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Anticancer Effects of Fibronectin Leucine Rich Transmembrane Protein 3 as a Novel Therapeutic Molecule in Lung Cancer and Lung Cancer-derived Stem Cell
Biomed Sci Letters 2023;29:336-343
Published online December 31, 2023;  https://doi.org/10.15616/BSL.2023.29.4.336
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Joong-Won Baek* and Pyung-Hwan Kim†,**

Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea
Correspondence to: Pyung-Hwan Kim. Department of Biomedical Laboratory Science, Konyang University, 158 Gwanjeodong-Ro, Seo-Gu, Daejeon 35365, Korea.
Tel: +82-42-600-8436, Fax: +82-42-600-8408, e-mail: kimph1010@konyang.ac.kr
*Graduate student, **Professor.
Received November 15, 2023; Revised December 6, 2023; Accepted December 7, 2023.
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
Lung cancer is one of the cancers with high mortality and incidence rates worldwide. Although, various anticancer research efforts are underway to completely treat cancer, the challenge against it remains in the inability to eliminate cancer stem cells (CSCs), leading to difficulties in curing the cancer and resulting in recurrence. As a result, there is a growing interest in the discovery of new biomarkers and therapeutic molecules that can simultaneously target both cancer cells and CSCs. From this point of view, we focused on fibronectin leucine rich transmembrane protein 3 (FLRT3), one of the genes known to be present in human lung cells and the discovery from our previous cancer proteomic analysis study. This study aimed to evaluate the potential of FLRT3 as a specific therapeutic biomarker for lung cancer and Lung Cancer-derived-Stem Cells (LCSC). Also, to estimate the biological function of FLRT3 in cancer and LCSC, short hairpin RNA (shRNA) was generated and showed the ability of the decreased-cell migration and cell proliferation of lung cancer through ERK signaling pathway when FLRT3 was knock-downed. In conclusion, our study is the first to report that FLRT3 has the potential as therapeutic biomarker for the treatment of lung cancer and LCSC.
Keywords : Lung cancer, Cancer Stem Cells (CSCs), FLRT3, Biological function, ERK signaling pathway
꽌 濡

룓븫 踰붿꽭怨꾩쟻쑝濡 留롮씠 諛쒖깮븯怨 엳쑝硫, 븳援뿉꽌룄 궓꽦뿉꽌 諛쒕퀝瑜 1쐞, 뿬꽦뿉꽌 諛쒕퀝瑜 4쐞瑜 李⑥븯怨 엳뼱 솚옄媛 留롮 吏덊솚씠떎(Barta et al., 2019; Jung et al., 2019). 룓븫쓽 諛쒖깮 썝씤뿉뒗 씉뿰, 끂솕 벑 떎뼇븳 슂씤뱾씠 엳吏留, 듅엳 理쒓렐쓽 肄붾줈굹-19 씠뒋濡 씤빐 肄붾줈굹 諛붿씠윭뒪 媛먯뿼 솚옄쓽 룓븫 諛쒕퀝 媛뒫꽦뿉 븳 留롮 뿰援 寃곌낵媛 蹂닿퀬맖뿉 뵲씪 븵쑝濡쒖쓽 룓븫 諛쒕퀝瑜좉낵 洹몃줈 씤븳 궗留앸쪧 삉븳 뜑슧 利앷븷 寃껋쑝濡 궗猷뚮맂떎(Calabr챵 et al., 2021). 븣臾몄뿉 룓븫쓽 吏꾨떒怨 移섎즺瑜 쐞븳 떎뼇븳 뿰援ш 吏꾪뻾릺怨 엳吏留 conventional 移섎즺 諛 떎뼇븳 븫 移섎즺踰 쟻슜 썑쓽 5뀈 궡 궙 깮議댁쑉(Najafi et al., 2021) 諛 븫 옱諛쒖쓽 썝씤쑝濡 븣젮吏 븫以꾧린꽭룷源뚯 셿踰쏀엳 젣嫄고븯吏 紐삵븯湲 븣臾몄뿉 빆븫 移섎즺 썑뿉룄 옱諛쒖씠 鍮덈쾲븯떎뒗 젏怨 룓븫쓽 議곌린 吏꾨떒쓣 쐞븳 깉濡쒖슫 諛붿씠삤留덉빱 諛쒓뎬쓽 遺옱濡 씤븳 룓븫 移섎즺뿉 븳 븳怨꾧 엳뼱 솕떎(Eramo et al., 2010; Lundin and Driscoll, 2013).

