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Effect of Tetrodotoxin on the Proliferation and Gene Expression of Human SW620 Colorectal Cancer Cells
Biomed Sci Letters 2022;28:42-49
Published online March 31, 2022;  https://doi.org/10.15616/BSL.2022.28.1.42
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Yun-Ho Bae*, Hun Kim* and Sung-Jin Lee†,**

Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea
Correspondence to: Sung-Jin Lee. Department of Applied Animal Science, College of Animal Life Sciences, Kangwon National University, Chuncheon 24341, Korea.
Tel: +82-33-250-8636, Fax: +82-33-259-5574, e-mail: sjlee@kangwon.ac.kr
*Graduate student, **Professor.
Received February 10, 2022; Revised March 28, 2022; Accepted March 29, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Tetrodotoxin (TTX) is a natural neurotoxin found in several species of puffer fish belonging to Tetraodon fugu genus and has been reported to affect processes such as proliferation, metastasis and invasion of various cancer cells. However, it was not revealed which genes were influenced by these reactions. In this experiment, it was examined in human SW620 colorectal cancer cells. The proliferation of SW620 cells was significantly reduced when treated with 0, 1, 10 and 100 μM TTX for 48 h. It was confirmed using Annexin V-propidium iodide staining that some apoptosis was induced. Differentially expressed genes (DEGs) affecting cell proliferation through RNA sequencing (RNA-seq) were selected. The expression change of DEGs was confirmed by conducting quantitative real-time polymerase chain reaction (qRT-PCR). As a result, the mRNA expression of FOS and WDR48 genes was found to be increased in the 100 μM TTX treatment group compared to the control group. On the other hand, the mRNA expression of ALKBH7, NDUFA13, RIPPLY3 and SELENOM genes was found to be reduced, and in the case of the ALKBH7 gene was identified to show significant differences. This experiment suggests that TTX can be used as an important fundamental data to elucidate the mechanism that inhibits the proliferation of SW620 cells.
Keywords : Tetrodotoxin (TTX), Human SW620 colorectal cancer cells, Cell proliferation, Differentially expressed gene (DEG), RNA sequencing (RNA-seq), Quantitative real-time polymerase chain reaction (qRT-PCR)
꽌 濡

Statistics Korea (2021)뿉꽌 젣怨듯븯뒗 븫 諛쒖깮 諛 궗留 쁽솴뿉 뵲瑜대㈃, 옣븫쓽 議곕컻깮瑜좎 理쒓렐 10뿬 뀈 룞븞 媛먯냼븯뒗 異붿꽭瑜 蹂댁씠怨 엳쑝굹, 2019뀈 湲곗 議곗궗留앸쪧 룓븫怨 媛꾩븫 떎쓬쑝濡 뿬쟾엳 넂 寃껋쑝濡 굹궗떎. 씠윭븳 옣븫 쟻깋쑁怨 媛怨듭쑁쓽 넂 꽠痍⑤웾怨 꽟쑀吏덉씠 뭾遺븳 怨쇱씪, 梨꾩냼, 怨〓Ъ 諛 쑀젣뭹쓽 궙 꽠痍⑤웾, 吏굹移 쓬二쇰웾 諛 슫룞 遺議 벑쓽 썝씤쑝濡 씤빐 諛쒖깮븳떎(Shannon et al., 1996; Corpet, 2011; Bouvard et al., 2015; Friedenreich et al., 2016; Na and Lee, 2017; Gianfredi et al., 2018).

吏湲덇퉴吏 닔닠쟻 젅젣닠, 솕븰슂踰, 硫댁뿭移섎즺 諛 諛⑹궗꽑移섎즺 벑怨 媛숈 뿬윭 移섎즺 諛⑸쾿뱾쓽 諛쒕떖濡 씤빐 옣븫 솚옄뱾쓽 5뀈 깮議댁쑉 利앷릺뿀쑝硫, 닔닠뿉 뵲瑜 궗留앸쪧怨 빀蹂묒쬆 媛먯냼릺뿀떎(David et al., 2010; Jeong, 2010). 븯吏留, 옣븫 移섎즺瑜 諛쏆 솚옄뱾쓽 떎닔媛 옱諛쒖뿉 븳 몢젮怨 썑쑀利앹쓣 寃り퀬 엳쑝硫, 닔닠쓣 븯뒗 룞븞 븫꽭룷媛 떎瑜 湲곌쑝濡 쟾씠릺嫄곕굹 移⑥쑄씠 맆 닔 엳怨, 븫쓽 꽦옣怨 삁愿떊깮 벑쓽 遺옉슜쑝濡 씤빐 蹂대떎 굹 移섎즺 諛⑸쾿쓣 李얜뒗 怨쇱젙씠 븘슂븯떎(Ceelen et al., 2014; Dariya et al., 2018).

