C/D-Box Snord105b Promotes Tumorigenesis in ... - Karger Publishers

Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 DOI: 10.1159/000488265 © 2018 The Author(s) www.karger.com/cpb online:March March19,19, 2018 Published online: 2018 Published by S. Karger AG, Basel and Biochemistry Published www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer Accepted: December 09, 2017

This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution IRUFRPPHUFLDOSXUSRVHVDVZHOODVDQ\GLVWULEXWLRQRIPRGLÀHGPDWHULDOUHTXLUHVZULWWHQSHUPLVVLRQ

Original Paper

C/D-Box Snord105b Promotes Tumorigenesis in Gastric Cancer via ALDOA/C-Myc Pathway Cong Zhang Lian-mei Zhao Bao-en Shan

Hao Wu

Guo Tian

Su-li Dai

Ri-yang Zhao

Research Centre, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China

Key Words Small nucleolar RNA • Snord105b • Gastric cancer • Carcinogenesis Abstract Background/Aims: Small nucleolar RNAs (snoRNAs) play an important role in carcinogenesis. ,QWKLVVWXG\ZHLGHQWLÀHGD&'ER[VQR51$VQRUGEDQGIXUWKHULQYHVWLJDWHGWKHIXQFWLRQ and mechanism of the snord105b in gastric cancer (GC). Methods: 7KH H[SUHVVLRQ OHYHO RI VQRUGE LQ *& WLVVXUHV VHUD DQG FHOO OLQHV ZHUH GHWHFWHG E\ T573&5 &HOO YLDELOLW\ ZDV assessed using MTS assay. Transwell and wound healing assay were performed to evaluate PLJUDWLRQ DQG LQYDVLRQ DQG SURWHLQ H[SUHVVLRQ ZDV H[DPLQHG E\ ZHVWHUQ EORWWLQJ &K,53 and MS analysis was used to seek for the special binding protein of snord105b. Results: The snord105b was upregulated and associated with tumor size, differentiation, and pathological stage in GC. Snord105b affected proliferation, migration and invasion in multiple GC cell lines. 7KH RQFRJHQLF DFWLYLW\ RI VQRUGE ZDV DOVR FRQÀUPHG ZLWK in vivo data. Mechanistically, VQRUGE VSHFLÀFDOO\ ERXQG WR $/'2$ DQG DIIHFWHG &P\F ZKLFK SOD\V D NH\ UROH LQ carcinogenesis and tumor development. Conclusion: Snord105b appears to be a novel oncogene and is clinically and functionally involved in the development of GC. Targeting snord105b and its pathway may provide new biomarkers or potential treatments for patients with GC. © 2018 The Author(s) Published by S. Karger AG, Basel

Introduction

GC is one of the most common cancers and is the third highest cause of cancer-related †‡ƒ–Š•™‘”Ž†™‹†‡Ǥ‡…ƒ—•‡•’‡…‹ϐ‹…„‹‘ƒ”‡”•ˆ‘”‡ƒ”Ž›†‹ƒ‰‘•‹•ƒ†‡ˆˆ‡…–‹˜‡–”‡ƒ–‡–• ƒ”‡ Žƒ…‹‰ǡ ’ƒ–‹‡–• ™‹–Š ƒ†˜ƒ…‡†  —•—ƒŽŽ› Šƒ˜‡ ƒ ’‘‘” ’”‘‰‘•‹• ȏͳǡ ʹȐǤ ‘™‡˜‡”ǡ –Š‡ ‡…Šƒ‹•• ‘ˆ  ƒ”‡ •–‹ŽŽ —…Ž‡ƒ”ǡ ƒ† ‘—” •–—†› ‹• †‡˜‘–‡† –‘ †‹•…‘˜‡”‹‰ •’‡…‹ϐ‹… „‹‘ƒ”‡”•ƒ†‘˜‡Ž‡–Š‘†•ˆ‘” †‹ƒ‰‘•‹•ƒ†–Š‡”ƒ’›Ǥ &=KDQJDQG/0=KDRFRQWULEXWHGHTXDOO\WRWKLVZRUN

Baoen Shan

Research Centre, the Fourth Hospital of Hebei Medical University, 12# Jiankang road, Shijiazhuang (China) Tel. +86311-86095283, E-Mail [email protected]

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Small nucleolar RNAs (snoRNAs) are a class of non-coding RNAs (ncRNAs) that are ‡”‹…Š‡† ‹ –Š‡ —…Ž‡‘Ž—• ƒ† ƒ’’”‘š‹ƒ–‡Ž› ͸ͲǦ͵ͲͲ – ‹ Ž‡‰–ŠǤ —ƒ •‘• ƒ”‡ ‡…‘†‡†„›‹–”‘•Ǥ‘•ǡ‹…‘„‹ƒ–‹‘™‹–Š•‘‡’”‘–‡‹•ǡƒ”‡ƒ„Ž‡–‘ˆ‘”•‘• ȋ•ƒŽŽ—…Ž‡‘Žƒ””‹„‘—…Ž‡‘’”‘–‡‹’ƒ”–‹…Ž‡•Ȍƒ†ƒ”‡•–ƒ„Ž‡‹’Žƒ•ƒǤƒ•‡†‘…‘•‡”˜‡† •‡“—‡…‡‡Ž‡‡–•ǡ•‘•ƒ”‡…Žƒ••‹ϐ‹‡†ƒ•Ȁ„‘š•‘•‘”Ȁ„‘š•‘•ǤŠ‡ „‡•–Ǧ—†‡”•–‘‘†ˆ—…–‹‘‘ˆ•‘•‹•–‘†‡ϐ‹‡–Š‡–ƒ”‰‡–•‹–‡•‘ˆ”•ˆ‘”ʹԢǦǦ”‹„‘•‡ ‡–Š›Žƒ–‹‘ƒ†’•‡—†‘—”‹†›Žƒ–‹‘ȏ͵ǡͶȐǤŠ—•ǡ•‘•’Žƒ›ƒ˜‹–ƒŽ”‘Ž‡‹–Š‡‰‡‡•‹•ƒ† ’”‘…‡••‹‰ ‘ˆ –Š‡ ”‹„‘•‘‡Ǥ ‘†‹ϐ‹…ƒ–‹‘ ‘ˆ –Š‡ ”‹„‘•‘‡ Šƒ• „‡‡ ‹’Ž‹…ƒ–‡† ‹ …ƒ…‡” †‡˜‡Ž‘’‡–ǡ‹†‹…ƒ–‹‰–Šƒ––Š‡…Žƒ••‹…ƒŽˆ—…–‹‘•‘ˆ•‘•‹‰Š–…‘–”‹„—–‡–‘…ƒ…‡” ‘……—””‡…‡ƒ††‡˜‡Ž‘’‡–ǡƒ†–Š‹•‹•ƒ‡™ϐ‹‡Ž†–Šƒ–”‡“—‹”‡•ˆ—”–Š‡””‡•‡ƒ”…Š‡•ȏͷǦ͹ȐǤ ‘”†ͳͲͷ„ ‹• ƒ ‡„‡” ‘ˆ –Š‡ Ȁ „‘š •‘ ˆƒ‹Ž› ƒ† ™ƒ• ϐ‹”•– †‹•…‘˜‡”‡† ‹ ʹͲͲ͸ȏͺȐǤ™‹‰–‘‹–•Š‘•–‰‡‡ǡ™Š‹…Š‹•‹˜‘Ž˜‡†‹Š—ƒ‹—‹–›ƒ†…ƒ…‡”ǡ ™‡‡š’Ž‘”‡–Š‡”‘Ž‡‘ˆ•‘”†ͳͲͷ„‹‘…‘‰‡‡•‹•ȏͻȐǤ –Š‹••–—†›ǡ™‡•Š‘™–Šƒ–•‘”†ͳͲͷ„ ‹•‡š’”‡••‡†ƒ–Š‹‰ŠŽ‡˜‡Ž•‹–Š‡–‹••—‡•ƒ†•‡”ƒ‘ˆ ’ƒ–‹‡–•…‘’ƒ”‡†–‘–Š‘•‡‘ˆ–Š‡ Š‡ƒŽ–Š›†‘‘”•ƒ†‹•”‡“—‹”‡†ˆ‘”–Š‡†‡˜‡Ž‘’‡–‘ˆ Ǥ Materials and Methods Patients and healthy donors Fresh GC tissues and adjacent normal gastric tissues in patients with GC were collected from the Fourth ‘•’‹–ƒŽ‘ˆ‡„‡‹‡†‹…ƒŽ‹˜‡”•‹–›„‡–™‡‡ʹͲͳͷƒ†ʹͲͳ͸Ǥ ˆ‘”‡†…‘•‡–ˆ‘”–Š‡—•‡‘ˆ–Š‡•ƒ’Ž‡• ™ƒ•‘„–ƒ‹‡†ˆ”‘ƒŽŽ’ƒ–‹‡–•ǡƒ†ƒ’’”‘˜ƒŽ™ƒ•‘„–ƒ‹‡†ˆ”‘–Š‡Š‘•’‹–ƒŽǤŽŽ•ƒ’Ž‡•™‡”‡†‹ƒ‰‘•‡† „›ʹǦ͵„Ž‹†‡†’ƒ–Š‘Ž‘‰‹•–•ǤŠ‡‹…Ž—•‹‘…”‹–‡”‹ƒ”‡“—‹”‡†–Šƒ–’ƒ–‹‡–•Šƒ†’”‹ƒ”› ƒ–•–ƒ‰‡• Ǧ ǡŠƒ† ”‡…‡‹˜‡†•—”‰‡”›ƒ•–Š‡‹‹–‹ƒŽ–”‡ƒ–‡–ƒ†Šƒ†…‘’Ž‡–‡…Ž‹‹…‘’ƒ–Š‘Ž‘‰‹…ƒŽ†ƒ–ƒǤ

Cells culture and transfection Š‡ …‡ŽŽŽ‹‡• Ǧ͹ͻͲͳǡ ǦͺͲ͵ǡ Ǧͺʹ͵ǡ Ǧʹ͹ǡƒ† ™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡‡•‡ƒ”…Š ‡–‡”‘ˆ–Š‡ ‘”–Š‘•’‹–ƒŽ‘ˆ‡„‡‹‡†‹…ƒŽ‹˜‡”•‹–›ǤŠ‡‹‘”–ƒŽ‹œ‡†‘”ƒŽ‰ƒ•–”‹…‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ Ž‹‡ Ǧͳ ™ƒ• ‹†Ž› ’”‘˜‹†‡† „› ”‘ˆ‡••‘”‡ Ǥ ŽŽ –Š‡ …‡ŽŽ Ž‹‡• ™‡”‡ …—Ž–—”‡† ‹   ͳ͸ͶͲ ȋ ‹„…‘Ȍ •—’’Ž‡‡–‡†™‹–ŠͳͲΨˆ‡–ƒŽ…ƒŽˆ•‡”—ȋ Ȍƒ†’‡‹…‹ŽŽ‹ƒ†•–”‡’–‘›…‹ȋ ˜‹–”‘‰‡Ȍƒ†‹…—„ƒ–‡†ƒ– ͵͹鋐ͷΨʹǤ‡ŽŽ•™‡”‡–”ƒ•‹‡–Ž›‘…‡††‘™™‹–Š•‘”†ͳͲͷ„Ǧ•‹ȋ‹„‘‹‘Ȍ‘”–”ƒ•ˆ‡…–‡†™‹–Š–Š‡ ‘˜‡”‡š’”‡••‹‘’Žƒ•‹†‘ˆ•‘”†ͳͲͷ„ȋ ˜‹–”‘‰‡ȌǤ

RNA isolation and quantitative real-time PCR (q-PCR) ‘–ƒŽ  ™ƒ• ‡š–”ƒ…–‡† ˆ”‘ –Š‡ …‡ŽŽ• ‘” –‹••—‡• —•‹‰ ƒ  œ‘Ž •‘Ž—–‹‘ ȋ”‘‡‰ƒȌǤ ‡˜‡”•‡ –”ƒ•…”‹’–‹‘ ”‡ƒ…–‹‘• ™‡”‡ ’‡”ˆ‘”‡† ™‹–Š  ”‡˜‡”•‡ –”ƒ•…”‹’–ƒ•‡ ȋ”‘‡‰ƒȌ ƒ……‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ…–—”‡”ǯ• ‹•–”—…–‹‘•Ǥ Ǧ ™ƒ• …‘†—…–‡† —•‹‰ ƒ  ”‡‡  ‹– ȋ”‘‡‰ƒȌ ™‹–Š ƒ ‡ƒŽǦ ‹‡›•–‡ȋ ͹ͷͲͲȌǤ ‡‡Ǧ•’‡…‹ϐ‹…“Ǧ’”‹‡”•™‡”‡ƒ•ˆ‘ŽŽ‘™•ǣ•‘”†ͳͲͷ„ˆ‘”™ƒ”†