븫以꾧린꽭룷뒗 醫낆뼇쓣 삎꽦븷 닔 엳뒗 뒫젰씠 엳뒗 以꾧린 꽭룷濡, 醫낆뼇 궡뿉 1~2% 젙룄 냼닔 옍옱븯硫 룊긽떆뿉뒗 쑕吏湲 긽깭濡 議댁옱븯湲 븣臾몄뿉 諛쒓껄씠 뼱졄떎. 肉먮쭔 븘땲씪 빆븫젣뿉 븳 빆꽦씠 겕湲 븣臾몄뿉 빆븫젣 닾빟 썑뿉룄 셿쟾엳 젣嫄곕릺吏 븡븘 븫쓽 옱諛쒓낵 쟾씠쓽 二 썝씤쑝濡 뿬寃⑥쭊떎(Hardavella et al., 2016; Cojoc et al., 2015; Suresh et al., 2016). 삉븳, 븫쓽 議곌린 吏꾨떒 뼢썑 솚옄쓽 5뀈 깮議댁쑉쓣 70% 씠긽 利앷떆궗 닔 엳떎뒗 蹂닿퀬媛 엳뼱 議곌린 吏꾨떒쓽 뿭븷뿉 븳 以묒슂꽦씠 뜑슧 몢릺怨 엳쑝硫, 議곌린 吏꾨떒 臾쇱쭏쓣 諛쒓뎬븯뒗 뿰援щ 넻빐 븫쓽 議곌린 移섎즺瑜 넻븳 셿移 諛 씠瑜 씠슜븳 쓳슜뿰援ш 吏꾪뻾릺怨 엳떎(Huang et al., 2022).

씠뿉 슦由щ뒗 슦由ъ쓽 씠쟾 떎醫 븫쓽 떒諛깆껜 遺꾩꽍쓣 넻빐 룞젙맂 떎뼇븳 떒諛깆쭏 以 룓븫怨 룓븫쑝濡쒕꽣 쑀옒븳 븫以꾧린꽭룷瑜 룞떆뿉 몴쟻쑝濡 븯怨 룓븫쓽 吏꾨떒 諛 移섎즺 臾쇱쭏濡쒖꽌 옉슜븷 닔 엳뒗 깉濡쒖슫 諛붿씠삤留덉빱 쑀슚 臾쇱쭏씤 Fibronectin leucine rich transmembrane protein 3 (FLRT3)瑜 諛쒓뎬븿怨 룞떆뿉 씠 떒諛깆쭏씠 移섎즺 臾쇱쭏濡쒖꽌쓽 媛뒫꽦씠 엳뒗吏瑜 룊媛빐 蹂댁븯떎.

옱猷 諛 諛⑸쾿

꽭룷 諛곗뼇

븫 꽭룷뒗 궗엺 룓븫 꽭룷 A549, 궗엺 媛꾩븫 꽭룷 Huh7, 궗엺 쑀諛⑹븫 꽭룷 MCF7瑜, 븫以꾧린꽭룷濡쒕뒗 A549濡쒕꽣 쑀옒븳 룓븫 以꾧린꽭룷 Lung Cancer Stem Cell (LCSC), Huh7濡쒕꽣 쑀옒븳 媛꾩븫 以꾧린꽭룷 Liver Cancer Stem Cell (LiCSC), MCF7濡쒕꽣 쑀옒븳 쑀諛⑹븫 以꾧린꽭룷 Breast Cancer Stem Cell (BCSC)瑜 씠슜븯떎. A549, Huh7, MCF7 American Type Culture Collection (ATCC, VA, USA)濡쒕꽣 援щℓ븯떎. 븫 꽭룷二쇰 諛곗뼇븯湲 쐞븳 諛곗뒗 1% penicillin-streptomycin (PS) (Hyclone, UT, USA)怨 10% fetal bovine serum (FBS) (Hyclone, UT, USA)瑜 泥④븳 Dulbecc처s Modified Eagl챕s Medium (DMEM) (Hyclone, UT, USA)瑜 씠슜븯떎. 븫以꾧린꽭룷二쇰뒗 DMEM/F-12 Nutrient Mixture Ham (DMEM/F-12) (Welgene, Gyeongsan-si, Gueongsangbuk-do, Korea) 諛곗뿉 1% PS, 10% FBS, 5 關g/mL insulin (Invitrogen, CA, USA), 20 ng/mL EGF (Gibco, Life Technologies, Grand Island, NY, USA), 20 ng/mL b-FGF (Gibco, Life Technologies, Grand Island, NY, USA), 1% B27 (Invitrogen, CA, USA)瑜 泥④븯뿬 6-well ultra-low attachment plates (Corning, NY, USA)뿉꽌 14씪 룞븞 諛곗뼇븯떎. 븫 꽭룷瑜 泥섏쓬 well 떦 2 횞 104 留뚰겮 seeding 븯뿬 7씪 룞븞 諛곗뼇븳 뮘, subculture瑜 吏꾪뻾븯뿬 珥 14씪 룞븞 諛곗뼇븯떎(Koh et al., 2022). 꽭룷뱾 紐⑤몢 CO2 냽룄瑜 5%濡, 삩룄瑜 37꼦 쑀吏븯뒗 諛곗뼇湲곗뿉꽌 諛곗뼇븯떎.