蹂듭뼱룆쑝濡 븣젮吏 tetrodotoxin (TTX) Tetraodon쓽 蹂듭뼱 냽뿉 냽븯뒗 紐뉖챺 醫낆쓽 蹂듭뼱뱾濡쒕꽣 諛쒓껄릺뒗 泥쒖뿰 떊寃쎈룆씠硫, 굹듃瑜 씠삩 梨꾨꼸쓽 李⑤떒젣濡 븣젮졇 엳떎(Hagen et al., 2008). 씤媛꾩뿉 븳 TTX쓽 理쒖냼移섏궗웾 빟 10,000 MU씠硫, 씠뒗 빟 2 mg뿉 빐떦릺뒗 뼇씠떎(Noguchi and Ebesu, 2001). 씠泥섎읆 TTX뒗 씤媛꾩씠 꽠痍⑦븷 寃쎌슦 궗留앹뿉 씠瑜 젙룄쓽 留밸룆씠吏留, 洹뱀냼웾쓽 寃쎌슦뿉뒗 媛뺣젰븳 吏꾪넻 슚怨쇰 굹궡뒗 寃껋쑝濡 븣젮졇 엳떎(Lyu et al., 2000; Marcil et al., 2006; González-Cano et al., 2021). 삉븳, 떎뼇븳 븫뱾뿉 븳 꽑뻾 뿰援щ뱾쓣 넻빐 TTX媛 븫꽭룷쓽 利앹떇, 쟾씠 諛 移⑥쑄 벑쓽 怨쇱젙뱾뿉 쁺뼢쓣 誘몄튇떎뒗 寃껋씠 엯利앸릺뿀떎(Cho et al., 2015; Yang et al., 2017). 씠 뜑遺덉뼱 씠윭븳 怨쇱젙뱾뿉 愿뿬븯뒗 굹듃瑜 씠삩 梨꾨꼸씠 臾댁뾿씤吏 솗씤븯湲 쐞븳 뿰援щ뱾씠 쁽옱 吏꾪뻾 以묒뿉 엳떎(Xiao, 2014).

븯吏留, 옣湲곌컙뿉 嫄몄퀜 留롮 뿰援щ뱾씠 吏꾪뻾릺뿀쓬뿉룄 遺덇뎄븯怨 TTX瑜 移섎즺젣濡 궗슜븯湲 쐞븳 젙蹂대뱾 븘吏 遺議깊븯硫, TTX쓽 泥섎━媛 뼱뼡 쑀쟾옄뱾쓽 諛쒗쁽뿉 蹂솕瑜 씪쑝궡쑝濡쒖뜥 씠윭븳 諛섏쓳뱾쓣 쑀룄븯뒗吏뿉 븳 뿰援 삉븳 씠猷⑥뼱吏吏 븡븯떎. 뵲씪꽌, 蹂 떎뿕뿉꽌뒗 SW620 꽭룷뿉 TTX瑜 泥섎━븯쓣 떆 愿뿬븯뒗 硫붿빱땲利섏씠 臾댁뾿씤吏瑜 솗씤븯湲 쐞빐 쑀꽭룷 遺꾩꽍(FACS), RNA 뿼湲곗꽌뿴 遺꾩꽍(RNA-seq) 諛 떎떆媛 젙웾쟻 PCR (qRT-PCR)쓣 吏꾪뻾븿쑝濡쒖뜥 븫뿉 븳 移섎즺젣濡쒖뜥 TTX쓽 솢슜 媛뒫꽦쓣 룊媛븯떎.

옱猷 諛 諛⑸쾿

Tetrodotoxin (TTX)

TTX뒗 Abcam (Cambridge, UK)뿉꽌 援ъ엯븯쑝硫, sodium citrate (BioShop, Burlington, ON, Canada)瑜 pH 4.8, 20 mM쓽 냽룄濡 3李 硫멸퇏닔뿉 슜빐떆耳 뼸뼱吏 buffer뿉 끃뿬 10 mM 냽룄쓽 옣븸쓣 留뚮뱾뿀떎. 留뚮뱾뼱吏 옣븸 aliquot븯뿬 -20℃뿉꽌 蹂닿븯떎.