Ǧ  

 ǡ ”‡˜‡”•‡    

ǡ     Ǣ ƒ† ͸ ˆ‘”™ƒ”†



 Ǧƒ†”‡˜‡”•‡     ǦǤŠ‡’”‹‡”ˆ‘”͸™ƒ• –Š‡•ƒ‡ƒ•–Š‡”‡˜‡”•‡•‡“—‡…‡Ǥ‡š’”‡••‹‘Ž‡˜‡Ž•™‡”‡‘”ƒŽ‹œ‡†–‘͸‡š’”‡••‹‘Ž‡˜‡Ž•Ǥ‡Žƒ–‹˜‡ ‡š’”‡••‹‘Ž‡˜‡Ž•™‡”‡…ƒŽ…—Žƒ–‡†—•‹‰–Š‡ʹǦȟȟ– method. Cell proliferation assay Š‡ƒ••ƒ›™ƒ•’‡”ˆ‘”‡†ƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘–‘‡šƒ‹‡–Š‡’”‘Ž‹ˆ‡”ƒ–‹‘ ‘ˆ …‡ŽŽ•ǤŠ‡…‡ŽŽ•ȋͳͲ͵Ȍ™‡”‡’Žƒ–‡†‹ͻ͸Ǧ™‡ŽŽ’Žƒ–‡•™‹–Š͸”‡’Ž‹…ƒ–‡•Ȁ‰”‘—’ǤŠ‡…‡ŽŽ•™‡”‡‹…—„ƒ–‡† ˆ‘” Ͳǡ ʹͶǡ Ͷͺǡ ͹ʹ ƒ† ͻ͸ Š‘—”• ƒˆ–‡” –”ƒ•ˆ‡…–‹‘Ǥ – ‡ƒ…Š ‘ˆ –Š‡ †‡•‹”‡† –‹‡ ’‘‹–•ǡ –Š‡  •‘Ž—–‹‘ ȋ”‘‡‰ƒȌ™ƒ•ƒ††‡†ȋʹͲɊȀ™‡ŽŽȌ‹–‘‡ƒ…Š™‡ŽŽƒ†‹…—„ƒ–‡†ˆ‘”ʹŠƒ˜‘‹†‹‰Ž‹‰Š–ƒ–͵͹ιǡˆ‘ŽŽ‘™‡†„› ‡ƒ•—”‡‡–‘ˆ–Š‡ƒ„•‘”„ƒ…‡ƒ–Ͷͻʹ™‹–Šƒ‹…”‘’Žƒ–‡”‡ƒ†‡”Ǥ

Transwell migration and invasion assay ‘” –Š‡ –”ƒ•™‡ŽŽ ‹‰”ƒ–‹‘ ƒ••ƒ›•ǡ ͳ έ ͳͲͷ  Ǧ͹ͻͲͳ …‡ŽŽ• ‘”   …‡ŽŽ• ™‡”‡ •‡‡†‡† ‹ –Š‡ —’’‡” …Šƒ„‡”ȋʹͶǦ™‡ŽŽ‹•‡”–ǢͺɊ’‘”‡•‹œ‡Ǣ‘”‹‰‘•–ƒ”ǡƒ„”‹†‰‡ǡǡȌ—•‹‰ͲǤʹŽ‘ˆ‡†‹—

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

™‹–Š‘—–•‡”—ǡƒ†ͲǤ͸Ž‘ˆ‡†‹—™‹–ŠʹͲΨ ™ƒ•ƒ††‡†–‘–Š‡Ž‘™‡”…Šƒ„‡”ƒ•ƒ…Š‡‘ƒ––”ƒ…–ƒ–Ǥ ˆ–‡” ƒ ͳͷ –‘ ʹͲ Š ‹…—„ƒ–‹‘ǡ ‘Ǧ‹‰”ƒ–‹‰ …‡ŽŽ• ‹ –Š‡ —’’‡” •—”ˆƒ…‡ ‘ˆ –Š‡ ‡„”ƒ‡ ™‡”‡ ”‡‘˜‡† ™‹–Š ƒ …‘––‘ •™ƒ„ǡ ƒ† –Š‡ …‡ŽŽ• –Šƒ– ’‡‡–”ƒ–‡† –Š‡ Ž‘™‡” •—”ˆƒ…‡ ‘ˆ –Š‡ ‡„”ƒ‡ ™‡”‡ •–ƒ‹‡† ™‹–Š …”›•–ƒŽ˜‹‘Ž‡–ǤŠ‡—„‡”‘ˆ…‡ŽŽ•‹˜ƒ†‹‰–Š‡‡„”ƒ‡™ƒ•…‘—–‡†™‹–Šƒ‹…”‘•…‘’‡‹ͷ”ƒ†‘Ž› •‡Ž‡…–‡†ϐ‹‡Ž†•Ǥƒ–ƒ™‡”‡‘„–ƒ‹‡†ˆ”‘͵‹†‡’‡†‡–‡š’‡”‹‡–•Ǥ ‘”–Š‡…‡ŽŽ‹˜ƒ•‹‘ƒ••ƒ›•ǡƒ’”‘–‘…‘Ž •‹‹Žƒ”–‘–Š‡…‡ŽŽ‹‰”ƒ–‹‘ƒ••ƒ›™ƒ•—•‡†‡š…‡’––Šƒ––Š‡–”ƒ•™‡ŽŽ—‹–•™‡”‡’”‡…‘ƒ–‡†™‹–ŠʹͲͲɊ‰ȀŽ ƒ–”‹‰‡Žȋ‡›‘–‹‡‹‘–‡…Š‘Ž‘‰›Ȍƒ†‹…—„ƒ–‡†‘˜‡”‹‰Š–Ǥ

Cell invasion assay ‘” –Š‡ …‡ŽŽ ‹˜ƒ•‹‘ ƒ••ƒ›•ǡ ƒ ’”‘–‘…‘Ž •‹‹Žƒ” –‘ –Š‡ …‡ŽŽ ‹‰”ƒ–‹‘ ƒ••ƒ› ™ƒ• —•‡† ‡š…‡’– –Šƒ– –Š‡ –”ƒ•™‡ŽŽ —‹–• ™‡”‡ ’”‡…‘ƒ–‡† ™‹–Š ʹͲͲ ρ‰ Ȁ Ž ƒ–”‹‰‡Ž ȋ‡›‘–‹‡ ‹‘–‡…Š‘Ž‘‰›Ȍ ƒ† ‹…—„ƒ–‡† ‘˜‡”‹‰Š–Ǥ‡ŽŽ•–Šƒ–‹˜ƒ†‡†–Š”‘—‰Š–Š‡ƒ–”‹‰‡Žƒ†”‡ƒ…Š‡†–Š‡Ž‘™‡”•—”ˆƒ…‡‘ˆ–Š‡ϐ‹Ž–‡”™‡”‡…‘—–‡† ™‹–Šƒ‹…”‘•…‘’‡‹ͷ”ƒ†‘ϐ‹‡Ž†•Ǥƒ–ƒ™‡”‡‘„–ƒ‹‡†ˆ”‘͵‹†‡’‡†‡–‡š’‡”‹‡–•Ǥ

Wound healing experiments ‡ŽŽ•ȋͷhͳͲͷȌ™‡”‡•‡‡†‡†‹͸Ǧ™‡ŽŽ’Žƒ–‡•ǤŠ‡–Š‡…‡ŽŽ…‘ϐŽ—‡…‡”‡ƒ…Š‡†ƒ’’”‘š‹ƒ–‡Ž›ͺͲΨƒ– ʹͶŠƒˆ–‡”–”ƒ•ˆ‡…–‹‘ǡ•…”ƒ–…Š™‘—†•™‡”‡ƒ†‡„›•…”ƒ’‹‰–Š‡…‡ŽŽŽƒ›‡”ƒ…”‘••‡ƒ…Š…—Ž–—”‡’Žƒ–‡—•‹‰ ƒ’‹’‡––‡–‹’Ǥ‘‡•—”‡–Š‡•ƒ‡”‡‰‹‘™ƒ•‡˜ƒŽ—ƒ–‡†ǡ–Š‡™‡ŽŽ•™‡”‡ƒ”‡†ƒ…”‘••–Š‡™‘—†‡†ƒ”‡ƒǤ Š‡‡†‹—™ƒ•”‡’Žƒ…‡†™‹–Š•‡”—Ǧˆ”‡‡‡†‹—–‘‹Š‹„‹–…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘ǤŠƒ•‡Ǧ…‘–”ƒ•–‹ƒ‰‡•™‡”‡ ”‡…‘”†‡†”ƒ†‘Ž›™‹–Šƒ‹˜‡”–‡†Ǧ‹…”‘•…‘’‡ƒ––Š‡–‹‡‘ˆ™‘—†‹‰ƒ†ʹͶƒ†ͶͺŠƒˆ–‡”™ƒ”†•ǤŠ‡ experiments were performed in triplicate.

Clonogenic assay ‘‹˜‡•–‹‰ƒ–‡–Š‡ƒ„‹Ž‹–›‘ˆ…‡ŽŽ•–‘ˆ‘”…‘Ž‘‹‡•ǡͳέͳͲ͵…‡ŽŽ•™‹–Šƒ†™‹–Š‘—–•‘”†ͳͲͷ„‘…‡† †‘™ƒ†–”ƒ•ˆ‡…–‡†™‹–Š‡‹–Š‡”•‘”†ͳͲͷ„‘”ͷͳͳ™‡”‡•‡‡†‡†‹–‘͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ†‹…—„ƒ–‡†ˆ‘”ͳͲ †ƒ›•™‹–Š‡†‹—…Šƒ‰‡†‡˜‡”›͵ǦͶ†ƒ›•Ǥ‘Ž‘‹‡•™‡”‡ϐ‹š‡†™‹–ŠͳΨˆ‘”ƒŽ†‡Š›†‡ˆ‘”ͳͲ‹ǡ•–ƒ‹‡† ™‹–Š ͲǤͲͷΨ …”›•–ƒŽ ˜‹‘Ž‡– ȋ‹‰ƒ Š‡‹…ƒŽ ‘’ƒ›ǡ ‘—‹•ǡ ǡ Ȍ ˆ‘” ͳͲ ‹ ƒ– ”‘‘ –‡’‡”ƒ–—”‡ǡ ™ƒ•Š‡† –™‹…‡ ™‹–Š ’Š‘•’Šƒ–‡Ǧ„—ˆˆ‡”‡† •ƒŽ‹‡ ȋȌǡ ƒ† ‘„•‡”˜‡† ƒ† …‘—–‡† —†‡” ƒ ‹…”‘•…‘’‡ ȋŽ›’—•ǡ‘›‘ǡ ƒ’ƒȌǤ

‘’”‡Š‡•‹˜‡‹†‡–‹ϔ‹…ƒ–‹‘‘ˆǦ„‹†‹‰’”‘–‡‹•„›ƒ•••’‡…–”‘‡–”›ȋŠ ǦȌƒƒŽ›•‹• ‘”–Š‡Š ǦƒƒŽ›•‹•ǡ„”‹‡ϐŽ›ǡ…‡ŽŽ•ȋʹέͳͲ͹ Ǧ͹ͻͲͳ…‡ŽŽ•Ȍ™‡”‡…”‘••Ž‹‡†™‹–ŠͳΨˆ‘”ƒŽ†‡Š›†‡ ˆ‘”ͳͲ‹ǡ‡“—‹Ž‹„”ƒ–‡†‹‰Ž›…‹‡„—ˆˆ‡”ˆ‘”ͷ‹ƒ†Ž›•‡†–‘‘„–ƒ‹–Š‡’”‘–‡‹•Ǥƒ”‰‡–•‘™‡”‡ ‡š–”ƒ…–‡† „› „‹‘–‹›Žƒ–‡†Ǧƒ–‹•‡•‡Ǧ‘Ž‹‰‘•ǡ ƒ† ƒ••‘…‹ƒ–‡† ’”‘–‡‹• ™‡”‡ ‡Ž—–‡† ™‹–Š „‹‘–‹Ǧ„‡ƒ†• ƒ† •‡’ƒ”ƒ–‡†„›‡Ž‡…–”‘’Š‘”‡•‹•Ǥƒ…Š•‹œ‡ˆ”ƒ…–‹‘™ƒ••—„Œ‡…–‡†–‘ƒ•••’‡…–”‘‡–”›ȋȌ‹†‡–‹ϐ‹…ƒ–‹‘Ǥ Š‡–ƒ”‰‡–’”‘–‡‹•™‡”‡•‡’ƒ”ƒ–‡†„›Ǥ

