Prognostic value of snoRNA U50A and its regulatory function in breast cancer Yao-Lung Kuo1, Jie-Ning Li2,3, Yi-Ting Chen2,4, Ming-Yang Wang5, Pai-Sheng Chen2,3 1Department of Surgery, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan and Dou-Liou Branch, , Tainan, Taiwan 2Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan 3Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan 4Department of medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan  5Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan

SnoRNAs classification pseudouridylation 介紹SNORNA主要分成兩類 C/D box以及H/ACA box，分別有不同的二級結構，並會對rRNA(細胞內最多含量的non-codingRNA)進行不同的修飾。 C/D box進行2’-O-methylation 的修飾 H/ACA box進行pseudouridylation的修飾 C/D box snoRNAs: guide 2’-O-methylation of rRNAs . H/ACA box snoRNAs: guide pseudouridylation rRNAs . Nat Rev Cancer. 2012 Jan 19;12(2):84-8. doi: 10.1038/nrc3195.

SnoRNAs biogenesis Host gene Pre-snoRNA Mature snoRNA 介紹SNORNA 生合成 SNORNA大多位於其host gene的intron，host gene切割後，會形成pre-snoRNA，再經由切割形成mature snoRNA Nat Rev Cancer. 2012 Jan 19;12(2):84-8. doi: 10.1038/nrc3195.

SnoRNAs is highly conserved SNORNA在各個物種之間是很conserved的 BMC Evol Biol. 2012 Sep 15;12:183. doi: 10.1186/1471-2148-12-183.

Background Re-expression SNORD50A(U50A) inhibits tumorigenesis property of breast cancer cell lines. J Genet Genomics. 2009 Aug;36(8):447-54. doi: 10.1016/S1673-8527(08)60134-4. U50A is a potential tumor suppressor in multiple human cancers using genomic analysis. Through binding to K-Ras, U50A suppresses K-Ras activity and resulted in decreased tumor growth in vivo. 之前有人發現u50在breast cancer cell line是會抑制tumorigenesis property (並未解開mechanism) 近年來，利用genomic analysis的方法，u50a確定在很多的human cancer中為potential tumor suppressor 同一篇也證明了mechanism: u50a會抑制KRAS activity，並且在動物實驗上會抑制tumor growth 所以我們想研究u50a(potential tumor suppressor )，是否在乳癌當中可做為prognostic marker Nat Genet. 2016 Jan;48(1):53-8. doi: 10.1038/ng.3452. Epub 2015 Nov 23.

Higher U50A expression is correlated with better overall survival of breast cancer patients HR=0.43 Numbers:123 High expression level of u50a 與better patient overall survival有關 U50以median區分

U50A expression is increased in stage Ι of breast cancer * **** U50 expression在stage1比較高，與stage2.3相比

Elevated level of U50A is observed in tumors which size are below 20mm Tumor size * size小於20mm的Tumor ,u50比較高

Elevated level of U50A is correlated with better overall survival in hormone-receptor positive patients Hormone-receptor positive patients High expression level of u50a 與better patient overall survival有關，在hormone receptor+patient當中 U50以average區分 p=0.037* HR=0.34 Numbers:73

U50A expression is not correlate with overall survival in hormone-receptor negative patients HR=0.79 Numbers:31 u50a expression level與patient overall survival無關，在hormone receptor-patient當中 U50以average區分

Role of estrogen in breast cancer 說明estrogen會induce cell proliferation，在breast cancer中為重要的因子 http://www.darngoodlemonade.com/index.php/2012/01/03/the-cranky-to-pain-ratio/

U50A is downregulated in estrogen-treated MCF-7 * ** (normalized to GAPDH) Relative RNA level ** Treat estrogen之下，u50a會下降 U50A

Biogenesis of U50A Host gene (SNHG5) Pre-snoRNA (pre-U50A) Mature snoRNA (U50A) 接下來要看u50a的biogenesis的上游會不會受到estrogen的影響 Nat Rev Cancer. 2012 Jan 19;12(2):84-8. doi: 10.1038/nrc3195.

Estrogen treatment has no effect on U50A biogenesis NS (normalized to GAPDH) Relative RNA level Pre-U50A NS 在treat estrogen之下，pre-u50a以及SNHG5皆沒有下降，由此可知Estrogen-mediated u50a downregulation不是調控u50a的biogenesis (Pre-snoRNA) (Host gene) SNHG5

Estrogen signaling pathway 因為estrogen會由ER傳導signaling pathway，所以想知道estrogen調控u50a會不會經由ER https://www.qiagen.com/us/shop/genes-and-pathways/pathway-details/?pwid=166

Estrogen-mediated U50A downregulation is ERα -independent ** **** ** **** U50A ** **** GFP 5uM Estrogen GFP shERα#1 5uM Estrogen shERα#2 5uM Estrogen shERα#3 5uM Estrogen ** NS (normalized to GAPDH) Relative RNA level 先確定ER有KD(左圖) 在Estrogen treatment之下，u50有確定下降，在KD ER之後，U50還是有下降，表示Estrogen-mediated u50a downregulation 是ER-independent的 ERα

U50A level shows no correlation with ER expression NS 在ER+/-patient中，U50expression沒有差異

Conclusion Higher U50A expression level is correlated with better overall survival of breast cancer patients. U50A is a prognostic marker for hormone receptor positive breast cancer. Estrogen-mediated U50A downregulation is ERα-independent pathway.