꽭룷 諛곗뼇

SW620 꽭룷뒗 븳援꽭룷二쇱뻾(Seoul, Korea)뿉꽌 援ъ엯븯뿬 떎뿕뿉 궗슜븯떎. 꽭룷뒗 10% fetal bovine serum (FBS, Gibco-BRL, Grand Island, NY, USA), 100 units/mL penicillin 諛 100 mg/mL streptomycin (Welgene, Daegu, Korea)씠 븿쑀맂 Roswell Park Memorial Institute 1640 (RPMI 1640, Welgene) 諛곗瑜 궗슜븯뿬 37℃, 5% CO2 議곌굔쓽 꽭룷諛곗뼇湲곗뿉꽌 諛곗뼇븯떎. SW620 꽭룷瑜 씠슜븳 떎뿕뱾 媛뺤썝븰援 깮紐낆쑄由ъ쐞썝쉶쓽 떖쓽硫댁젣듅씤(KNUIRB-2022-03-010)쓣 諛쏆븯떎.

MTT assay

MTT assay뒗 CellTiter 96® Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, WI, USA)瑜 궗슜븯쑝硫, Lee 벑 (2018)씠 궗슜븳 諛⑸쾿쓣 씪遺 닔젙븯뿬 吏꾪뻾븯떎. 癒쇱, 1李 諛곗뼇뿉꽌 뼸 꽭룷瑜 96-well cell culture plate (SPL Life Sciences, Seoul, Korea)뿉 2×104 cells/well쓽 냽룄濡 seeding븯뿬 24떆媛 룞븞 諛곗뼇븯떎. 꽭룷媛 plate뿉 遺李⑸릺硫 諛곗瑜 젣嫄고븯怨, TTX媛 0, 1, 10 諛 100 μM쓽 냽룄濡 븿쑀맂 깉濡쒖슫 諛곗瑜 泥④븯뿬 48떆媛 룞븞 諛곗뼇븯떎. 諛곗뼇 48떆媛 썑뿉 떎떆 諛곗瑜 젣嫄고븯怨, 깉濡쒖슫 諛곗 50 μL dye solution 15 μL瑜 泥④븯뿬 떎떆 4떆媛 룞븞 諛곗뼇븯떎. 洹 썑, solubilization/stop solution 100 μL瑜 媛 well뿉 泥④븳 떎쓬 1떆媛 룞븞 異붽濡 諛곗뼇븯떎. SpectraMax M4 microplate reader (Molecular Devices, San Jose, CA, USA)濡 570nm쓽 뙆옣뿉꽌 씉愿묐룄瑜 痢≪젙븿쑝濡쒖뜥 SW620 꽭룷쓽 利앹떇쓣 怨꾩궛븯떎.

Annexin V-propidium iodide staining쓣 넻븳 apoptosis 愿李

떎뼇븳 FACS 湲곗닠 以 annexin V-propidium iodide staining 諛⑸쾿쓣 넻빐 apoptosis瑜 愿李고븯떎. 떎뿕 eBioscienceTM Annexin V Apoptosis Detection Kit FITC (eBioscience, Ltd., San Diego, CA, USA)瑜 궗슜븯뿬 吏꾪뻾븯떎. 癒쇱, 꽭룷瑜 6-well cell culture plate (SPL Life Sciences)뿉 2×105 cells /well쓽 냽룄濡 seeding븯뿬 24떆媛 룞븞 諛곗뼇븯떎. 꽭룷媛 plate뿉 遺李⑸릺硫 諛곗瑜 젣嫄고븯怨, TTX媛 0 諛 100 μM쓽 냽룄濡 븿쑀맂 깉濡쒖슫 諛곗瑜 泥④븯뿬 48떆媛 룞븞 諛곗뼇븯떎. 48떆媛 룞븞 諛곗뼇쓣 븳 썑 꽭룷瑜 harvest븳 떎쓬 썝떖遺꾨━븯뿬 긽벑븸쓣 젣嫄고븯怨, Dulbeccós phosphate -buffered saline (DPBS, Welgene)쑝濡 꽭泥숈쓣 吏꾪뻾븯떎. 洹 썑, 1X annexin V binding buffer뿉 遺쑀떆궓 떎쓬 annexin V FITC propidium iodide staining solution쓣 泥④븳 썑 븫떎뿉꽌 15遺 룞븞 諛섏쓳떆耳곕떎. 諛섏쓳떆궓 꽭룷뒗 BD FACSAria II (Bectom Dickinson, San Jose, CA, USA)瑜 궗슜븯뿬 annexin V-positive apoptotic cell쓽 鍮꾩쑉쓣 痢≪젙븯떎.