‡•–‡”„Ž‘–ƒƒŽ›•‹• ‘–‡•––Š‡•‡–ƒ”‰‡–’”‘–‡‹•ǡ–”ƒ•ˆ‡…–‡†…‡ŽŽ•™‡”‡Ž›•‡†‹Ž›•‹•„—ˆˆ‡”ǡƒ†–Š‡’”‘–‡‹…‘…‡–”ƒ–‹‘• ™‡”‡‡ƒ•—”‡†—•‹‰ƒ’”‘–‡‹ƒ••ƒ›‹–ȋŠ‡”‘ȌǤ”‘–‡‹‡š–”ƒ…–•™‡”‡•‡’ƒ”ƒ–‡†„›ͳͲΨǦ ǡ –”ƒ•ˆ‡””‡† –‘   ‡„”ƒ‡•ǡ ƒ† ‹…—„ƒ–‡† ™‹–Š ƒ–‹„‘†‹‡• ƒ‰ƒ‹•– ǡ Ǧ…ƒ†Š‡”‹ǡ ‹‡–‹ǡ Ǧƒ†Š‡”‹ȋ”‘–‡‹–‡…ŠȌƒ†Ǧ›…ȋ‡ŽŽ‹‰ƒŽ‹‰‡…Š‘Ž‘‰›ȌǤ”‘–‡‹‡š’”‡••‹‘Ž‡˜‡Ž•™‡”‡‘”ƒŽ‹œ‡† –‘–Š‡‡†‘‰‡‘—• …‘–”‘ŽǤ–‹Ǧ”ƒ„„‹– ‰ ȋ‡ŽŽ‹‰ƒŽ‹‰‡…Š‘Ž‘‰›Ȍ™ƒ•—•‡†ƒ•–Š‡•‡…‘†ƒ”› ƒ–‹„‘†›ǡ ƒ† –Š‡ ƒ–‹‰‡Ǧƒ–‹„‘†› ”‡ƒ…–‹‘ ™ƒ• ˜‹•—ƒŽ‹œ‡† „› †‡–‡…–‹‘ ™‹–Š ƒ ‘†›••‡› ƒ••ƒ› ȋǡ ‹ŽŽ‹’‘”‡ǡ‹ŽŽ‡”‹…ƒǡȌǤ

 ƒ••ƒ›  ™ƒ•’‡”ˆ‘”‡†—•‹‰ƒƒ‰ƒ ‹–ȋ‹ŽŽ‹’‘”‡ǡȌƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•Ǥ  „”‹‡ˆǡ ƒ ƒ–‹„‘†› ƒ‰ƒ‹•–  ™ƒ• —•‡†Ǥ Š‡ …‘Ǧ’”‡…‹’‹–ƒ–‡† • ™‡”‡ ƒ†•‘”„‡† ™‹–Š ƒ‰‡–‹… „‡ƒ†• ƒ† †‡–‡…–‡† „› ǦǤ ‘–ƒŽ  ȋ‹’—–Ȍ ƒ† –Š‡ …‘–”‘Ž ƒ–‹Ǧ”ƒ„„‹– ‰  ƒ–‹„‘†› ™‡”‡ —•‡† •‹—Ž–ƒ‡‘—•Ž›–‘†‡‘•–”ƒ–‡–Šƒ––Š‡†‡–‡…–‡†•‹‰ƒŽ™ƒ•ˆ”‘••’‡…‹ϐ‹…ƒŽŽ›„‹†‹‰–‘–Š‡ ’”‘–‡‹ȋα͵ˆ‘”‡ƒ…Š‡š’‡”‹‡–ȌǤ

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Animal experiments ƒŽ‡ Ȁ… ƒ–Š›‹… —†‡ ‹…‡ ȋͶǦ͸ ™‡‡• ‘Ž†Ȍ ™‡”‡ ‘„–ƒ‹‡† ˆ”‘ –Š‡ ‹–ƒŽ ‹˜‡” ƒ„‘”ƒ–‘”› ‹ƒŽ‡…Š‘Ž‘‰›‘Ǥ–†Ǥǡ‡‹Œ‹‰Ǥ‘‡˜ƒŽ—ƒ–‡–Š‡‡ˆˆ‡…–‘ˆ•‘”†ͳͲͷ„‹—†‡‹…‡ǡ™‡…‘•–”—…–‡† Ǧ ͹ͻͲͳ…‡ŽŽ•™‹–Š•–ƒ„Ž‡•‘”†ͳͲͷ„Ǧ‘…†‘™‡š’”‡••‹‘ǤŽŽ‹…‡™‡”‡‹Œ‡…–‡†•—„…—–ƒ‡‘—•Ž›‹–‘‡‹–Š‡” •‹†‡‘ˆ–Š‡ϐŽƒƒ”‡ƒ™‹–ŠͳέͳͲ͹…‡ŽŽ•™‹–Š•‘”†ͳͲͷ„•–ƒ„Ž›†‘™”‡‰—Žƒ–‡†‘”™‹–Š…‘–”‘Ž Ǧ͹ͻͲͳ …‡ŽŽ•–‘‡•–ƒ„Ž‹•Š–Š‡ š‡‘‰”ƒˆ–‘†‡ŽǤ—‘”‰”‘™–Š™ƒ•‘„•‡”˜‡††ƒ‹Ž›ǡƒ†–Š‡–—‘”˜‘Ž—‡•™‡”‡ …ƒŽ…—Žƒ–‡†ȋαͲǤͷέŽ‡‰–Šέ™‹†–ŠʹȌ‹‹…‡‡˜‡”›–Š”‡‡†ƒ›•Ǥˆ–‡”Ͷ™‡‡•ǡ–Š‡‹…‡™‡”‡•ƒ…”‹ϐ‹…‡†ǡƒ† –Š‡–—‘”•™‡”‡‡š‡”…‹•‡†ƒ†•—„Œ‡…–‡†–‘‹—‘Š‹•–‘…Š‡‹…ƒŽƒƒŽ›•‹•‘ˆ‹͸͹ǡǡǦ›…ƒ† ”‡Žƒ–‡†’”‘–‡‹•ǤŽŽ ƒ‹ƒŽ ‡š’‡”‹‡–•™‡”‡’‡”ˆ‘”‡†ƒ– –Š‡‹ƒŽ ƒ„‘”ƒ–‘”›‡–‡”‘ˆ–Š‡ ‘—”–Š ‘•’‹–ƒŽ‘ˆ‡„‡‹‡†‹…ƒŽ‹˜‡”•‹–›Ǥ

–ƒ–‹•–‹…ƒŽƒƒŽ›•‹• ŽŽ–Š‡•–ƒ–‹•–‹…ƒŽƒƒŽ›•‡•™‡”‡’‡”ˆ‘”‡†—•‹‰˜‡”•‹‘ͳ͵ǤͲ•‘ˆ–™ƒ”‡ǤŽŽ†ƒ–ƒƒ”‡’”‡•‡–‡†ƒ• –Š‡‡ƒά•–ƒ†ƒ”††‡˜‹ƒ–‹‘ˆ”‘–Š”‡‡‹†‡’‡†‡–‡š’‡”‹‡–•–Šƒ–™‡”‡‡ƒ…Š‡ƒ•—”‡†‹–”‹’Ž‹…ƒ–‡Ǥ ‡Ǧ™ƒ› ƒƒŽ›•‹• ‘ˆ ˜ƒ”‹ƒ…‡ ƒ† –—†‡–ǯ• –Ǧ–‡•– ™‡”‡ ’‡”ˆ‘”‡† ˆ‘” …‘’ƒ”‹•‘ ƒ• †‡•…”‹„‡†Ǥ  …Š‹Ǧ •“—ƒ”‡–‡•–™ƒ•—•‡†–‘ƒƒŽ›œ‡…ƒ–‡‰‘”‹…ƒŽ˜ƒ”‹ƒ„Ž‡•ǤP ˜ƒŽ—‡‘ˆŽ‡••–ŠƒͲǤͲͷ™ƒ•…‘•‹†‡”‡†•–ƒ–‹•–‹…ƒŽŽ› •‹‰‹ϐ‹…ƒ–ȋȗ˜ƒŽ—‡δͲǤͲͷȌǡƒ†ƒŽŽ•–ƒ–‹•–‹…ƒŽ–‡•–•™‡”‡–™‘Ǧ–ƒ‹Ž‡†Ǥ

Results

‘”†ͷͶͻ„‹•‘˜‡”‡š’”‡••‡†‹Š—ƒ‰ƒ•–”‹……ƒ…‡”–‹••—‡•ƒ†…‡ŽŽŽ‹‡• ‘‹†‡–‹ˆ›™Š‡–Š‡”•‘”†ͳͲͷ„Šƒ•ƒ”‡‰—Žƒ–‘”›”‘Ž‡‹–Š‡’”‘‰”‡••‹‘‘ˆŠ—ƒ ǡ ™‡ϐ‹”•–†‡–‡…–‡†–Š‡‡š’”‡••‹‘’”‘ϐ‹Ž‡‘ˆ•‘”†ͳͲͷ„‹ –‹••—‡•ˆ”‘’ƒ–‹‡–•„›“Ǧ

Fig. 1.‘”†ͳͲͷ„™ƒ•—’”‡‰—Žƒ–‡†‹ –‹••—‡•ǡ•‡”ƒƒ†…‡ŽŽŽ‹‡•ǤȋȌŠ‡‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„‹

•ƒ’Ž‡•ƒ†ƒ†Œƒ…‡–‘”ƒŽ‰ƒ•–”‹…–‹••—‡•™ƒ•ƒƒŽ›œ‡†„›“ǦȋαͳͲͻǡȗδͲǤͲͷȌǤ‘”†ͳͲͷ„ ‡š’”‡••‹‘ Ž‡˜‡Ž• ™‡”‡ ‘”ƒŽ‹œ‡† –‘ ͸Ǥ ȋȌ ‘™ ƒ† Š‹‰Š ‡š’”‡••‹‘ ‘ˆ •‘”†ͳͲͷ„ ‹  –‹••—‡• ƒ† ƒ†Œƒ…‡– ‘”ƒŽ ‰ƒ•–”‹… –‹••—‡• ™ƒ• ƒƒŽ›œ‡† „ƒ•‡† ‘ –Š‡ †ƒ–ƒ ƒ„‘˜‡Ǥ ȋȌ ‘”†ͳͲͷ„ ‡š’”‡••‹‘ ™ƒ• ƒƒŽ›œ‡† „› “Ȃ ‹ –Š‡ •‡”— ‘ˆ  ’ƒ–‹‡–• ƒ† …‘’ƒ”‡† ™‹–Š –Šƒ– ‘ˆ Š‡ƒŽ–Š› †‘‘”• ȋ α ͸ͺǡ ȗδͲǤͲͷȌǤȋǦȌ“Ǧƒ†ǦƒƒŽ›•‹••Š‘™‡†–Šƒ––Š‡‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„™ƒ•Š‹‰Š‡”‹–Š‡