Transfection

FLRT3쓽 諛쒗쁽쓣 議곗젅븯湲 쐞빐 linear short hairpin RNA (shRNA) scrambled shRNA (shScr) (Genolution, Seoul, Korea)쓣 젣옉븯怨, shRNA series瑜 A549 LCSC뿉 transfection 븯떎. 꽭룷뒗 6-well plate뿉 2 횞 105 留뚰겮 諛곗뼇븳 뮘, LipofectamineTM 3000 Transfection Reagent (Invitrogen, CA, USA)瑜 씠슜븯뿬 FBS媛 뾾뒗 諛곗뿉 manufacturer's instructions 濡 닔뻾븯떎. 4떆媛 룞븞 transfection쓣 吏꾪뻾븳 뮘, FBS媛 룷븿맂 諛곗濡 援먰솚빐二쇨퀬 24떆媛 룞븞 諛곗뼇븯뿬 꽭룷 궡뿉꽌 shRNA媛 異⑸텇엳 諛쒗쁽븯룄濡 븯떎. Transfection씠 맂 꽭룷濡쒕꽣 RNA瑜 異붿텧븯怨 conventional PCR쓣 吏꾪뻾븯뿬 FLRT3쓽 諛쒗쁽쓣 솗씤븯떎. Transfection뿉 궗슜맂 shRNA series sequence뒗 Table 1怨 媛숇떎.

shRNA series sequence

shRNA name shRNA sequence (5'-3')
shFLRT3 #1 UCAAGUAGCACCUCUAUCA UCUC UGAUAGAGGUGCUACUUGA UU
shFLRT3 #2 CAGAUAACCACUGCAAUAC UCUC GUAUUGCAGUGGUUAUCUG UU
shFLRT3 #3 CAAUCGAGAGCAAGAGAAA UCUC UUUCUCUUGCUCUCGAUUG UU
shFLRT3 #4 GGAAAUCAGGGAAACUUCU UCUC AGAAGUUUCCCUGAUUUCC UU
shFLRT3 #5 CAGUGAAAGCAGUAGUAAC UCUC GUUACUACUGCUUUCACUG UU
shScr CCUCGUGCCGUUCCAUCAGGUAG UCUC CCUCGUGCCGUUCCAUCAGGUAG UU


RNA 異붿텧 諛 conventional polymerase chain reaction (PCR)

24떆媛 룞븞 transfection쓣 닔뻾븳 꽭룷濡쒕꽣 TRIzol (Invitrogen, CA, USA)쓣 씠슜븯뿬 RNA瑜 異붿텧븯떎. NanodropTM (Thermo Fisher Scientific, Waltham, MA, USA)쑝濡 RNA 뼇쓣 젙웾븯怨 2X RT Pre-Mix (Solgent, Daejeon, South Korea)濡 complementary DNA (cDNA)瑜 빀꽦븯떎. 洹 떎쓬, manufacturer's protocol뿉 뵲씪 2X Taq PCR Pre-Mix (Solgent, Daejeon, South Korea)瑜 닔뻾븯떎. 빀꽦씠 걹궃 깦뵆 2% 븘媛濡쒖뒪 寃(Vivantis, Molecular Biology Grade, USA)濡 遺꾨━븳 뮘, chemiluminescence detection system (VilberLourmat, Everhardzell, Germany)쑝濡 씠誘몄瑜 珥ъ쁺븯떎. PCR쓣 닔뻾븯湲 쐞빐 궗슜븳 primer sequence뒗 Table 2 媛숇떎.

Primer sequence

Primer Forward (5'-3') Reverse (5'-3')
GAPDH AGGGCTGCTTTTAACTCTGGT CCCCACTTGATTTTGGAGGGA
FLRT3 GTAACAGGGGAACHCAGTGA AAAGGTAACCAGGGCCACAG


Wound healing scratch assay瑜 솢슜븳 꽭룷 씠룞뒫 솗씤

媛곴컖쓽 shRNA濡 transfection씠 맂 꽭룷瑜 24-well plate뿉 5 횞 104/well 留뚰겮 seeding 븯떎. 꽭룷媛 plate쓽 80% 젙룄 옄씪룄濡 諛곗뼇븳 뮘, 200 關L 硫멸퇏맂 뙆씠렖 똻쓣 씠슜븯뿬 씪젙븯寃 뒪겕옒移섎 궡뿀떎. 뒪겕옒移섎 궦 0떆媛 썑 12떆媛 썑쓽 궗吏꾩쓣 쁽誘멸꼍(Nikon eclipse Ts2R)쓣 씠슜븯뿬 珥ъ쁺븳 썑, 0떆媛꾧낵 12떆媛꾩뿉꽌쓽 꽭룷 씠룞 젙룄瑜 Image J 봽濡쒓렇옩(National Institute of Health, USA)쑝濡 젙웾븯뿬 遺꾩꽍븯떎.

MTT assay瑜 솢슜븳 꽭룷 利앹떇뒫 솗씤

븵꽑 궡슜怨 룞씪븯寃 transfection쓣 吏꾪뻾븳 썑, 96-well plate뿉 8 횞 103/well 留뚰겮 꽭룷瑜 seeding 븳 뮘, 떆媛 議곌굔(0, 24, 48, 72떆媛)뿉 留욊쾶 諛곗뼇븯떎. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent; Sigma, UK)瑜 well 떦 100 關L (2 mg/mL)濡 4떆媛 룞븞 泥섎━븳 뮘 깮꽦맂 formazan쓣 dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) 100 關L뿉 20遺 룞븞 끃떎. 洹 썑 microplate reader (Versamax microplate reader, Associates of cape cod incorporated, MA, USA)濡 540 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 0떆媛꾩쓽 씉愿묐룄 寃곌낵瑜 湲곗쑝濡 꽭룷 利앹떇 젙룄瑜 鍮꾧탳븯떎.