RNA 異붿텧 諛 sequencing 遺꾩꽍

RNA-seq쓣 쐞빐 100 mm cell culture dish (SPL Life Sciences)뿉 2×106 cells/dish쓽 냽룄濡 꽭룷瑜 seeding븳 썑 24떆媛 룞븞 諛곗뼇븳 썑 꽭룷媛 dish뿉 遺李⑸릺硫 諛곗瑜 젣嫄고븯怨, 48떆媛 룞븞 0 諛 100 μM 냽룄쓽 TTX媛 븿쑀맂 깉濡쒖슫 諛곗瑜 泥④븯뿬 諛곗뼇븯떎. 洹 떎쓬, 꽭룷瑜 harvest븳 썑 썝떖遺꾨━븯뿬 긽벑븸쓣 젣嫄고븯怨, DPBS濡 꽭泥숈쓣 吏꾪뻾븯떎. Total RNA뒗 TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA)瑜 궗슜븯뿬 異붿텧븯쑝硫, 씠뿉 븳 뿼湲곗꽌뿴 遺꾩꽍 LAS Biotechnology (Seoul, Korea)뿉 쓽猶고븯뿬 닔뻾븯떎.

qRT-PCR쓣 넻븳 mRNA 諛쒗쁽 遺꾩꽍

Total RNA뒗 쐞 룞씪븳 議곌굔쑝濡 꽭룷瑜 諛곗뼇븳 썑 TRIzol® Reagent瑜 궗슜븯뿬 異붿텧븯떎. 異붿텧맂 RNA쓽 냽룄 닚룄뒗 NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA)濡 痢≪젙븯떎. iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA)瑜 궗슜븯뿬 500 ng쓽 total RNA濡쒕꽣 cDNA瑜 빀꽦븯쑝硫, PowerUPTM SYBRTM Green Master Mix (Thermo Fisher Scientific) 0.4 pM쓽 湲곕뒫꽦 쑀쟾옄뿉 븳 듅씠쟻 봽씪씠癒몃뱾쓣 궗슜븯뿬 20 μL쓽 reaction volume뿉꽌 qRT-PCR쓣 吏꾪뻾븯떎. 궗슜뻽뜕 봽씪씠癒몃뱾뿉 븳 젙蹂대뒗 Table 1뿉 젣떆븯떎. 遺꾩꽍 Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA)瑜 궗슜븯뿬 닔뻾븯떎. ALKBH7 쑀쟾옄瑜 젣쇅븳 씪諛섏쟻씤 qRT-PCR쓽 諛섏쓳 議곌굔 떎쓬怨 媛숇떎: 珥덇린 蹂꽦떒怨 50℃뿉꽌 2遺, 95℃뿉꽌 2遺 썑 蹂꽦떒怨 95℃뿉꽌 15珥 諛 寃고빀怨 떊옣떒怨 60℃뿉꽌 1遺 怨쇱젙 40쉶 諛섎났. ALKBH7 쑀쟾옄쓽 qRT-PCR쓽 諛섏쓳 議곌굔 떎쓬怨 媛숇떎: 珥덇린 蹂꽦떒怨 50℃뿉꽌 2遺, 95℃뿉꽌 2遺 썑 蹂꽦떒怨 95℃뿉꽌 15珥 諛 寃고빀떒怨 62℃뿉꽌 30珥덉 떊옣떒怨 72℃뿉꽌 30珥 怨쇱젙 40쉶 諛섎났. 몢 寃쎌슦 紐⑤몢 빐由ш끝꽑쓽 議곌굔 95℃뿉꽌 15珥, 60℃뿉꽌 1遺 諛 95℃뿉꽌 15珥덈 吏꾪뻾븯쑝硫, 愿떖 엳뒗 쑀쟾옄뱾쓽 mRNA 諛쒗쁽쓣 젙洹쒗솕븯湲 쐞븳 湲곗쑝濡 GAPDH 쑀쟾옄쓽 몴以怨≪꽑쓣 궗슜븯떎. 빐떦 쑀쟾옄뱾쓽 긽쟻씤 mRNA 諛쒗쁽 2-ΔΔCt 諛⑸쾿쓣 궗슜븯뿬 怨꾩궛븯떎.