…‡ŽŽŽ‹‡•…‘’ƒ”‡†™‹–Š–Š‡‘”ƒŽ‰ƒ•–”‹…‹‘”–ƒŽ‹œ‡†‡’‹–Š‡Ž‹ƒŽ…‡ŽŽŽ‹‡ ǦͳǤ

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

 ȋ α ͳͲͻȌǤ • •Š‘™ ‹ ‹‰Ǥ ͳ ƒ† Table 1. ‘”ƒƒŽ›•‹•‘ˆ…‘””‡Žƒ–‹‘„‡–™‡‡•‘”†ͳͲͷ„ ‹‰Ǥͳǡ–Š‡–”ƒ•…”‹’–Ž‡˜‡Ž‘ˆ•‘”†ͳͲͷ„ Ž‡˜‡Ž•ƒ†…Ž‹‹…ƒŽˆ‡ƒ–—”‡•ǡ–Š‡ɖʹǦ–‡•–•™‡”‡—•‡†ǤȗŠ‡ ™ƒ••‹‰‹ϐ‹…ƒ–Ž›—’”‡‰—Žƒ–‡†‹ –‹••—‡ ˜ƒŽ—‡•Šƒ†•–ƒ–‹•–‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡• compared with the corresponding normal Expression level of snord105b Parameters Total P-value Low High ‰ƒ•–”‹… –‹••—‡ ˆ”‘ –Š‡ •ƒ‡ †‘‘” ȋ δ Age/year ͲǤͲͷȌǤ ††‹–‹‘ƒŽŽ›ǡ –Š‡ —’”‡‰—Žƒ–‹‘ ‘ˆ 5 43 5 38 ‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„‹–Š‡•‡”ƒ‘ˆ  Differentiation 49 16 33 0.015* ’ƒ–‹‡–•ȋα͸ͺȌ„›“ǦǤ••Š‘™‹ Well Poor 60 8 52 ‹‰Ǥͳǡ–Š‡–”ƒ•…”‹’–Ž‡˜‡Ž•‘ˆ•‘”†ͳͲͷ„ Stage 21 8 13 0.014* ™‡”‡•‹‰‹ϐ‹…ƒ–Ž›—’”‡‰—Žƒ–‡†‹ •‡”ƒ ĉ-Ċ 88 11 77 ċ …‘’ƒ”‡†™‹–Š–Šƒ–ˆ”‘Š‡ƒŽ–Š›†‘‘”• Cancer thrombus yes 52 8 44 0.162 ȋα͵ͲǡδͲǤͲͷȌǤ no 57 15 42 ‡ ˆ—”–Š‡” ‹˜‡•–‹‰ƒ–‡† –Š‡ ‡š’”‡••‹‘ Ž‡˜‡Ž ‘ˆ •‘”†ͳͲͷ„ ‹ –Š‡ Š—ƒ  …‡ŽŽ Ž‹‡•  ǦͺͲ͵ǡ  Ǧͺʹ͵ǡ  Ǧ͹ͻͲͳǡ   ƒ†  Ǧʹ͹Ǥ ‘’ƒ”‡† –‘ Ǧ ͳǡ –Š‡ Ž‡˜‡Ž ‘ˆ •‘”†ͳͲͷ„ ™ƒ• ƒ”‡†Ž› ‹…”‡ƒ•‡† ‹ ƒŽŽ ͷ  …‡ŽŽ Ž‹‡• ȋ ‹‰Ǥ ͳ ƒ† ͳȌǤ Š‡•‡ ”‡•—Ž–• •—‰‰‡•–‡† –Šƒ– –Š‡ ‹…”‡ƒ•‡† ‡š’”‡••‹‘ ‘ˆ •‘”†ͳͲͷ„ ‹‰Š– „‡ ƒ ‰‡‡”ƒŽ characteristic of GC.

‘”†ͷͶͻ„’”‘‘–‡•–Š‡’”‘Ž‹ˆ‡”ƒ–‹‘ǡ‹‰”ƒ–‹‘ǡ‹˜ƒ•‹‘ƒ†…Ž‘‘‰‡‹…‹–›‘ˆ …‡ŽŽ• in vitro ‘…Žƒ”‹ˆ›–Š‡ˆ—…–‹‘‘ˆ•‘”†ͳͲͷ„‹ ǡ™‡–”ƒ•ˆ‡…–‡† ƒ† Ǧ͹ͻͲͳ…‡ŽŽ•™‹–Š •‘”†ͳͲͷ„•‹‘”‘˜‡”‡š’”‡••‹‘’Žƒ•‹†Ǥ‘’ƒ”‡†–‘–Š‡…‘–”‘Ž‰”‘—’ǡ–Š‡‡š’”‡••‹‘ ‘ˆ•‘”†ͳͲͷ„™ƒ••‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡†ǡ™‹–ŠƒŽ‘••‘ˆƒ’’”‘š‹ƒ–‡Ž›ͷͲΨ‹ Ǧ͹ͻͲͳ …‡ŽŽ•ƒ†ͶͲΨ‹ …‡ŽŽ•ǡ‘”ƒ”‡†Ž›‹…”‡ƒ•‡†„›ƒ’’”‘š‹ƒ–‡Ž›–™‘–‹‡•‹ Ǧ͹ͻͲͳ ƒ†ˆ‘—”–‹‡•‹ …‡ŽŽ•ǡƒ•‡ƒ•—”‡†„›“ǦƒƒŽ›•‹•ȋ ‹‰ǤʹȌǤ —”–Š‡”‘”‡ǡ ȋ ‹‰ǤʹȌǡ–”ƒ•™‡ŽŽȋ ‹‰Ǥʹƒ†ʹȌƒ†™‘—†ǦŠ‡ƒŽ‹‰ƒ••ƒ›•ȋ ‹‰ǤʹȌ†‡‘•–”ƒ–‡†–Šƒ– •‘”†ͳͲͷ„ ’”‘‘–‡†  …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ ‹‰”ƒ–‹‘ ƒ† ‹˜ƒ•‹‘ǡ ƒ† ‘…‹‰ †‘™ •‘”†ͳͲͷ„ ‹Š‹„‹–‡† –Š‡ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ ‹‰”ƒ–‹‘ ƒ† ‹˜ƒ•‹‘ ‘ˆ  …‡ŽŽ•Ǥ  ƒ††‹–‹‘ǡ –Š‡ •‘”†ͳͲͷ„Ǧ‘…†‘™  …‡ŽŽ• •Š‘™‡† ƒ †‡…”‡ƒ•‡† ƒ„‹Ž‹–› ˆ‘” …‘Ž‘› ˆ‘”ƒ–‹‘ǡ „—– –Š‡ •‘”†ͳͲͷ„Ǧ‘˜‡”‡š’”‡••‹‰  …‡ŽŽ• ˆ‘”‡† ‘”‡ …Ž‘‡• –Šƒ –Š‡ …‘–”‘Ž• ȋ ‹‰Ǥ ʹ ȌǤ Ž–‘‰‡–Š‡”ǡ –Š‡•‡ †ƒ–ƒ …Ž‡ƒ”Ž› †‡‘•–”ƒ–‡ –Šƒ– •‘”†ͳͲͷ„ ’Žƒ›• ƒ ˜‹–ƒŽ ’ƒ”– ‹  …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘ƒ†…Ž‘‘‰‡‹…‹–›Ǥ

‘”†ͷͶͻ„’”‘‘–‡•–Š‡‰”‘™–Š‘ˆ–”ƒ•’Žƒ–‡†–—‘”•‹˜‹˜‘ ‘ ˜ƒŽ‹†ƒ–‡ –Š‡ ‡ˆˆ‡…– ‘ˆ •‘”†ͳͲͷ„ ‘ –Š‡ –—‘”‹‰‡‡•‹• ‘ˆ  …‡ŽŽ• in vivoǡ ™‡ ‡•–ƒ„Ž‹•Š‡†•ŠǦ•‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳ…‡ŽŽ•ǡ™Š‹…ŠŠƒ˜‡•‘”†ͳͲͷ„•–ƒ„Ž›‘…‡††‘™ ‹ Ǧ͹ͻͲͳ…‡ŽŽ•ȋ ‹‰Ǥ͵Ǧ͵ȌǤŠ‡ǡ•ŠǦ•‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳ‘”•ŠǦ…Ǧ Ǧ͹ͻͲͳ …‡ŽŽ•™‡”‡‹Œ‡…–‡†‹–‘–Š‡ϐŽƒ•‘ˆ—†‡‹…‡–‘‡•–ƒ„Ž‹•Š–Š‡ƒ‹ƒŽ‘†‡Ž•Ǥˆ–‡”ʹͷ†ƒ›•ǡ –Š‡•‹œ‡ƒ†™‡‹‰Š–‘ˆ–Š‡•ŠǦ•‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳ…‡ŽŽ•‹–Š‡•—„…—–ƒ‡‘—•š‡‘‰”ƒˆ–• ™‡”‡ ‹˜‡•–‹‰ƒ–‡†Ǥ —” ”‡•—Ž–• †‡‘•–”ƒ–‡† –Šƒ– –Š‡ †‘™”‡‰—Žƒ–‹‘ ‘ˆ •‘”†ͳͲͷ„ •‹‰‹ϐ‹…ƒ–Ž›‹Š‹„‹–‡†–Š‡‰”‘™–Š‘ˆ–—‘”•…‘’ƒ”‡†™‹–Š–Š‡•ŠǦ……‡ŽŽ•ȋ ‹‰Ǥ͵Ǧ͵ ȌǤ ƒƒŽ›•‹• •Š‘™‡† –Šƒ– –Š‡ ‡š’”‡••‹‘ Ž‡˜‡Ž ‘ˆ •‘”†ͳͲͷ„ ‹ –—‘” –‹••—‡ ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› †‘™”‡‰—Žƒ–‡†…‘’ƒ”‡†™‹–Š–Šƒ–‹–Š‡•ŠǦ…‰”‘—’ȋ ‹‰Ǥ͵Ǧ͵ ȌǤ‘”‡‘˜‡”ǡŠ‹•–‘…Š‡‹…ƒŽ ƒƒŽ›•‹• …‘ϐ‹”‡† –Šƒ– –Š‡ ‡š’”‡••‹‘ Ž‡˜‡Ž• ‘ˆ „‘–Š –Š‡ …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ ƒ”‡” ‹Ǧ͸͹ ƒ†–Š‡ƒ”‡”•‹‡–‹ƒ†Ǧ…ƒ†Š‡”‹™‡”‡•‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡†‹–Š‡•ŠǦ •‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳš‡‘‰”ƒˆ–•…‘’ƒ”‡†™‹–Š–Š‡•ŠǦ…Ǧ Ǧ͹ͻͲͳš‡‘‰”ƒˆ–•ǡ„—––Š‡ ‡š’”‡••‹‘ ‘ˆ Ǧ…ƒ†Š‡”‹ ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› —’”‡‰—Žƒ–‡† ȋ ‹‰Ǥ ͵ Ǧ͵ȌǤ ƒ‡ –‘‰‡–Š‡”ǡ –Š‡•‡ †ƒ–ƒ‹†‹…ƒ–‡–Šƒ–‘…†‘™‘ˆ•‘”†ͳͲͷ„…ƒ‹Š‹„‹––—‘”‰”‘™–Šin vivo.

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Fig. 2. ‘”†ͳͲͷ„ ’”‘‘–‡† –Š‡ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ ‹‰”ƒ–‹‘ǡ ‹˜ƒ•‹‘ ƒ† …‘Ž‘› ˆ‘”ƒ–‹‘ ‘ˆ  …‡ŽŽ•Ǥ ȋȌ Š‡‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„™ƒ•†‡–‡…–‡†„›“Ǧ‹ Ǧ͹ͻͲͳ…‡ŽŽ•ƒ† …‡ŽŽ•–”ƒ•ˆ‡…–‡†™‹–Š –Š‡ •‹ ‘” ‘˜‡”‡š’”‡••‹‘ ’Žƒ•‹† ˆ‘” ʹͶ Šǡ ȗδͲǤͲͷǤ ȋȌ ”‘Ž‹ˆ‡”ƒ–‹‘ ‘ˆ  …‡ŽŽ• ™‹–Š •‘”†ͳͲͷ„ ‘…†‘™‘”‘˜‡”‡š’”‡••‹‘™ƒ•ƒ••‡••‡†„›–Š‡ƒ••ƒ›ǡȗδͲǤͲͷǤŠ‡‡ˆˆ‡…–‘ˆ•‘”†ͳͲͷ„‘…†‘™ ‘”‘˜‡”‡š’”‡••‹‘‘–Š‡‹‰”ƒ–‹‘ȋȌƒ†‹˜ƒ•‹‘ȋȌ‘ˆ …‡ŽŽ•™ƒ•‹˜‡•–‹‰ƒ–‡†—•‹‰–Š‡”ƒ•™‡ŽŽ ƒ† ƒ–”‹‰‡Ž ƒ••ƒ›ǡ ”‡•’‡…–‹˜‡Ž›Ǥ ˜‡”ƒ‰‡ …‘—–• ™‡”‡ …‘ŽŽ‡…–‡† ˆ”‘ ϐ‹˜‡ ”ƒ†‘ ‹…”‘•…‘’‹… ϐ‹‡Ž†•ǡ ȗδͲǤͲͷǤȋȌŠ‡™‘—†ǦŠ‡ƒŽ‹‰ƒ••ƒ›•Š‘™‡†–Š‡‡ˆˆ‡…–‘ˆ•‘”†ͳͲͷ„‘…†‘™‘”‘˜‡”‡š’”‡••‹‘‘…‡ŽŽ ‘„‹Ž‹–›ǡȗδͲǤͲͷǤȋ Ȍ‘…‹‰†‘™•‘”†ͳͲͷ„‹Š‹„‹–‡†–Š‡…‘Ž‘›Ǧˆ‘”ƒ–‹‘ƒ„‹Ž‹–›‘ˆ …‡ŽŽ•ǡ™Š‹Ž‡ ‘˜‡”‡š’”‡••‹‰•‘”†ͳͲͷ„’”‘‘–‡†–Š‹•ƒ„‹Ž‹–›ǤŠ‡—„‡”‘ˆ…‘Ž‘‹‡•™ƒ•…ƒŽ…—Žƒ–‡†ȋαͷȌƒ†’Ž‘––‡† ‘ƒŠ‹•–‘‰”ƒǡȗδͲǤͲͷǤ