떒諛깆쭏 寃異 Western Blot 遺꾩꽍

shRNA瑜 씠슜븯뿬 FLRT3쓽 諛쒗쁽쓣 議곗젅븳 꽭룷뿉꽌 EzRIPA Lysis Kit (RIPA buffer and inhibitors) (DAWINBIO Inc, Gyeonggido, Koera)瑜 씠슜븯뿬 떒諛깆쭏쓣 異붿텧븯떎. 異붿텧맂 떒諛깆쭏 BCA protein assay reagent (Thermo Fisher Scientific, Waltham, MA, USA)쓣 씠슜븯뿬 20 關g쑝濡 젙웾븳 뮘, 10% sulfate polyacrylamide gel electrophoresis (SDS-PAGE)濡 遺꾨━븯떎. 遺꾨━맂 떒諛깆쭏 PVDF membrane (Milipore Co., NA, USA)쑝濡 씠룞떆궓 썑, 5% 깉吏 遺꾩쑀 0.1% Tween 20쓣 븿쑀븳 TBS-T瑜 궗슜븯뿬 1떆媛 룞븞 釉붾줈궧쓣 닔뻾븯떎. 씠썑, 1李 빆泥 p-ERK (1:1,000) (sc-7383, Santa Cruz., CA, USA) 棺-actin (1:1,000) (sc-4778, Santa Cruz., CA, USA)쓣 4꼦뿉꽌 븯猷 룞븞 諛섏쓳떆耳곕떎. TBS-T濡 membrane쓣 10遺 룞븞 3踰 꽭泥숉븳 썑, horseradish peroxidase (HRP) 寃고빀븳 2李 빆泥 (1:10,000) (31430, Invitrogen, CARLSBAD, CA)瑜 1떆媛 룞븞 諛섏쓳떆耳곕떎. 諛섏쓳씠 걹궃 membrane chemiluminescent ECL reagent (Thermo Fisher, Waltham, MA, USA)瑜 씠슜븯뿬 떒諛깆쭏쓣 寃異쒗븯怨 Image J 봽濡쒓렇옩쓣 씠슜븯뿬 냽룄瑜 遺꾩꽍븯떎.

넻怨 遺꾩꽍

SPSS (SPSS Inc,. IL, USA)瑜 씠슜븳 one-way ANOVA瑜 떎떆븯뿬 吏묐떒 媛꾩쓽 李⑥씠瑜 궡렣蹂댁븯떎. 쑀쓽 닔以 *P < 0.05, **P < 0.01, ***P < 0.001濡 븯떎.

寃 怨

떎뼇븳 꽭룷뿉꽌쓽 FLRT3쓽 諛쒗쁽 솗씤

룓뿉 議댁옱븳떎怨 븣젮吏 FLRT3 (Chen et al., 2009)媛 떎젣 룓븫뿉꽌 듅씠쟻쑝濡 諛쒗쁽븯뒗吏 솗씤븯湲 쐞빐 뿬윭 醫낅쪟쓽 븫 꽭룷 諛 洹몃줈遺꽣 쑀옒븳 븫以꾧린꽭룷(LCSC)濡쒕꽣 FLRT3쓽 諛쒗쁽쓣 鍮꾧탳븯떎(Fig. 1). 궗엺 븫 꽭룷 A549, Huh7, MCF7怨 媛 븫쑝濡쒕꽣 쑀옒맂 븫以꾧린꽭룷 LCSC, LiCSC, BCSC濡쒕꽣 RNA瑜 異붿텧븯怨 conventional PCR쓣 닔뻾븯뿬 FLRT3쓽 諛쒗쁽쓣 솗씤븳 寃곌낵, A549 LCSC뿉꽌 떎瑜 꽭룷뱾뿉 鍮꾪빐 FLRT3媛 넂寃 諛쒗쁽릺怨 엳쓬쓣 솗씤븷 닔 엳뿀쑝硫(Fig. 1A), 씠쓽 젙웾 洹몃옒봽(Fig. 1B)뿉꽌룄 룞씪븳 寃쏀뼢씠 솗씤릺뿀떎. 씠뒗 FLRT3媛 떎젣 룓븫쓣 쐞븳 몴쟻臾쇱쭏濡쒖꽌쓽 媛뒫꽦씠 엳쓬쓣 떆궗븳떎.

Fig. 1. The confirmation of FLRT3 expression in various cell lines and cancer stem cells.
RNA was extracted from human cancer cell lines and cancer stem cell lines, followed by cDNA synthesis, and PCR was conducted to verify the expression of FLRT3. (A) The results of FLRT3 expression by PCR. (B) The quantified data of the expressed FLRT3. FLRT3 from each cell was quantified relative to GAPDH expression.