Information and sequence of functional primers for qRT-PCR

Genes Sequence Tm (℃) Product size (bp) Accession No.
FOS F: GGGCAAGGTGGAACAGTTAT 60 138 NM_005252.4
R: AGTTGGTCTGTCTCCGCTTG
NDUFA13 F: TTGCCACTGTTACAGGCAGA 60 119 NM_015965.7
R: AACACAGACTCCCCCACCTT
RIPPLY3 F: GCAGAACTTGATGCTGTCCA 60 159 NM_001317777.1
R: GCCTCTAGCCTCTGCACAC
SELENOM F: AGAGGAAACTTCGGACCACG 60 128 NM_080430.4
R: CGGCTCTCAGCAAGAGAAGT
WDR48 F: AGAAGTACAACCGAAATGGAGTC 60 169 NM_020839.4
R: ACAATGTCGTTTACCCAATCAGT
ALKBH7 F: GGCTTCCGAGAGACAGAGAA 62 171 NM_032306.4
R: GCAGAACTTGATGCTGTCCA
GAPDH F: CTGCACCACCAACTGCTTAG 60~62 108 NM_002046.7
R: GTCTTCTGGGTGGCAGTGAT


넻怨 遺꾩꽍

紐⑤뱺 떎뿕뱾 꽭 踰덉뵫 諛섎났 吏꾪뻾븯쑝硫, 씠뿉 븳 寃곌낵뒗 룊洹 ± 몴以삤李(SEM)濡 怨꾩궛븯떎. TTX 誘몄쿂由ш뎔(議곌뎔)怨 TTX 泥섎━援 媛꾩쓽 李⑥씠뒗 SAS software ver. 9.4 (SAS Institute, Cary, NC, USA)쓽 遺꾩궛遺꾩꽍(ANOVA), 씪諛섏꽑삎紐⑦삎(GLM) 諛 Duncan 떎以묐쾾쐞寃젙쓣 궗슜븯뿬 寃곗젙븯쑝硫, P-value媛 0.05 誘몃쭔씪 寃쎌슦 넻怨꾩쟻쑝濡 쑀쓽誘명븯떎怨 뙋젙븯떎. 룄몴뒗 GraphPad Prism ver. 7.0 (GraphPad Software, San Diego, CA, USA)쓣 궗슜븯뿬 옉꽦븯떎.

寃곌낵 諛 怨좎같

TTX媛 SW620 꽭룷쓽 利앹떇뿉 誘몄튂뒗 쁺뼢쓣 솗씤븯湲 쐞빐 TTX瑜 48떆媛 룞븞 媛곴컖 0, 1, 10 諛 100 μM 냽룄濡 泥섎━븳 썑 MTT assay瑜 吏꾪뻾븯쑝硫, 洹 寃곌낵瑜 Fig. 1뿉 젣떆븯떎. SW620 꽭룷쓽 利앹떇 1 μM 냽룄뿉꽌 88.35% (P<0.05), 10 μM 냽룄뿉꽌 85.36% (P<0.01) 諛 100 μM 냽룄뿉꽌 74.56% (P<0.001)濡 냽룄쓽議댁쟻쑝濡 쑀쓽誘명븯寃 媛먯냼릺뒗 寃껋쑝濡 솗씤릺뿀떎.

Fig. 1. Effect of TTX on the proliferation of SW620 cells in vitro using MTT assay. SW620 cells have cultured with 0, 1, 10 and 100 μM TTX for 48 h. Data are expressed as percentage of 0 μM (control), are the mean ± standard error of means (SEM) of three separate experiments. *P<0.05 vs control, **P<0.01 vs control, ***P<0.001 vs control.