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Fig. 3. ‘…‹‰ †‘™ •‘”†ͳͲͷ„ ‹Š‹„‹–‡†  …‡ŽŽ ‰”‘™–Š ‹ ˜‹˜‘Ǥ Š‡ •–ƒ„Ž‡ ƒ† Ž‘™Ǧ‡š’”‡••‹‘ ‘ˆ •‘”†ͳͲͷ„™ƒ•†‡–‡…–‡†‹ Ǧ͹ͻͲͳ …‡ŽŽ•—•‹‰‹—‘ϐŽ—‘”‡•…‡…‡‹…”‘•…‘’›ȋȌƒ†ȋǦȌ ƒˆ–‡” –”ƒ•ˆ‡…–‹‘ ™‹–Š ƒ Ž‡–‹˜‹”—• Šƒ”„‘”‹‰ –Š‡ ˆ—ŽŽǦŽ‡‰–Š Š—ƒ •‘”†ͳͲͷ„ •‡“—‡…‡ǡ ȗδͲǤͲͷ ȋǣ ‡‰ƒ–‹˜‡ …‘–”‘ŽȌǤ ȋǦȌ Š‡ ‰”‘™–Š ‘ˆ •ŠǦ•‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳ …‡ŽŽ• ȋŽ‡ˆ–Ȍ ƒ† •ŠǦ…Ǧ Ǧ͹ͻͲͳ …‡ŽŽ• ȋ”‹‰Š–Ȍ –Šƒ– ™‡”‡ ‹Œ‡…–‡† •—„…—–ƒ‡‘—•Ž› ‹–‘ —†‡ ‹…‡ ȋ α ͹ȌǤ ȋ Ȍ —‘” •‹œ‡ ƒ† ȋ Ȍ –—‘” ™‡‹‰Š– ™‡”‡‡ƒ•—”‡†ǤȋǦ ȌƒƒŽ›œ‡†–Š‡‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„‹–Š‡–‹••—‡•‘ˆ—†‡‹…‡ˆ”‘†‹ˆˆ‡”‡– ‰”‘—’•Ǥȋ ǦȌ‡’”‡•‡–ƒ–‹˜‡‹—‘Š‹•–‘…Š‡‹…ƒŽ‹ƒ‰‡••Š‘™‹‰–Š‡‹–‡•‹–›‘ˆ‹Ǧ͸͹•–ƒ‹‹‰ƒ†–Š‡ ‡š’”‡••‹‘‘ˆǦƒ••‘…‹ƒ–‡†’”‘–‡‹•ȋ„”‘™Ȍ‹–—‘”•ˆ”‘–Š‡–™‘‰”‘—’•Ǥ

‘”†ͷͶͻ„‹•ƒ••‘…‹ƒ–‡†™‹–Š–Š‡’”‘–‡‹ ‡…‡–Ž›ǡ •–—†‹‡• Šƒ˜‡ •Š‘™ –Šƒ– ‹–‡”ƒ…–‹‰ ™‹–Š ’”‘–‡‹• ‹• ‘‡ ‘ˆ –Š‡ ‹’‘”–ƒ– ‘Ž‡…—Žƒ” ‡…Šƒ‹•• ˆ‘” ‘…‘†‹‰ • –‘ ’‡”ˆ‘” –Š‡‹” ˆ—…–‹‘•Ǥ  „‹‘–‹ǦŽƒ„‡Ž‡† Š ƒ••ƒ›ǡˆ‘ŽŽ‘™‡†„›ƒ•••’‡…–”‘‡–”‹…ƒƒŽ›•‹•ǡ™ƒ•’‡”ˆ‘”‡†–‘‹†‡–‹ˆ›–Š‡ ’”‘–‡‹•–Šƒ–‹‰Š–‹–‡”ƒ…–™‹–Š•‘”†ͳͲͷ„Ǥ••Š‘™‹ ‹‰ǤͶȋ”‡†ƒ””‘™Ȍǡ‘‡’”‘–‡‹ „ƒ†™ƒ•ˆ‘—†–‘•’‡…‹ϐ‹…ƒŽŽ›‹–‡”ƒ…–™‹–Š„‹‘–‹›Žƒ–‡†•‘”†ͳͲͷ„ǤŠ‡„ƒ†™ƒ•–Š‡…—– ‘—–ǡ †‹‰‡•–‡† ™‹–Š –”›’•‹ ƒ† •—„Œ‡…–‡† –‘ ƒ•• •’‡…–”‘‡–”›Ǥ Š‡ ”‡•—Ž–• ”‡˜‡ƒŽ‡† –Šƒ– ƒ› ’”‘–‡‹• „‹† –‘ •‘”†ͳͲͷ„Ǥ ‘‰ –Š‡•‡ ’”‘–‡‹•ǡ  ƒ––”ƒ…–‡† ‘—” ƒ––‡–‹‘Ǥ ‡…ƒ—•‡ ‹– ™ƒ• ƒ ’”‘–‡‹ ™‹–Š ƒ Š‹‰Š ƒ„—†ƒ…‡ ‹ –Š‡ ƒ•• •’‡…–”‘‡–”› ƒ••ƒ›ǡ ƒ† ‹– Šƒ† „‡‡ …Ž‡ƒ”Ž› ƒ••‘…‹ƒ–‡† ™‹–Š –—‘” ‹˜ƒ•‹‘ ƒ† ‡–ƒ•–ƒ•‹•Ǥ ‡ ˆ—”–Š‡” ˜‡”‹ϐ‹‡† –Š‹•

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Fig. 4. ‘”†ͳͲͷ„ ™ƒ• ƒ••‘…‹ƒ–‡† ™‹–Š –Š‡  ’”‘–‡‹Ǥ ȋȌ †‡–‹ϐ‹…ƒ–‹‘ ‘ˆ –Š‡ ’”‘–‡‹• –Šƒ– ƒ••‘…‹ƒ–‡† ™‹–Š •‘”†ͳͲͷ„Ǥ ‘–ƒŽ ’”‘–‡‹ ™ƒ• ‡š–”ƒ…–‡† ˆ”‘  Ǧ͹ͻͲͳ …‡ŽŽ• ƒ† —–‹Ž‹œ‡† ‹ ƒ „‹‘–‹›Žƒ–‡†  Š  ƒ••ƒ›Ǥ Š‡ „ƒ†• •’‡…‹ϐ‹… –‘ •‘”†ͳͲͷ„ ™‡”‡ •—„Œ‡…–‡† –‘ ƒ•• •’‡…–”‘‡–”‹… ƒƒŽ›•‹•Ǥ ȋȌ ‡•–‡” „Ž‘– ƒƒŽ›•‹•‘ˆ–Š‡‹–‡”ƒ…–‹‘‘ˆ•‘”†ͳͲͷ„™‹–ŠǤ ȋǦȌ ‡š’‡”‹‡–•™‡”‡’‡”ˆ‘”‡†—•‹‰–Š‡ ‘” ‰ ƒ–‹„‘†›–‘’”‘„‡ …‡ŽŽ‡š–”ƒ…–•ǡ ƒ†–Š‡Ž‡˜‡Ž•‘ˆ–Š‡…‘Ǧ’”‡…‹’‹–ƒ–‡†•™‡”‡ †‡–‡”‹‡†„›ǡȗδͲǤͲͷǤ

Fig. 5.‘”†ͳͲͷ„–”ƒ’’‡† in the nucleus and promote tumori‰‡‡•‹•–Š”‘—‰Š–Š‡ȀǦ›… ’ƒ–Š™ƒ›Ǥ ȋȌ —‘ϐŽ—‘”‡•…‡…‡ •Š‘™‡† –Šƒ–  ™ƒ• ƒ‹Ž› Ž‘…ƒ–‡† ‹ —…Ž‡—•Ǥ ‘…†‘™ ‘ˆ •‘”†ͳͲͷ„ ‹Š‹„‹–‡† –Š‡ ‡š’”‡••‹‘ ‘ˆ Ǥ ȋǦȌ Š‡ ‡š’”‡••‹‘ ‘ˆ ǡ Ǧ›… ƒ† ʹ ‹  Ǧ͹ͻͲͳ …‡ŽŽ• ƒ†   …‡ŽŽ• –”ƒ•ˆ‡…–‡† ™‹–Š •‹• ‘” ‘˜‡”‡š’”‡••‹‘’Žƒ•‹†•ˆ‘”ͶͺŠ™‡”‡ ƒ••‡••‡† „› ™‡•–‡” „Ž‘–Ǥ ȋȌ Š‡ ‡š’”‡••‹‘‘ˆ‹–Š‡–‹••—‡• of nude mice. (E-F) Representa–‹˜‡ ‹—‘Š‹•–‘…Š‡‹…ƒŽ ‹ƒ‰‡• •Š‘™‹‰ –Š‡ ‡š’”‡••‹‘ Ž‡˜‡Ž• ‘ˆ  ƒ† Ǧ›… ‹ –—‘”• ˆ”‘ the two groups of nude mice.

‹–‡”ƒ…–‹‘ ‹†‡’‡†‡–Ž› „› ™‡•–‡” „Ž‘– ƒƒŽ›•‹•ǡ ƒ† –Š‡ ”‡•—Ž–• •Š‘™‡† –Šƒ–  ™ƒ• …Ž‡ƒ”Ž› †‡–‡…–‡† ‹ –Š‡ ’”‘–‡‹ …‘’Ž‡š‡• ‡š–”ƒ…–‡† „› •‘”†ͳͲͷ„ „—– ‘– ƒ–‹•‡•‡ •‘”†ͳͲͷ„ȋ ‹‰ǤͶȌǤ ƒ††‹–‹‘ǡƒ‹—‘’”‡…‹’‹–ƒ–‹‘ȋ Ȍƒ••ƒ›™ƒ•’‡”ˆ‘”‡† –‘ ˜ƒŽ‹†ƒ–‡ –Š‡ ‹–‡”ƒ…–‹‘ „‡–™‡‡ •‘”†ͳͲͷ„ ƒ† Ǥ • ‡š’‡…–‡†ǡ ™‡ ‘„•‡”˜‡† ƒ •‹‰‹ϐ‹…ƒ–‡”‹…Š‡–‘ˆ•‘”†ͳͲͷ„‹–Š‡ ”‡Žƒ–‹˜‡–‘–Š‡ ‰ …‘–”‘Ž ȋ ‹‰Ǥ Ͷƒ†ͶȌǡ•Š‘™‹‰–Šƒ–ƒ••‘…‹ƒ–‡†™‹–Š•‘”†ͳͲͷ„Ǥ

‘”†ͷͶͻ„ ƒ› ’Žƒ› ƒ ”‘Ž‡ ‹ ’”‘‘–‹‰ –—‘”‹‰‡‡•‹• –Š”‘—‰Š –Š‡ ȀǦ›… pathway  ȋŽ†‘Žƒ•‡ Ȍ ‹• …‘•‹†‡”‡† ƒ• ƒ …ƒ”…‹‘‰‡‹… ‰‡‡ ƒ† ‹• Š‹‰ŠŽ› ‡š’”‡••‡† ‹ ƒ›–—‘”•Ǥ”‡…‡–”‡’‘”–•Š‘™‡†–Šƒ–”‡‰—Žƒ–‡†Ǧ›…–‘’”‘‘–‡–Š‡‰”‘™–Š ƒ† ‡–ƒ•–ƒ•‹• ‘ˆ ’ƒ…”‡ƒ–‹… …ƒ…‡” …‡ŽŽ• ȏͳͲȐǤ ‘ ‹†‡–‹ˆ› –Š‡ ‡…Šƒ‹•• ƒ••‘…‹ƒ–‡† ™‹–Š •‘”†ͳͲͷ„ ‹  …‡ŽŽ•ǡ ™‡ –‡•–‡† –Š‡ ‡š’”‡••‹‘ Ž‡˜‡Ž• ‘ˆ  ƒ† Ǧ›…Ǥ Š‡ ‹—‘ϐŽ—‘”‡•…‡…‡ƒ””ƒ›•Š‘™‡†–Šƒ––Š‡™ƒ•Ž‘…ƒ–‡†‹–Š‡—…Ž‡—•ǡƒ†‘…‹‰ †‘™•‘”†ͳͲͷ„‹Š‹„‹–‡†–Š‡‡š’”‡••‹‘‘ˆȋ ‹‰ǤͷȌǤ —”–Š‡”‘”‡ǡƒ••Š‘™‹ ‹‰Ǥ ͷƒ†ͷǡ‘…‹‰†‘™•‘”†ͳͲͷ„‹Š‹„‹–‡†–Š‡‡š’”‡••‹‘‘ˆǡǦ›…ƒ†ʹǡ ƒ†‘˜‡”‡š’”‡••‹‰•‘”†ͳͲͷ„’”‘‘–‡†–Š‡‡š’”‡••‹‘‘ˆ–Š‡•ƒ‡–Š”‡‡’”‘–‡‹•‹ 