FLRT3쓽 諛쒗쁽쓣 슚怨쇱쟻쑝濡 議곗젅븷 닔 엳뒗 shRNA 젣옉 諛 꽑젙

룓븫 꽭룷 룓븫쑀옒 븫以꾧린꽭룷뿉꽌 FLRT3쓽 諛쒗쁽 議곗젅씠 븫쓽 깮臾쇳븰쟻 湲곕뒫뿉 誘몄튂뒗 쁺뼢쓣 룊媛븯湲 쐞빐 FLRT3쓽 諛쒗쁽쓣 븯뼢 議곗젅븷 닔 엳뒗 shRNA series瑜 젣옉븯떎. 젣옉맂 shRNA series瑜 꽭룷뿉 24떆媛 룞븞 transfection 떆궓 썑 FLRT3쓽 諛쒗쁽쓣 솗씤븯떎(Fig. 2). 泥섎━맂 shRNA series 以 룓븫꽭룷씤 A549뿉꽌뒗 shFLRT3 #3怨 shFLRT3 #5媛(Fig. 2A), 룓븫쑀옒 븫以꾧린꽭룷씤 LCSC뿉꽌뒗 shFLRT3 #1怨 shFLRT3 #3媛 FLRT3 諛쒗쁽쓣 뼲젣븯뒗 슚怨쇨 媛옣 겙 寃껋쓣 솗씤븷 닔 엳뿀떎(Fig. 2B). 뵲씪꽌, shFLRT3 #3(씠븯 shFLRT3)媛 몢 븫꽭룷뿉꽌 FLRT3쓽 諛쒗쁽쓣 뼲젣븯뒗 슚怨쇨 媛옣 쎇뼱궃 寃껋쓣 솗씤븷 닔 엳뿀쑝硫, 븵쑝濡쒖쓽 떎뿕 紐⑤몢 씠 shFLRT3쓣 泥섎━븯뿬 깮臾쇳븰쟻 湲곕뒫 룊媛瑜 닔뻾븯떎.

Fig. 2. Selection of the most effective shRNA for regulating FLRT3 expression.
(A-B) Optimal shRNA decision for FLRT3 gene knock-down in transcriptomic level of cancer and cancer stem cells, respectively. (C-D) The quantitative analysis of FLRT3 expression in cancer and cancer stem cell. Data shown represent the mean ± SD (n=3). ***P < 0.001, **P < 0.01 compared with shScr.

룓븫怨 븫以꾧린꽭룷뿉꽌쓽 shRNA FLRT3뿉 쓽븳 媛먯냼맂 꽭룷 씠룞뒫怨 利앹떇뒫쓽 蹂솕

shFLRT3瑜 씠슜븯뿬 A549 LCSC뿉꽌 FLRT3 諛쒗쁽 媛먯냼瑜 쑀룄븳 뮘, 꽭룷쓽 깮臾쇳븰쟻 湲곕뒫 蹂솕瑜 룊媛븯떎. 븫쓽 二쇱슂 깮臾쇳븰쟻 듅吏뺤쑝濡쒕뒗 꽭룷 씠룞뒫怨 꽭룷 利앹떇뒫쓣 뱾 닔 엳떎(Shiga et al., 2015). 븫뿉꽌 꽭룷 씠룞뒫 븫 꽭룷쓽 떎瑜 議곗쭅씠굹 옣湲곕줈쓽 씠룞쓣 珥덈옒븯뿬 떎瑜 遺쐞濡쒖쓽 쟾씠瑜 媛뒫븯寃 븷 肉먮쭔 븘땲씪 (Krakhmal et al., 2015), 븫 꽭룷 利앹떇 떎뼇븳 꽭룷二쇨린 솢꽦뿉 愿젴맂 떒諛깆쭏뱾쓽 깮궛쓣 쑀룄븯뿬 鍮좊Ⅸ 꽭룷 꽦옣쓣 쑀룄븿쑝濡쒖뜥 利앷맂 꽭룷 利앹떇쓣 굹궦떎(Schiliro and Firestein, 2021).

癒쇱, 슦由щ뒗 shFLRT3媛 泥섎━릺뿀쓣 븣쓽 꽭룷 씠룞뒫쓣 솗씤븯湲 쐞븯뿬 wound healing assay瑜 吏꾪뻾븯떎(Fig. 3). Short hairpin FLRT3 (shFLRT3)媛 泥섎━맂 꽭룷瑜 24-well plate뿉 seeding 븳 뮘, 200 關L 硫멸퇏맂 뙆씠렖 똻쓣 씠슜븯뿬 洹좎씪븯寃 뒪겕옒移섎 궡뿀떎. 씠썑, 0떆媛꾧낵 12떆媛 뮘쓽 꽭룷 씠룞 젙룄瑜 쁽誘멸꼍쑝濡 궗吏 珥ъ쁺븯怨(Fig. 3A-B) Image J 봽濡쒓렇由쇱쓣 씠슜븯뿬 젙웾븳 洹몃옒봽(Fig. 3C-D)濡 옱李 솗씤븯떎. Fig. 3쓽 寃곌낵뿉꽌 蹂 닔 엳벏씠, A549 LCSC뿉꽌 紐⑤몢 12떆媛꾩㎏쓽 寃곌낵뿉꽌 媛곴컖쓽 꽭룷뿉 shFLRT3媛 泥섎━릺뿀쓣 븣 0떆媛꾩㎏쓽 寃곌낵뿉 鍮꾪빐 꽭룷 씠룞 젙룄媛 겕寃 媛먯냼븳 寃껋쓣 愿李고븷 닔 엳뿀떎.

Fig. 3. Decreased cell migration ability by shFLRT3 treatment in cancer and cancer stem cell.
To investigate the impact of reduced FLRT3 expression on cell migration ability, a wound healing assay was conducted. (A-B) Bright image of the migrated cell. (C-D) The calculated space by image J at 0 and 12 hrs. Data shown represent the mean ± SD (n=3). ***P < 0.001, **P < 0.01, and *P < 0.05 compared with 0 h.