TTX쓽 泥섎━뿉 쓽빐 SW620 꽭룷쓽 利앹떇씠 媛먯냼릺뒗 寃껋쑝濡 솗씤릺뿀湲 븣臾몄뿉 꽭룷利앹떇쓽 媛먯냼媛 apoptosis쓽 쑀룄 뿰愿씠 엳쓣 寃껋쑝濡 湲곕릺뼱 annexin V-propidium iodide staining쓣 吏꾪뻾븯떎. 48떆媛 룞븞 TTX瑜 0 諛 100 μM쓽 냽룄濡 泥섎━븳 썑 annexin V-positive apoptotic cell뱾쓽 鍮꾩쑉쓣 痢≪젙븯떎. 議곌뎔뿉 鍮꾪빐 100 μM 냽룄쓽 TTX 泥섎━援곗뿉꽌 apoptosis媛 떆옉맂 꽭룷뱾쓽 鍮꾩쑉 5.205%뿉꽌 6.605%濡, apoptosis媛 吏꾪뻾릺뼱 二쎌 꽭룷뱾쓽 鍮꾩쑉 7.335%뿉꽌 8.23%濡 利앷릺뒗 寃껋쑝濡 솗씤릺뿀쑝굹, 쑀쓽誘명븳 李⑥씠뒗 굹굹吏 븡븯떎(Fig. 2). 뵲씪꽌, TTX쓽 泥섎━뒗 apoptosis瑜 쑀룄븯뿬 SW620 꽭룷쓽 利앹떇뿉 쁺뼢쓣 誘몄튂湲곕뒗 븯吏留, 씠蹂대떎뒗 떎瑜 硫붿빱땲利섏뿉 쓽븳 쁺뼢쓣 뜑 留롮씠 諛쏅뒗 寃껋쑝濡 뙋떒븯뿬 RNA-seq쓣 吏꾪뻾븿쑝濡쒖뜥 TTX瑜 泥섎━븯쓣 떆 굹굹뒗 李⑤벑諛쒗쁽 쑀쟾옄(differentially expressed gene, DEG)뱾쓣 솗씤븯떎.

Fig. 2. Effect of TTX on annexin V-positive apoptotic cell numbers in SW620 cells. SW620 cells were treated with TTX (0 and 100 μM) for 48 h. Apoptosis was measured by annexin V and propidium iodide double staining. Apoptotic cells were analyzed by flow cytometry.

議곌뎔怨 TTX 泥섎━援곗쓽 RNA-seq쓣 넻빐 솗씤맂 쑀쟾옄뱾쓽 닔뒗 珥 19,389媛쒖떎. 씠 以, q-value ≤ 0.01쓽 議곌굔뿉꽌 珥 16媛쒖쓽 쑀쓽誘명븳 李⑥씠瑜 굹궡뒗 DEG뱾씠 솗씤릺뿀떎. 議곌뎔뿉 鍮꾪빐 100 μM 냽룄쓽 TTX 泥섎━援곗뿉꽌 5媛쒖쓽 긽뼢議곗젅릺뒗 DEG뱾(FOS, ZFAND4, FAM8A1, WDR48ZC3HAVIL)怨 11媛쒖쓽 븯뼢議곗젅릺뒗 DEG뱾(HBA1, CAMK2B, CCDC88B, RIPPLY3, ALKBH7, TMEM256, SELENOM, NDUFA13, HBA2, CLDND2TNNI2)씠 쑀쓽誘명븳 李⑥씠瑜 굹궡뒗 寃껋쑝濡 솗씤릺뿀떎(Table 2).

UP- and down-regulated genes in 100 μM TTX-treated SW620 cells compared with control

Official gene symbol Full description Log2FC P-value q-value
Up
FOS Fos proto-oncogene, AP-1 transcription factor subunit
[Source:HGNC Symbol;Acc:HGNC:3796] 1.102 0.00005 0.00295
ZFANDA Zinc finger AN1-type containing 4
[Source:HGNC Symbol;Acc:HGNC:23504] 1.082 0.00005 0.00295
FAM8A1 Family with sequence similarity 8 member A1
[Source:HGNC Symbol;Acc:HGNC:16372] 1.03 0.00005 0.00295
WDR48 WD repeat domain 48
[Source:HGNC Symbol;Acc:HGNC:30914] 1.008 0.00005 0.00295
ZC3HAV1L Zinc finger CCCH-type containing, antiviral 1 like
[Source:HGNC Symbol;Acc:HGNC:22423] 1.648 0.00025 0.00976
DOWN
HBA1 Hemoglobin subunit alpha 1
[Source:HGNC Symbol;Acc:HGNC:4823] -3.312 0.00005 0.00295
CAMK2B Calcium/Calmodulin dependent protein kinase Ⅱ beta
[Source:HGNC Symbol;Acc:HGNC:1461] -2.189 0.00005 0.00295
CCDC88B Coiled-coil domain containing 88B
[Source:HGNC Symbol;Acc:HGNC:26757] -1.128 0.00005 0.00295
RIPPLY3 Ripply transcriptional repressor 3
[Source:HGNC Symbol;Acc:HGNC:3047] -1.039 0.00005 0.00295
ALKBH7 AlkB homolog 7
[Source:HGNC Symbol;Acc:HGNC:21306] -1.027 0.00005 0.00295
TMEM256 Transmembrane protein 256
[Source:HGNC Symbol;Acc:HGNC:28618] -1.014 0.00005 0.00295
SELENOM Selenoprotein M
[Source:HGNC Symbol;Acc:HGNC:30397] -1.01 0.00005 0.00295
NDUFA13 NADH:ubiquinone oxidoreductase subunit A13
[Source:HGNC Symbol;Acc:HGNC:17194] -1.007 0.00005 0.00295
HBA2 Hemoglobin subunit alpha 2
[Source:HGNC Symbol;Acc:HGNC:4824] -6.617 0.00015 0.0068
CLDND2 Claudin domain containing 2
[Source:HGNC Symbol;Acc:HGNC:28511] -1.392 0.00015 0.0068
TNNI2 Troponin I2, fast skeletal type
[Source:HGNC Symbol;Acc:HGNC:11946] -1.128 0.0002 0.00832