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

Fig. 6. ‘”†ͳͲͷ„ functioned through its association with Ǥ ȋȌ Š‡ ‡š’”‡••‹‘ ‘ˆ  ™ƒ• †‡–‡…–‡† „› “Ȃ ‹  cells transfected ™‹–Š –Š‡ ‘˜‡”‡špression plasmid  ˆ‘” ʹͶ Šǡ ȗδͲǤͲͷǤ ȋȌ Š‡ ‡š’”‡••‹‘ ‘ˆ  ™ƒ• †‡–‡…–‡† „› ™‡•–‡”„Ž‘– ‹ GC cells transfected ™‹–Š –Š‡ ‘˜‡”‡špression plasmid  ˆ‘” ʹͶ ŠǤ ȋȌ Š‡ ‡š’”‡••‹‘ ‘ˆ  ™ƒ• †‡–‡…–‡† „› “Ȃ in GC cells transfected with the •‘”†ͳͲͷ„ •‹ ‘” ‘˜‡”‡š’”‡••‹‘ ’Žƒ•‹†•‘”†ͳͲͷ„ ˆ‘” ʹͶ Šǡ ȗδͲǤͲͷǤ ȋȌ ”‘Ž‹ˆ‡”ƒ–‹‘ ‘ˆ GC cells with snor†ͳͲͷ„ ‘…†‘™ ƒ†  ‘˜‡”expression was as•‡••‡† „› –Š‡  ƒ••ƒ›ǡ ȗδͲǤͲͷǤ Š‡ ‡ˆˆ‡…–‘ˆ•‘”†ͳͲͷ„ ‘…†‘™ ƒ† ‘˜‡”‡š’”‡•sion on the migration (E and F) and ‹˜ƒ•‹‘ ȋ  ƒ† Ȍ ‘ˆ  …‡ŽŽ• ™ƒ• ‹˜‡•–‹‰ƒ–‡† —•‹‰ –Š‡”ƒ•™‡ŽŽƒ†ƒ–”‹‰‡Žƒ••ƒ›ǡ”‡•’‡…–‹˜‡Ž›Ǥ˜‡”ƒ‰‡…‘—–•™‡”‡…‘ŽŽ‡…–‡†ˆ”‘ϐ‹˜‡”ƒ†‘‹…”‘•…‘’‹… ϐ‹‡Ž†•ǡȗδͲǤͲͷǤȋ ƒ† ȌŠ‡™‘—†ǦŠ‡ƒŽ‹‰ƒ••ƒ›•Š‘™‡†–Š‡‡ˆˆ‡…–‘ˆ•‘”†ͳͲͷ„‘…†‘™ƒ† ‘˜‡”‡š’”‡••‹‘‘…‡ŽŽ‘„‹Ž‹–›ǡȗδͲǤͲͷǤȋȌ‡•–‡”„Ž‘–ƒ••ƒ›•Š‘™‡†–Š‡‡š’”‡••‹‘‘ˆǦ›…‹–Š‡  …‡ŽŽ•–”ƒ•ˆ‡…–‡†™‹–Š•‘”†ͳͲͷ„‘…†‘™ƒ†‘˜‡”‡š’”‡••‹‘ǤȋȌŠ‡•…Š‡ƒ‘ˆ–Š‡‡…Šƒ‹•‘ˆ•‘”†ͳͲͷ„Ǥ

…‡ŽŽŽ‹‡•Ǥ‡•–‡”„Ž‘––‹‰ƒ†Š‹•–‘…Š‡‹…ƒŽƒƒŽ›•‹•‘ˆƒ‹ƒŽ–‹••—‡•ˆ—”–Š‡”…‘ϐ‹”‡† –Šƒ––Š‡‡š’”‡••‹‘Ž‡˜‡Ž•‘ˆƒ†Ǧ›…™‡”‡•‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡†‹–Š‡•ŠǦ •‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳš‡‘‰”ƒˆ–•…‘’ƒ”‡†™‹–Š–Š‡•ŠǦ…Ǧ Ǧ͹ͻͲͳš‡‘‰”ƒˆ–•ȋ ‹‰Ǥ ͷǦͷ ȌǤŠ‡”‡•—Ž–•†‘…—‡–‡†–Šƒ–•‘”†ͳͲͷ„‹‰Š–ƒˆˆ‡…––Š‡†‡˜‡Ž‘’‡–‘ˆ –Š”‘—‰Š –Š‡ȀǦ›…’ƒ–Š™ƒ›Ǥ

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

‘”†ͷͶͻ„ˆ—…–‹‘‡†–Š”‘—‰Š‹–•ƒ••‘…‹ƒ–‹‘™‹–Š ‘‡š’Ž‘”‡™Š‡–Š‡”–Š‡•‘”†ͳͲͷ„‡†‹ƒ–‡†„‹‘Ž‘‰›ˆ—…–‹‘‹ …‡ŽŽ•†‡’‡†‡†‘ ‹–•ƒ••‘…‹ƒ–‹‘™‹–Šǡ™‡…‘†—…–‡† Ǧ͹ͻͲͳƒ† ™‹–Š‘˜‡”‡š’”‡••‹‘ ȋ ‹‰Ǥ ͸ȌǤ Š‡ ”‡•—Ž–• ™‡”‡ …‘ϐ‹”‡† „› ™‡•–‡”Ǧ„Ž‘– ȋ ‹‰Ǥ ͸ȌǤ Š”‘—‰Š “ ƒ””ƒ›ǡ ™‡ ˆ‘—†•‘”†ͳͲͷ„Šƒ†‘‡ˆˆ‡…–‘‡š’”‡••‹‘ȋ ‹‰Ǥ͸ȌǤ‡–Š‡…Š‡…‡†‹ˆ•‘”†ͳͲͷ„ ˆ—…–‹‘•–Š”‘—‰Š‘”‘–Ǥ‘…‹‰†‘™•‘”†ͳͲͷ„•—’’”‡••‡†–Š‡‰”‘™–Šȋ ‹‰Ǥ ͸Ȍǡ ‹‰”ƒ–‹‘ ƒ† ‹˜ƒ•‹‘ ‘ˆ  …‡ŽŽ•ǡ ™Š‹Ž‡ Šƒ† ‘ ‡ˆˆ‡…– ‹  —’”‡‰—Žƒ–‡† …‡ŽŽ• ȋ ‹‰Ǥ͸Ǧ͸ ȌǤ —”–Š‡”‘”‡ǡ–Š‡‹Š‹„‹–‡†‘…‘‰‡‡Ǧ›…‘ˆ–Š‡•‘”†ͳͲͷ„Ǧ‘…†‘™  …‡ŽŽ•™ƒ•”‡•…—‡†„›–Š‡—’Ǧ”‡‰—Žƒ–‡†‡š’”‡••‹‘‘ˆȋ ‹‰Ǥ͸ȌǤ„‘˜‡ƒŽŽǡ–Š‡•‡†ƒ–ƒ •—‰‰‡•–‡†–Šƒ––Š‡‡…Šƒ‹•„›™Š‹…Š•‘”†ͳͲͷ„‡š‡”–‡†‹–•ˆ—…–‹‘‹ …‡ŽŽ•‹‰Š– „‡ †—‡ –‘ –Š‡ ‡š‹•–‡…‡ ‘ˆ ƒ •‘”†ͳͲͷ„Ȁ ƒ••‘…‹ƒ–‹‘ ƒš‹•ǡ ƒ† –Š‡ •…Š‡ƒ ˆ‘” –Š‹• ‡…Šƒ‹•™ƒ••Š‘™‹ ‹‰Ǥ͸Ǥ Discussion