떎쓬쑝濡쒕뒗, FLRT3媛 꽭룷뱾쓽 利앹떇뿉 엳뼱 뼱뼚븳 쁺뼢쓣 誘몄튂뒗吏瑜 룊媛븯湲 쐞븯뿬 shFLRT3媛 泥섎━맂 媛곴컖쓽 꽭룷瑜 96-well plate뿉 seeding 븳 뮘, 젙빐吏 떆媛 議곌굔뿉 留욎떠 MTT assay瑜 吏꾪뻾븯뿬 꽭룷 利앹떇뒫쓣 룊媛빐 蹂댁븯떎. 洹 寃곌낵, Fig. 4뿉꽌 蹂대뒗 寃껋쿂읆, A549 only LCSC only뒗 떆媛 쓽議댁쟻쑝濡 꽭룷 利앹떇씠 利앷븯뒗 寃껋쓣 愿李고븷 닔 엳뿀怨 씠윭븳 寃쏀뼢 shScr씠 泥섎━맂 꽭룷뱾뿉꽌룄 쑀궗븳 寃곌낵瑜 蹂댁떎. 븯吏留, shRNA濡 FLRT3쓽 諛쒗쁽쓣 媛먯냼떆궓 A549 LCSC뿉꽌 떎瑜 洹몃9뱾뿉 鍮꾪빐 媛먯냼맂 꽭룷 利앹떇뒫쓣 蹂댁엫쓣 솗씤븷 닔 엳뿀떎.

Fig. 4. Decreased cell proliferation ability by the downregulation of FLRT3.
(A) Lung cancer and (B) Lung cancer-derived stem cell. Data shown represent the mean ± SD (n=3). ***P < 0.001, and *P < 0.05 compared with 0 h.

씠瑜 넻빐 FLRT3쓽 諛쒗쁽 쑀룄뒗 룓븫怨 룓븫쑀옒 以꾧린꽭룷쓽 깮臾쇳븰쟻 湲곕뒫쓣 媛먯냼떆궓떎뒗 궗떎쓣 솗씤븯떎.

ERK signaling pathway瑜 넻븳 FLRT3쓽 깮臾쇳븰쟻 湲곕뒫 議곗젅 湲곗쟾

떎쓬쑝濡쒕뒗 븵꽑 FLRT3쓽 諛쒗쁽 쑀룄瑜 넻븳 깮臾쇳븰쟻 湲곕뒫뱾씠 꽭룷쓽 뼱뼡 떊샇湲곗쟾뿉 쁺뼢쓣 諛쏅뒗吏瑜 솗씤빐 蹂닿퀬옄 shRNA媛 泥섎━맂 꽭룷뱾뿉꽌쓽 꽭룷 떊샇 쟾떖 泥닿퀎瑜 룊媛빐 蹂댁븯떎.

븫꽭룷쓽 몴쟻 솢꽦솕맂 떊샇 쟾떖 寃쎈줈씤 extracellular signal-regulated kinase (ERK) signaling pathway뒗 꽭룷 媛 삉뒗 꽭룷 궡 떊샇 쟾떖뿉 愿뿬븯硫, 꽭룷쓽 떎뼇븳 湲곕뒫쓣 議곗젅븯뒗 以묒슂븳 떊샇 쟾떖 泥닿퀎濡 븣젮졇 엳떎(Ullah et al., 2022). 듅엳 ERK signaling pathway뿉 쓽븳 꽭룷 씠룞怨 利앹떇, 꽭룷 궗硫몄쓽 뿰愿꽦뿉 빐꽌뒗 씠誘 留롮 뿰援ш 릺뼱 엳떎(Degirmenci et al., 2020; Ren et al., 2019). 뵲씪꽌, 슦由щ뒗 븵꽑 깮臾쇳븰쟻 湲곕뒫 룊媛뿉꽌 FLRT3쓽 諛쒗쁽 議곗젅씠 꽭룷 씠룞怨 利앹떇뿉 쁺뼢쓣 以 닔 엳쓬쓣 솗씤븯怨, 씠寃껋씠 ERK 떊샇 쟾떖 湲곗쟾쓣 넻빐 議곗젅씠 媛뒫븳吏뿉 빐 western blot 遺꾩꽍쓣 씠슜븯뿬 protein level뿉꽌 솗씤빐 蹂댁븯떎.

Fig. 5뿉꽌 愿李고븷 닔 엳벏씠, shFLRT3媛 泥섎━맂 A549 LCSC뒗 떎瑜 洹몃9뱾怨쇰뒗 떖由 p-ERK 떒諛깆쭏쓽 媛먯냼맂 諛쒗쁽쓣 쑀룄븯쑝硫, 씠뒗 FLRT3쓽 諛쒗쁽 궙 ERK signaling pathway쓽 솢꽦쓣 쑀룄븳떎뒗 寃껋쓣 쓽誘명븯硫, 씠뒗 怨 꽭룷쓽 媛먯냼맂 꽭룷 씠룞 諛 利앹떇뒫쓣 珥덈옒븳떎뒗 寃껋쓣 쓽誘명븯硫, ERK 떊샇 쟾떖 湲곗쟾쓣 넻빐 깮臾쇳븰쟻 湲곕뒫 蹂솕瑜 珥덈옒븳떎뒗 寃껋쓣 굹궦떎.