삉븳, 紐⑤뱺 DEG뱾뿉 븳 쑀쟾옄 삩넧濡쒖(gene ontology, GO) 湲곕뒫 遺꾩꽍쓣 吏꾪뻾븳 寃곌낵, DEG뿉 뭾遺븳 寃껋쑝濡 諛앺吏 GO term biological process쓽 寃쎌슦 'oxygen transport', 'gas transport', 'hydrogen peroxide catabolic process', 'antibiotic catabolic process', 'bicarbonate transport', 'response to inorganic substancé 諛 'hydrogen peroxide metabolic process'媛, cellular component쓽 寃쎌슦뿉뒗 'haptoglobin-hemoglobin complex', 'hemoglobin complex', 'endocytic vesicle lumen' 諛 'cytosolic small ribosomal subunit'씠, molecular function쓽 寃쎌슦뿉뒗 'haptoglobin binding', 'oxygen carrier activity', 'oxygen binding', 'molecular carrier activity', 'peroxidase activity' 諛 'oxidoreductase activity, acting on peroxide as acceptor'濡 솗씤릺뿀떎(Fig. 3). 븯吏留, 蹂 떎뿕뿉꽌뒗 뼱뼡 쑀쟾옄뱾쓽 諛쒗쁽 蹂솕媛 꽭룷利앹떇쓽 뼲젣瑜 쑀룄븯뒗吏瑜 솗씤븯뒗 寃껋뿉 以묒젏쓣 몢뿀湲 븣臾몄뿉 쑀쓽誘명븳 李⑥씠瑜 굹궡뒗 寃껋쑝濡 솗씤맂 16媛쒖쓽 DEG뱾 以 諛쒗쁽씠 利앷릺뿀쓣 떆 꽭룷利앹떇쓣 솢꽦 떆궎뒗 寃껋쑝濡 븣젮吏 쑀쟾옄(FOS, RIPPLY3SELENOM) 꽭룷利앹떇쓣 뼲젣떆궎뒗 寃껋쑝濡 븣젮吏 쑀쟾옄(ALKBH7, NDUFA13WDR48)瑜 꽑蹂꾪븳 썑 RNA-seq 寃곌낵瑜 寃利앺븯湲 쐞빐 qRT-PCR쓣 吏꾪뻾븯떎.

Fig. 3. GO functional analysis of DEGs in SW620 cells. GO analysis based on the treatment of TTX (0 and 100 μM) for 48 h was performed by g:Profiler ver. 0.6.7. The 10 singular enrichment analysis terms related to biological process (red), cellular component (blue) and molecular function (green) are presented. All adjusted statistically significant values of the GO terms were -log10 converted.

TTX瑜 0 諛 100 μM쓽 냽룄濡 泥섎━븯뿬 qRT-PCR쓣 吏꾪뻾븳 寃곌낵, Fig. 4 5뿉 젣떆맂 諛붿 媛숈씠 RNA-seq 寃곌낵 쑀궗븯寃 FOSWDR48 쑀쟾옄쓽 mRNA 諛쒗쁽 利앷릺뒗 寃껋쑝濡 솗씤릺뿀쑝硫, ALKBH7, NDUFA13, RIPPLY3SELENOM 쑀쟾옄쓽 mRNA 諛쒗쁽 媛먯냼릺뒗 寃껋쑝濡 솗씤릺뿀떎. 삉븳, ALKBH7, FOSWDR48 쑀쟾옄쓽 쑀쟾옄쓽 寃쎌슦 쑀쓽誘명븳 李⑥씠瑜 굹궡뒗 寃껋쑝濡 솗씤릺뿀떎.

