? ?????? ???? ??? ???? ????. ? ????? ?? ????D1 ? CDK6? ???? p21? ?? ??? ?????. MCF7-ADR ???? AHCY? ????? ? p21? ?????? p53? ??? ??? (? 3a). ? ????? AHCY ???? ??? p21 ??? p53?? ????? ????.
?? 4: ??? ?? ??? ??? ??? AHCY ?? ?? ???? ??
??? ???? AHCY? ??? ???? ???? ??????? *adenosine dialdehyde; AdOx)? ??? ???? ??? ??? ????? ???? ?? 31 . AHCY? ??? ???? ??? ??? ???? ??? ??? ???? ??? ? ????? AHCY ??? ???? ???? ???? ??? ? ?? ?? ????? ???? ???? ?????. ? 4a? ??, MCF7-ADR/shAHCY ??? ???? ?????. ????, MCF7-ADR ????? AHCY ?? ??? ?? ? ??? ??? 24?? ??? ??? ??? ?? ????? (? 4b).
?? 5: 6?? ? 8?? AHCY +/- ????? ??? ??? ????.
?? ? ??? (????) ???? ? ?? (????) ???? ?????? ???? ??? ? ????? ??? ?? (Whole-mount) ?? ???? AHCY ??? (+/+)? ???? AHCY ??? (+/-) ???? ??? ???? ???? ?????. ??? ??? ?? ??? ???? ??? ??? (Terminal end buds; TEBs)? ???? 32 ,33 . AHCY +/- ????? ???? ?? 6? ? 8?? ??? ???? ????? ?? ??? (? 5). ? ??? AHCY? ???? ???? ?? ??? ??? ???? ????, ??? ? ??? ???? ?????.
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