‹•‘‡–Š‡‘•–ˆ”‡“—‡–…ƒ—•‡•‘ˆ…ƒ…‡”Ǧ”‡Žƒ–‡††‡ƒ–ŠǤŠ‡—†‡”•–ƒ†‹‰‘ˆ  ’ƒ–Š‘‰‡‡•‹•‹˜‘Ž˜‡•ƒ›—–ƒ–‹‘•ƒ†ƒ’Ž‹ϐ‹…ƒ–‹‘•‘ˆ–—‘”•—’’”‡••‘”‰‡‡•ƒ† ‘…‘‰‡‡•ȏͳͳǦͳ͵ȐǤ‘™‡˜‡”ǡ–Š‡‡…Šƒ‹•‘ˆ †‡˜‡Ž‘’‡–”‡ƒ‹•Žƒ”‰‡Ž›—‘™Ǥ ……——Žƒ–‹‰•–—†‹‡•ƒ”‡‹˜‡•–‹‰ƒ–‹‰•‘•ǡ™Š‹…Šƒ”‡ƒ‹†‘ˆ‘…‘†‹‰Ǥ ‡•’‹–‡–Š‡…‘‘ƒ••—’–‹‘–Šƒ–•‘•Šƒ˜‡‘Ž›…‡ŽŽ—Žƒ”Š‘—•‡‡‡’‹‰ˆ—…–‹‘•ǡ‹ –Š‡’ƒ•–ˆ‡™›‡ƒ”•ǡ‹†‡’‡†‡–”‡’‘”–•—•‹‰†‹˜‡”•‡•–”ƒ–‡‰‹‡•Šƒ˜‡ˆ‘…—•‡†‘–Š‡˜ƒ”‹‘—• ”‘Ž‡•‘ˆ•‘•‹–Š‡…‘–”‘Ž‘ˆ„‹‘Ž‘‰‹…ƒŽ’”‘…‡••ǡ‡•’‡…‹ƒŽŽ›‹–Š‡‘…‘‰‡‡•‹•ǡ•—…Šƒ• „”‡ƒ•–…ƒ…‡”ǡ’”‘•–ƒ–‡…ƒ…‡”ǡŠ‡’ƒ–‘…‡ŽŽ—Žƒ”…ƒ”…‹‘ƒǡ…‘Ž‘”‡…–ƒŽ…ƒ…‡”ƒ†Ž—‰…ƒ…‡” ȏͳͶǦͳͺȐǤ –Š‡” •–—†‹‡• Šƒ˜‡ •—’’‘”–‡† –Š‡ …‘…‡’– –Šƒ– •‘• Šƒ˜‡ ™‹†‡” ˆ—…–‹‘•Ǥ ‘•‹‰Š–ƒŽ•‘„‡‹˜‘Ž˜‡†‹–Š‡”‡‰—Žƒ–‹‘‘ˆ‰‡‡‡š’”‡••‹‘„›‰‹˜‹‰”‹•‡–‘‘–Š‡” ”‡‰—Žƒ–‘”›•’‡…‹‡•ǡ•—…Šƒ•‹•ȏͳͻǦʹͳȐǤ ”‡…‡–›‡ƒ”•ǡ‘”‡ƒ†‘”‡•–—†‹‡•ƒ”‡ …‘˜‡”‰‹‰‘–Š‡ϐ‹‡Ž†‘ˆ•‘•ǡƒ†–Š‡•’‡…‹ϐ‹…‡…Šƒ‹••‘ˆ•‘•”‡“—‹”‡ˆ—”–Š‡” •–—†›Ǥ –Š‹•ƒ”–‹…Ž‡ǡ™‡…‘ϐ‹”‡†–Šƒ––Š‡‡š’”‡••‹‘‘ˆ•‘”†ͳͲͷ„‹•‘„˜‹‘—•Ž›—’Ǧ”‡‰—Žƒ–‡† ‹  –‹••—‡•ǡ •‡”ƒ ƒ† …‡ŽŽ Ž‹‡• …‘’ƒ”‡† ™‹–Š ‘”ƒŽ –‹••—‡•ǡ Š‡ƒŽ–Š› †‘‘”• ƒ† ‹‘”–ƒŽ‹œ‡† ‰ƒ•–”‹… ‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ Ž‹‡ǡ ”‡•’‡…–‹˜‡Ž›Ǥ —”–Š‡”‘”‡ǡ …Ž‹‹…ƒŽ †ƒ–ƒ ‹†‹…ƒ–‡† –Šƒ– –Š‡ ‡š’”‡••‹‘ ‘ˆ •‘”†ͳͲͷ„ ™ƒ• ’‘•‹–‹˜‡Ž› …‘””‡Žƒ–‡† ™‹–Š –—‘” •‹œ‡ǡ •–ƒ‰‡ ƒ† †‹ˆˆ‡”‡–‹ƒ–‹‘Ǥ ‘…†‘™ ‘ˆ •‘”†ͳͲͷ„ †‡…”‡ƒ•‡† –—‘” …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ǡ ‹‰”ƒ–‹‘ǡ ‹˜ƒ•‹‘ƒ†…‘Ž‘›ˆ‘”ƒ–‹‘‹˜‹–”‘ƒ†Š‹†‡”‡†–Š‡‰”‘™–Š‘ˆš‡‘‰”ƒˆ–•‹ —†‡ ‹…‡ ‘†‡Ž•ǡ ƒ† –Š‡ ‘˜‡”‡š’”‡••‹‘ ‘ˆ •‘”†ͳͲͷ„ ’”‘‘–‡† …‡ŽŽ ‰”‘™–Šǡ ‹‰”ƒ–‹‘ ƒ† ‹˜ƒ•‹‘‹˜‹–”‘ǤŠ‡•‡”‡•—Ž–••—’’‘”–‡†–Š‡’ƒ”–‹…‹’ƒ–‹‘‘ˆ•‘”†ͳͲͷ„‹–Š‡†‡˜‡Ž‘’‡– of GC. ǡ™Š‹…Š‹•ƒŠ‹‰ŠŽ›…‘•‡”˜‡†‰‡‡ƒ‘‰†‹ˆˆ‡”‡–•’‡…‹‡•ƒ†ƒ™‡ŽŽǦ•–—†‹‡† ‡„‡”‘ˆ–Š‡ƒŽ†‘Žƒ•‡ˆƒ‹Ž›ǡŠƒ•„‡‡”‡’‘”–‡†–‘„‡—’”‡‰—Žƒ–‡†‹˜ƒ”‹‘—•–›’‡•‘ˆ–—‘” …‡ŽŽ•ǡ‹…Ž—†‹‰‘”ƒŽ•“—ƒ‘—•…‡ŽŽ…ƒ”…‹‘ƒǡ‘•–‡‘•ƒ”…‘ƒǡŠ‡’ƒ–‘…‡ŽŽ—Žƒ”…ƒ”…‹‘ƒƒ† Ž—‰…ƒ…‡”ȏʹʹǦʹ͸ȐǤ—‡–ƒŽǤˆ‘—†–Šƒ–‹‰Š–’”‘‘–‡–Š‡’”‘‰”‡••‹‘‘ˆŽ—‰…ƒ…‡” –Š”‘—‰Šȏʹ͹ȐǤ ‹‡–ƒŽǤ”‡’‘”–‡†–Šƒ–”‡‰—Žƒ–‡•Ǧ›…–‘’”‘‘–‡–Š‡‰”‘™–Šƒ† metastasis of pancreatic cancer cellsȏͳͲȐǤ ‹”•–ǡ™‡†‡–‡…–‡†–Š‡‡š’”‡••‹‘Ž‡˜‡Ž‘ˆ „›™‡•–‡”„Ž‘––‹‰ǤŠ‡”‡•—Ž–••Š‘™‡†–Šƒ––Š‡‡š’”‡••‹‘‘ˆ™ƒ•†‡…”‡ƒ•‡†ƒˆ–‡” ‘…‹‰†‘™•‘”†ͳͲͷ„ǡƒ†™ƒ•‹…”‡ƒ•‡†„›—’Ǧ”‡‰—Žƒ–‹‰‘ˆ•‘”†ͳͲͷ„ǤŠ‡ǡ™‡ƒŽ•‘ ‹˜‡•–‹‰ƒ–‡† –Š‡  ƒ”‡”• ‹ –Š‹• •–—†›Ǥ —‘Š‹•–‘…Š‡‹…ƒŽ ƒ••ƒ›• •Š‘™‡† ƒ —’Ǧ ”‡‰—Žƒ–‹‘‘ˆ‡’‹–Š‡Ž‹ƒŽƒ”‡”•ȋǦƒ†Š‡”‹Ȍƒ†ƒ†‘™”‡‰—Žƒ–‹‘‘ˆ‡•‡…Š›ƒŽƒ”‡”• ȋǦ…ƒ†Š‡”‹ ƒ† ‹‡–‹Ȍ ‹ –Š‡ –‹••—‡• ‘ˆ —†‡ ‹…‡ ™‹–Š •ŠǦ•‘”†ͳͲͷ„Ǧ Ǧ͹ͻͲͳ …‡ŽŽ š‡‘‰”ƒˆ–•ǡ•—‰‰‡•–‹‰–Šƒ–•‘”†ͳͲͷ„‹‰Š–ƒˆˆ‡…–•–ƒ–—•„›„‹†‹‰–‘ǤǦ›… ‹•ƒ™‡ŽŽǦ‘™‘…‘‰‡‡Ǥ‡š–ǡ™‡–‡•–‡†–Š‡‡š’”‡••‹‘‘ˆǦ›…„›™‡•–‡”„Ž‘––‹‰ƒ† –Š‡”‡•—Ž–••Š‘™‡†–Šƒ–‘…‹‰†‘™•‘”†ͳͲͷ„ǡ–Š‡‡š’”‡••‹‘‘ˆǦ›…™ƒ•†‡…”‡ƒ•‡†ǡ ™Š‹Ž‡ —’”‡‰—Žƒ–‹‰ ‘ˆ •‘”†ͳͲͷ„ǡ –Š‡ ‡š’”‡••‹‘ ‘ˆ Ǧ›… ™ƒ• ‹…”‡ƒ•‡†Ǥ ƒ‡ –‘‰‡–Š‡”ǡ –Š‡•‡ ”‡•—Ž–• •—‰‰‡•–‡† –Šƒ– •‘”†ͳͲͷ„ ‹‰Š– ‹ϐŽ—‡…‡ –Š‡ ‰”‘™–Š ƒ† ‡–ƒ•–ƒ•‹• ‘ˆ 

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

…‡ŽŽ•–Š”‘—‰Š–Š‡ȀǦ›…’ƒ–Š™ƒ›ǡƒ†•‘”†ͳͲͷ„‹‰Š–„‡ƒ’‘–‡–‹ƒŽ–Š‡”ƒ’‡—–‹… target for GC. Acknowledgements

Š‹•™‘”™ƒ•’ƒ”–‹ƒŽŽ›•—’’‘”–‡†„›–Š‡ƒ–—”ƒŽ…‹‡…‡ ‘—†ƒ–‹‘‘ˆŠ‹ƒȋ ”ƒ– ‘Ǥ ͺͳ͸͹͵ͳͶʹǡ ͺͳͷͲʹͲ͵ʹǡ ͺͳ͹͹ʹͷͷͲȌǡ –Š‡ ƒ–—”ƒŽ …‹‡…‡ ‘—†ƒ–‹‘ ‘ˆ Š‹ƒ ‘ˆ ‡„‡‹ ”‘˜‹…‡ȋ ”ƒ–ͲǤʹͲͳͷʹͲ͸͵͹͸ȌǤ ǤǤƒ†Ǥ…ƒ””‹†‘—––Š‹••–—†›ǡƒƒŽ›œ‡††ƒ–ƒǡƒ†‹–‡”’”‡–‡†ƒ—•…”‹’–ǤǤǤƒ†

ǤǤ…‘ŽŽ‡…–‡†ƒ†ƒƒŽ›œ‡††ƒ–ƒǤǤƒ†Ǥ…‘ŽŽ‡…–‡††ƒ–ƒƒ†™”‘–‡ƒ—•…”‹’–ǢǤ †‡•‹‰‡† –Š‹• ƒ”–‹…Ž‡ ƒ† ‹–‡”’”‡–‡† ƒ—•…”‹’–ǡ ƒ† ϐ‹ƒŽ ƒ’’”‘˜ƒŽ ‘ˆ ƒ—•…”‹’–Ǥ ŽŽ ƒ—–Š‘”•”‡˜‹‡™‡†–Š‡ƒ—•…”‹’–Ǥ Disclosure Statement

Š‡ƒ—–Š‘”•Šƒ˜‡†‡…Žƒ”‡†–Šƒ–‘…‘ϐŽ‹…–‘ˆ‹–‡”‡•–‡š‹•–•Ǥ References ͳ ʹ ͵ Ͷ

ͷ

͸ ͹

ͺ ͻ

ͳͲ ͳͳ

ͳʹ

ͳ͵

ͳͶ ͳͷ

ƒ‘ǣ ƒ•–”‹……ƒ…‡”ǣ•‹ƒƒ†–Š‡™‘”Ž†Ǥ ƒ•–”‹…ƒ…‡”ǡʹͲͳ͹ǢʹͲǣͳǦʹǤ ‹‡ǡ—ǡ— ǡ‹ƒ‰ǡƒ‹ǡŠƒ‰ǡŠ‘— ǡƒǡ—ǡ ƒ‰ ǡ—ƒǡ‘—ǡ ƒǣ‰Ž‘„ƒŽ„—”†‡ ‘ˆ‰ƒ•–”‹……ƒ…‡”ǣ–Š‡ƒŒ‘”‹’ƒ…–‘ˆŠ‹ƒǤš’‡”–‡˜ ƒ•–”‘‡–ʹͲͳ͹Ǣͳͳǣ͸ͷͳǦ͸͸ͳǤ ƒ‘‘”ǡ‹ƒ‘ ǡ ‹ƒ‰ ǣƒŽŽ—…Ž‡‘Žƒ”•‹…ƒ…‡”Ǥ‹‘…Š‹‹‘’Š›•…–ƒʹͲͳʹǢͳͺʹ͸ǣͳʹͳǦͳʹͺǤ ‹ŽŽ‹ƒ• ǡ ƒ”œƒ‡Š ǣ”‡•‘•ƒ†•‘Š‘•–‰‡‡•‡™’Žƒ›‡”•‹…ƒ…‡”ǫƒ–‡˜ƒ…‡” ʹͲͳʹǢͳʹǣͺͶǦͺͺǤ ‘ƒ‘ ǡ‡‡œ‹ƒ‘ǡ…—œ‘ǡ”‘…‡ǣƒŽŽ‘Ǧ…‘†‹‰ƒ†…ƒ…‡”Ǥƒ”…‹‘‰‡‡•‹• ʹͲͳ͹Ǣ͵ͺǣͶͺͷǦͶͻͳǤ ŠƒŽƒŒǡƒ”ǣ•‘•…‘–”‹„—–‡–‘›‡Ž‘‹†Ž‡—ƒ‡‘‰‡‡•‹•Ǥƒ–‡ŽŽ‹‘ʹͲͳ͹Ǣͳͻǣ͹ͷͺǦ͹͸ͲǤ —ƒ‰ǡŠ‹ ǡ —‘ǡ—ƒ‰ǡ—ƒ‰ǡ‹‰ǡ—ǡŠƒ‰ǡ‹‰ ǡŠƒ‘ǡ ‹ƒ ǡ—ƒ‰ǡ‹ƒ‰ǡŠ‹ ǡƒ‘ǣ•‘‘†—Žƒ–‡•͵ǯ‡†’”‘…‡••‹‰ƒ†”‡‰—Žƒ–‡•–Š‡‡š’”‡••‹‘‘ˆƒ•—„•‡–‘ˆ•Ǥ —…Ž‡‹……‹†•‡•ʹͲͳ͹ǢͶͷǣͺ͸Ͷ͹Ǧͺ͸͸ͲǤ ƒ‰ ǡŠƒ‰ǡ—ƒ‰ǡŠ‘—ǡ—ƒ‰ǡŠƒ‰ǡŠ‡ǡ—ǣ•‘‡‡‡”ǣƒƒ†˜ƒ…‡† …‘’—–ƒ–‹‘ƒŽ’ƒ…ƒ‰‡ˆ‘”•…”‡‡‹‰‘ˆ‰—‹†‡ƒ†‘”’Šƒ•‘‰‡‡•‹–Š‡Š—ƒ‰‡‘‡Ǥ—…Ž‡‹… …‹†•‡•ʹͲͲ͸Ǣ͵ͶǣͷͳͳʹǦͷͳʹ͵Ǥ ϐ‹•–‡”ǡ‡‹Žǡ—ŠŽǣŠ‡–ƒ”‰‡–”‘–‡‹‡–‡”ƒ‡ϐ‹‡•ƒ‘˜‡Ž’ͷ͵Ǧ‹†‡’‡†‡–—…Ž‡‘Žƒ” –”‡••Ǧ‡•’‘•‡ƒ–Š™ƒ›Ǥ ‹‘Š‡ʹͲͳͷǢʹͻͲǣͳͲͻͲͷǦͳͲͻͳͺǤ