Fig. 5. The change of ERK signaling pathway by shFLRT3 treatment.
(A-B) The expression of p-ERK protein in shFLRT3-treated cancer and cancer stem cell, respectively. (C-D) The quantification of p-ERK protein expression. Data shown represent the mean ± SD (n=3). ***P < 0.001, and **P < 0.01 compared with shScr.
怨 李

궃移섏꽦 吏덈퀝 以 븫뿉 쓽븳 궗留앸쪧 븘吏곴퉴吏 1쐞濡 굹궡怨 엳쑝硫, 씠以 룓븫 珥덇린 吏꾨떒뿉 쓽븳 移섎즺슚怨쇨 넂쑝굹 3湲곕줈 젒뼱뱾硫댁꽌 移섎즺젣媛 뾾怨, 理쒓렐 肄붾줈굹-19 媛먯뿼蹂 씠뒋濡 씤빐 븵쑝濡쒖쓽 룓吏덊솚쓣 룷븿븳 룓븫 솚옄뒗 袁몄엳 利앷븷 寃껋쑝濡 삁痢〓맂떎(Barta et al., 2019). 뵲씪꽌 룓븫뿉 븳 愿떖씠 넂븘吏怨 룓븫 移섎즺뿉 븳 떎뼇븳 쟾왂쟻 젒洹쇱쑝濡 留롮 移섎즺뿰援 諛 뿰援ш 吏꾪뻾 以묒뿉 엳떎(Sharma et al., 2019). 洹몃윭굹, 빆븫 移섎즺 썑 醫낆뼇 궡뿉 냼닔 옍옱븯뒗 븫以꾧린꽭룷源뚯 셿쟾엳 젣嫄고븯吏 紐삵븯뿬 옍議댄븯뒗 븫以꾧린꽭룷濡 씤빐 븫쓽 옱諛쒓낵 떎瑜 옣湲곕굹 議곗쭅쑝濡쒖쓽 쟾씠瑜 빐寃고븯吏 紐삵븯뒗 븳怨꾧 뿬쟾엳 궓븘엳떎(Hardavella et al., 2016; Cojoc et al., 2015; Suresh et al., 2016). 삉븳, 룓븫쓽 議곌린 吏꾨떒 珥덇린 醫낆뼇쓽 諛쒓껄쓣 媛뒫븯寃 븯怨, 쟻젅븳 移섎즺瑜 떎떆븷 닔 엳뼱 뼢썑 솚옄쓽 5뀈 깮議댁쑉쓣 겕寃 利앷떆궗 닔 엳떎. 肉먮쭔 븘땲씪 議곌린 吏꾨떒쓣 븳 솚옄쓽 빆븫 移섎즺 썑 삁썑媛 뜑 醫뗪린 븣臾몄뿉 議곌린 吏꾨떒 以묒슂꽦씠 몢릺怨 엳떎(Huang et al., 2022). 蹂 뿰援щ뒗 씠윭븳 湲곗〈쓽 빆븫 移섎즺쓽 븳怨꾨 洹밸났븯怨 빆븫 移섎즺쓽 깉濡쒖슫 쟾왂쓣 젣떆븯湲 쐞빐 룓븫 꽭룷 듅씠쟻쑝濡 議댁옱븯뿬 룓븫쓽 議곌린 吏꾨떒 臾쇱쭏濡쒖꽌쓽 媛뒫꽦씠 넂쑝硫, 룓븫쑀옒 븫以꾧린꽭룷源뚯 몴쟻쑝濡 븷 닔 엳뒗 떊洹 諛붿씠삤留덉빱瑜 諛쒓뎬븯怨좎옄 븯떎.

Fibronectin leucine rich transmembrane protein 3 (FLRT3)뒗 FLRT 떒諛깆쭏 怨꾩뿴쓽 援ъ꽦 떒諛깆쭏濡 븣젮졇 엳쑝硫, 떊옣, 뇤, 痍뚯옣, 룓뿉 議댁옱븯硫 꽭룷 젒李 諛 닔슜泥댁뿉꽌 떊샇 쟾떖怨 愿젴븳 湲곕뒫쓣 븯뒗 쑀쟾옄濡 븣젮졇 엳떎. 媛숈 FLRT 떒諛깆쭏 怨꾩뿴쓽 援ъ꽦 떒諛깆쭏씤 FLRT2쓽 븫뿉 븳 湲곕뒫 뿰援щ릺뼱 엳쑝굹 FLRT3 븫뿉 븳 뿰援щ뒗 蹂닿퀬맂 諛붽 뾾떎(Chen et al., 2009; Lacy et al., 1999, Guo et al., 2020). 뵲씪꽌 FLRT3쓽 떎젣 룓븫 꽭룷 룓븫쑀옒 븫以꾧린꽭룷뿉꽌쓽 듅씠쟻 諛쒗쁽쓣 솗씤븯怨 룓븫 吏꾨떒 諛 移섎즺 臾쇱쭏濡쒖꽌 옉슜 媛뒫꽦쓣 룊媛븳 뿰援щ뒗 슦由 뿰援ш 理쒖큹씠떎.