Fig. 4. Quantification of the mRNA profile of up-regulated genes in RNA-seq. SW620 cells incubated with each concentration of TTX (0 and 100 μM) for 48 h. TTX exposure affected the mRNA abundance of certain genes in the cells. The mRNA level of both genes was normalized to the GAPDH gene. Data are expressed as the mean ± SEM. All experiments were repeated at least three times. ***P<0.001 vs control.

SW620 꽭룷뿉 TTX瑜 泥섎━븯쓣 떆 꽭룷利앹떇씠 뼲젣맂떎뒗 꽑뻾 뿰援(Xiao, 2014) 쑀궗븯寃 蹂 떎뿕뿉꽌룄 MTT assay 寃곌낵 꽭룷利앹떇씠 뼲젣릺뒗 寃껋쑝濡 솗씤릺뿀떎(Fig. 1). 븯吏留, 꽭룷利앹떇쓣 뼲젣븯뒗 硫붿빱땲利섏 RNA-seq怨 qRT-PCR 寃곌낵뿉꽌룄 紐낇솗븯寃 굹굹吏 븡븯떎. 떎留, 씠윭븳 寃곌낵媛 굹궃 씠쑀뒗 蹂 떎뿕뿉꽌 꽭룷利앹떇쓣 뼲젣떆궎뒗 寃껋쑝濡 솗씤맂 WDR48 쑀쟾옄쓽 湲곕뒫씠 꽭룷利앹떇쓣 솢꽦 떆궎뒗 寃껋쑝濡 솗씤맂 ALKBH7FOS 쑀쟾옄쓽 湲곕뒫蹂대떎 뜑 겕寃 옉슜뻽嫄곕굹 븳몢 媛쒖쓽 쑀쟾옄뱾뿉 쓽빐꽌媛 븘땲씪 뿬윭 媛쒖쓽 쑀쟾옄뱾씠 蹂듯빀쟻쑝濡 옉슜뻽湲 븣臾몄씤 寃껋쑝濡 뙋떒맂떎.

Fig. 5. Quantification of the mRNA profile of down-regulated genes in RNA-seq. SW620 cells were cultured by treatment with each concentration of TTX (0 and 100 μM) for 48 h. TTX exposure affected the mRNA abundance of certain genes in the cells. The mRNA level of all genes was normalized to the GAPDH gene. Data are expressed as the mean ± SEM. All experiments were repeated at least three times. **P<0.01 vs control.

寃곕줎쟻쑝濡, TTX쓽 泥섎━뒗 씤媛 SW620 옣븫 꽭룷쓽 利앹떇쓣 뼲젣떆궎뒗 뜲 슚怨쇨 엳쑝硫, 씠윭븳 꽭룷利앹떇쓽 뼲젣뒗 WDR48 쑀쟾옄쓽 諛쒗쁽 利앷瑜 넻븳 apoptosis쓽 쁺뼢룄 씪遺 엳吏留, 떎瑜 硫붿빱땲利섏뿉 쓽븳 쁺뼢룄 議댁옱븷 寃껋쑝濡 異붿젙맂떎.

뵲씪꽌, TTX媛 SW620 꽭룷쓽 利앹떇뿉 쁺뼢쓣 誘몄튂뒗 硫붿빱땲利섏쓣 젙솗븯寃 諛앺엳湲 쐞빐꽌뒗 異뷀썑 蹂대떎 뜑 떎뼇븳 DEG 遺꾩꽍 諛 mRNA 떒諛깆쭏 닔以뿉꽌쓽 諛쒗쁽 蹂솕瑜 異붽쟻씤 떎뿕뱾쓣 넻빐 솗씤븯뒗 怨쇱젙씠 븘슂븷 寃껋쑝濡 깮媛곷맂떎. 삉븳, 蹂 떎뿕씠 씠瑜 諛앺엳뒗 뜲 以묒슂븳 湲곗큹옄猷뚮줈뜥 솢슜맆 닔 엳쓣 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

This study was supported by National Research Foundation of Korea (Project number: 2017R1A2B2012125).

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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