‹ǡŠƒ‰ǡ‹— ǡ‹ǡ‹ƒ‰ǡŠ‹ǡ ‹ǡ‹ƒ‰ǡ—ǡ—ǡƒ‰ǡ—ǡ‹—ǡ‹—ǡ—

ǡ‹ǡ— ǣˆ—…–‹‘•ƒ•ƒ‘…‘‰‡‡‹–Š‡Š‹‰ŠŽ›‡–ƒ•–ƒ–‹…’ƒ…”‡ƒ–‹……ƒ…‡”Ǥƒ…‡”‡–– ʹͲͳ͸Ǣ͵͹Ͷǣͳʹ͹Ǧͳ͵ͷǤ ƒ‰ǡ‹—ǡŠƒ‘ǣ—–ƒ–‹‘ƒŽƒƒŽ›•‹•‘ˆ†”‹˜‡”‰‡‡•™‹–Š–—‘”•—’’”‡••‹˜‡ƒ†‘…‘‰‡‹…”‘Ž‡•‹ ‰ƒ•–”‹……ƒ…‡”Ǥ‡‡” ʹͲͳ͹Ǣͷǣ‡͵ͷͺͷǤ ‹ŒŽ•ƒ ǡƒ†ƒƒ†ƒǡƒǡ‡”‡—Ž‡ǣ‘Ž‡…—Žƒ”•—„–›’‡•‹…ƒ…‡”•‘ˆ–Š‡‰ƒ•–”‘‹–‡•–‹ƒŽ–”ƒ…–Ǥ ƒ–‡˜ ƒ•–”‘‡’ƒ–ʹͲͳ͹ǢͳͶǣ͵͵͵Ǧ͵ͶʹǤ ‹‡Ž‹•‹ǡ —‘ ǡ‡›‡”•‘ǣ •‡”–‹‘•ƒ†‡Ž‡–‹‘•ƒ”‰‡–‹‡ƒ‰‡Ǧ‡ϐ‹‹‰ ‡‡•‹—ƒ ƒ…‡”•Ǥ‡ŽŽʹͲͳ͹Ǣͳ͸ͺǣͶ͸ͲǦͶ͹ʹ‡ͶͳͶǤ ‘‰ǡ‘†”‹‰—‡œǡ —‘ǡ—ǡƒŽ„‘– ǡŠ‘—ǡ‡–”‘• ǡ‹ǡ‡••‡ŽŽƒǡ‹„‡Žǡ–‡˜‡•ǡ ƒŽŽ‡ǡ‘‰ ǣ‘ͷͲ‹•ƒ…ƒ†‹†ƒ–‡–—‘”Ǧ•—’’”‡••‘”‰‡‡ƒ–͸“ͳͶǤ͵™‹–Šƒ—–ƒ–‹‘ƒ••‘…‹ƒ–‡† ™‹–Š…Ž‹‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–’”‘•–ƒ–‡…ƒ…‡”Ǥ—‘Ž ‡‡–ʹͲͲͺǢͳ͹ǣͳͲ͵ͳǦͳͲͶʹǤ ‘‰ǡ —‘ǡ‘›† ǡ—ǡ‹ǡŠ‘—ǡ‘‰ ǣ ’Ž‹…ƒ–‹‘‘ˆ•‘ͷͲ‹Š—ƒ„”‡ƒ•–…ƒ…‡”Ǥ

 ‡‡– ‡‘‹…•ʹͲͲͻǢ͵͸ǣͶͶ͹ǦͶͷͶǤ

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Physiol Biochem 2018;45:2471-2482 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000488265 and Biochemistry Published online: March 19, 2018 www.karger.com/cpb Zhang et al.: Snord105b Promotes Tumorigenesis in Gastric Cancer

ͳ͸

ͳ͹

ͳͺ

ͳͻ ʹͲ ʹͳ ʹʹ

ʹ͵ ʹͶ ʹͷ ʹ͸ ʹ͹

—ǡŠƒ‰ǡƒ‰ǡƒǡ‡‰ǡ—ƒǣŽ‹‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡‘ˆȀ„‘š•ƒŽŽ—…Ž‡‘Žƒ”͹͸ƒ• ƒ‘…‘‰‡‡ƒ†ƒ’”‘‰‘•–‹…„‹‘ƒ”‡”‹Š‡’ƒ–‘…‡ŽŽ—Žƒ”…ƒ”…‹‘ƒǤŽ‹‡•‡’ƒ–‘Ž ƒ•–”‘‡–‡”‘ŽʹͲͳ͹ ‘•Š‹†ƒǡ‘†‡ǡ‡‰ǡŠ‹‰‡›ƒ•—ǡ‹›‘•Š‹ ǡ—”‡” ǡƒ‰ƒ•ƒƒǡƒǡƒƒ›ƒƒǡ —Œ‹™ƒ”ƒǡ

‘‡ŽǣʹͳǦ…‘‰‡‹…ƒŽŽ—…Ž‡‘Žƒ”ǡ™‹–Šƒ”‘‰‘•–‹…‹‘ƒ”‡”‘–‡–‹ƒŽ‹—ƒ ‘Ž‘”‡…–ƒŽƒ…‡”Ǥ‹‘‡†‹…‹‡ʹͲͳ͹Ǣʹʹǣ͸ͺǦ͹͹Ǥ ‡‹ǡ‹ƒ‘ ǡŠ‡ ǡ—ǡ‹—ǡ‹—ǡ‹ǡ ‹ǡ‘”•‡› ǡ ‹ƒ‰ǡƒ–œǡƒ‰ ǡ ‹ƒ‰ ǣƒŽŽ —…Ž‡‘Žƒ”Ͷʹƒ…–•ƒ•ƒ‘…‘‰‡‡‹Ž—‰–—‘”‹‰‡‡•‹•Ǥ…‘‰‡‡ʹͲͳʹǢ͵ͳǣʹ͹ͻͶǦʹͺͲͶǤ ƒ––‡”•‘ ǡ‘„‡”–• ǡ‹‰ǡ‘—•‡”‘˜ƒǡƒ”Š‹ŽŽǡ”—…‡ŽŽ‘ǡ‘Ž•ƒ ǡ”ƒ–Ž‡›ǡƒ—ˆƒ

ǡ‰—›‡ǡƒ–ƒƒǡ…Š‹ŽŽ‡” ǡ’‹……‹ƒ‹ ǡƒ’ƒ–ƒǡ‹ŽŽ‡”ǡŠ‡”ƒǡƒǡŠƒ‘ǡ ”‘”ƒǡ‘Ž‡›ǡ‡‹†ƒǡƒǡ‹ǡ‘”…Š‡”– ǣ—ƒ•‘Ǧͻ͵‹•’”‘…‡••‡†‹–‘ƒ‹…”‘Ǧ Ž‹‡–Šƒ–’”‘‘–‡•„”‡ƒ•–…ƒ…‡”…‡ŽŽ‹˜ƒ•‹‘Ǥ ”‡ƒ•–ƒ…‡”ʹͲͳ͹Ǣ͵ǣʹͷǤ ƒ”–‡•Ǧœ—‘˜ƒǡŽ˜‡†›ǡ ‡•–‡” ǣ‡›‘†‹…”‘Ǧ‘˜‡Ž•†‡”‹˜‡†ˆ”‘•ƒŽŽ‘Ǧ…‘†‹‰ ƒ†–Š‡‹”‹’Ž‹…ƒ–‹‘‹…ƒ…‡”Ǥƒ…‡”‡––ʹͲͳ͵Ǣ͵ͶͲǣʹͲͳǦʹͳͳǤ †‡”ǡ”‡ǡ ”‹‡†Žƒ†‡”ǡ‡‹–œ‹‰‡”ǡ‡‹ƒǡŠ‡ǡˆ‡ˆˆ‡”ǡƒŒ‡™•›ǡ‡‹•–‡” ǣ —ƒ•‘™‹–Š‹…”‘Ǧ‹‡ —…–‹‘•Ǥ‘Ž‡ŽŽʹͲͲͺǢ͵ʹǣͷͳͻǦͷʹͺǤ Šƒ‰ǡŠƒǡŠƒ‰ǡ‹ ǡƒ‰ ǡ—ǡ—ƒ‰ǡ—ǡ•‹ƒ‘ǣ ‡‡†„ƒ…”‡‰—Žƒ–‹‘‘ˆ ƒ…–‹˜ƒ–‡•–Š‡ ǦͳƒŽ’ŠƒȀͻƒš‹•–‘’”‘‘–‡Ž—‰…ƒ…‡”’”‘‰”‡••‹‘Ǥƒ…‡”Ž‡––ʹͲͳ͹ǢͶͲ͵ǣʹͺǦ ͵͸Ǥ ‘”‹•ƒ‹ǡƒ•Š‹”‘ǡƒ‡Šƒ•Š‹ǡ ƒ‰ƒ‹ǡ‹‘•Š‹–ƒǡ ——‘ƒǡƒ•ƒ•Š‹ƒǡƒ•—†ƒ ǡƒ—”ƒ‹ ǡ—„‘ǡ—‰—”—ƒǡŠ‹”ƒǡƒ‹„—…Š‹ǡ‹”ƒƒ™ƒǣ‘’ƒ”ƒ–‹˜‡’”‘–‡‘‹…•ƒƒŽ›•‹•‘ˆ‰ƒ•–”‹… …ƒ…‡”•–‡…‡ŽŽ•ǤŽ‘‘‡ʹͲͳͶǢͻǣ‡ͳͳͲ͹͵͸Ǥ ƒǡ‹ǡ—ǡ‹ƒ‘ǡ‘‰ǡ—ƒ‰ǡ ‹ƒ‰ǣ‹‰Š‡š’”‡••‹‘‘ˆŽ†‘Žƒ•‡’”‡†‹…–•’‘‘”•—”˜‹˜ƒŽ‹ ’ƒ–‹‡–•™‹–Š…Ž‡ƒ”Ǧ…‡ŽŽ”‡ƒŽ…‡ŽŽ…ƒ”…‹‘ƒǤŠ‡”Ž‹‹•ƒƒ‰ʹͲͳ͹Ǣͳ͵ǣʹ͹ͻǦʹͺͷǤ ƒ™ƒ‹ǡ‡—”ƒǡ—ƒƒ–ƒǡƒƒŠƒ•Š‹ǡƒ”ƒ‰—…Š‹ǡ‹•Š‹—”ƒ ǡƒ–ƒǡƒ–•—†ƒǡ ‡ƒ‰ƒǡ —”ƒ–ƒǡ‹œ—•Š‹ƒǡƒƒ‘–‘ǡ‘‹ǡ‘”‹ǣ ”—…–‘•‡Ǧ„‹•’Š‘•’Šƒ–‡ƒŽ†‘Žƒ•‡‹•ƒ‡›”‡‰—Žƒ–‘” ‘ˆŠ›’‘š‹…ƒ†ƒ’–ƒ–‹‘‹…‘Ž‘”‡…–ƒŽ…ƒ…‡”…‡ŽŽ•ƒ†‹˜‘Ž˜‡†‹–”‡ƒ–‡–”‡•‹•–ƒ…‡ƒ†’‘‘”’”‘‰‘•‹•Ǥ – …‘ŽʹͲͳ͹ǢͷͲǣͷʹͷǦͷ͵ͶǤ Šƒ‰ ǡ‹ ǡ—‘ǡŠ—ƒ‰ǡ‘‰ǡŠƒ‰

ǡ—‹ ǡ—‹ǣŽ‡˜ƒ–‡†–”ƒ•…”‹’–‹‘ƒŽŽ‡˜‡Ž•‘ˆ ƒŽ†‘Žƒ•‡ȋȌƒ••‘…‹ƒ–‡•™‹–Š…‡ŽŽ…›…Ž‡Ǧ”‡Žƒ–‡†‰‡‡•‹’ƒ–‹‡–•™‹–Šƒ†•‡˜‡”ƒŽ•‘Ž‹† –—‘”•Ǥ‹‘†ƒ–ƒ‹ʹͲͳ͹ǢͳͲǤ —ǡ —ƒǡƒ‘ǡ‘‰ǡƒ‰ǡ ‘‰ǡ‹—ǡ‹ǡ‘—ǡŠƒ‘ ǣ ”—…–‘•‡Ǧ„‹•’Š‘•’Šƒ–‡ ƒŽ†‘Žƒ•‡ƒ‹•ƒ’‘–‡–‹ƒŽ‡–ƒ•–ƒ•‹•Ǧƒ••‘…‹ƒ–‡†ƒ”‡”‘ˆŽ—‰•“—ƒ‘—•…‡ŽŽ…ƒ”…‹‘ƒƒ†’”‘‘–‡•Ž—‰ …‡ŽŽ–—‘”‹‰‡‡•‹•ƒ†‹‰”ƒ–‹‘ǤŽ‘‘‡ʹͲͳͶǢͻǤ

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