뿬윭 醫낆쓽 븫 꽭룷 洹몃줈遺꽣 쑀옒븳 븫以꾧린꽭룷뿉꽌쓽 FLRT3쓽 諛쒗쁽 솗씤쓣 넻빐 蹂 FLRT3 떒諛깆쭏씠 룓븫 듅씠쟻쑝濡 諛쒗쁽맂떎뒗 寃껋쓣 엯利앺븯怨(Fig. 1), 씠瑜 넻빐 FLRT3쓽 룓븫뿉꽌쓽 깮臾쇳븰쟻 湲곕뒫 寃利 떎뿕쓣 닔뻾븯떎. 씠뒗 異뷀썑 엫긽 쟻슜쓣 쐞븳 룓븫 듅씠쟻 吏꾨떒留덉빱濡쒖꽌쓽 媛뒫꽦씠 넂쓬쓣 떆궗븳떎. 뜑슧씠, 룓븫肉먮쭔 븘땲씪 룓븫쑀옒 븫以꾧린꽭룷뿉 븳 씠쟾 뿰援щ뱾뿉꽌 蹂닿퀬媛 뾾뿀뜕 븫以꾧린꽭룷뿉꽌쓽 깮臾쇳븰쟻 湲곕뒫 룊媛瑜 넻빐 FLRT3媛 떎吏덉쟻쑝濡 룓븫뿉꽌 옉슜맆 닔 엳뒗 듅씠쟻 吏꾨떒 臾쇱쭏씠씪뒗 寃껋쓣 異붽쟻쑝濡 엯利앺븯떎. 젣옉맂 shRNA FLRT3 泥섎━ 꽭룷뱾쓽 깮臾쇳븰쟻 룊媛씤 꽭룷 씠룞뒫, 꽭룷 利앹떇뒫 寃곌낵瑜 蹂댁븯쓣 븣 씠뒗 吏꾨떒留덉빱濡쒖꽌쓽 媛뒫꽦肉먮쭔 븘땲씪 移섎즺 썑蹂 臾쇱쭏濡쒖꽌룄 媛뒫꽦 엳쓬씠 Fig. 3怨 4瑜 넻빐 엯利앸릺뿀떎. 씠윭븳 寃곌낵瑜 굹궡뒗 썝씤쑝濡 FLRT3媛 꽭룷 利앹떇뿉 愿젴릺뼱 엳뒗 ERK pathway瑜 솢꽦솕떆궎뒗吏瑜 룊媛빐 蹂댁븯怨, Fig. 5뿉꽌 솗씤븷 닔 엳벏씠 FLRT3쓽 諛쒗쁽 蹂솕媛 븫쓽 깮臾쇳븰쟻 湲곕뒫怨 愿젴씠 源딆 ERK 떊샇 쟾떖 泥닿퀎瑜 寃잛쑝濡 p-ERK 떒諛깆쭏쓽 諛쒗쁽쓣 媛먯냼떆궡쑝濡쒖뜥 깮臾쇳븰쟻 湲곕뒫쓣 議곗젅븯뒗 湲곗쟾쑝濡 利앸챸븯떎.

蹂 뿰援 寃곌낵瑜 醫낇빀빐 蹂댁븯쓣 븣, FLRT3뒗 룓븫 듅씠쟻쑝濡 諛쒗쁽븯뒗 臾쇱쭏濡 룓븫쓽 議곌린 吏꾨떒씠 媛뒫븯硫, 룓븫쑀옒 븫以꾧린꽭룷뿉꽌룄 諛쒗쁽븯뿬 븫以꾧린꽭룷瑜 몴쟻쑝濡 븯吏 紐삵븯뒗 湲곗〈쓽 빆븫 移섎즺쓽 븳怨꾨 洹밸났븷 닔 엳뒗 몴쟻 臾쇱쭏濡 옉슜븷 닔 엳쓣 寃껋쑝濡 궗猷뚮맂떎. 삉븳, FLRT3쓽 諛쒗쁽 議곗젅씠 룓븫 꽭룷 룓븫쑀옒 븫以꾧린꽭룷쓽 씠룞뒫怨 利앹떇뒫쓣 ERK 떊샇 쟾떖 泥닿퀎瑜 넻빐 媛먯냼떆궗 닔 엳쓬쓣 꽭怨 理쒖큹濡 뿰援ы븯쑝硫, 씠瑜 넻빐 FLRT3뒗 룓븫 듅씠 몴쟻 臾쇱쭏濡쒖꽌쓽 媛뒫꽦肉먮쭔 븘땲씪 移섎즺 臾쇱쭏濡쒖꽌룄 옉슜븷 닔 엳쓬쓣 쓽誘명븳떎. 뵲씪꽌 FLRT3 諛붿씠삤留덉빱쓽 솢슜 떊洹 빆븫 移섎즺젣쓽 媛쒕컻 諛 吏꾨떒 臾쇱쭏濡쒖꽌쓽 媛移섎 엯利앺븯뿬 룓븫 移섎즺쓽 깉濡쒖슫 쟾왂쟻 젒洹쇱쑝濡 솢슜맆 닔 엳쓣 寃껋씠떎.

ACKNOWLEDGEMENT

Following are results of a study on the "Leaders in Industry-university Cooperation 3.0" Project and Basic Science Research Program (2020R1F1A1053663) supported by the Ministry of Education and National Research Foundation of Korea.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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