MicroRNA-196b Inhibits Cell Growth and Metastasis of Lung Cancer ...

Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 DOI: 10.1159/000481559 © 2017 The Author(s) online:September 27, 2017 www.karger.com/cpb Published online: Published by S. Karger AG, Basel and Biochemistry Published www.karger.com/cpb September 27, 2017

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Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2 Accepted: June 23, 2017

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Original Paper

MicroRNA-196b Inhibits Cell Growth and Metastasis of Lung Cancer Cells by Targeting Runx2 Xiaoxue Baia Lin Menga Shucheng Huac

Huijie Suna

Zhuo Lib

Xiufang Zhangc

Cadre’s Ward, The First Hospital of Jilin University, Changchun, bDepartment of Endocrinology, The First Hospital of Jilin University, Changchun, cDepartment of Respiratory Medicine, The First Hospital of Jilin University, Changchun, China

a

Key Words 0L5E‡/XQJFDQFHU‡5XQ[‡3,.$.7*6.DŽSDWKZD\‡6PDGSDWKZD\‡-1.SDWKZD\ Abstract Background/Aims: Lung cancer is one of the most common causes of cancer related deaths worldwide. The role of several microRNAs (miRNAs) including miR-196b in different cancers has already been established. The study was aimed to explore the role of miR-196b in lung cancer and its possible underlying mechanism. Methods: Human lung cancer cell line A549 was transfected with miR-196b mimic, miR-196b inhibitor and corresponding controls. Then cell viability, migration, invasion, and apoptosis of A549 lung cancer cells either with overexpression RU ZLWK VXSSUHVVLRQ RI PL5E ZHUH HVWLPDWHG VHTXHQWLDOO\ 1H[W GXDO OXFLIHUDVH DFWLYLW\ assay was performed to clarify whether Runx2 was a direct target of miR-196b. Finally, the expressions of main factors associated with epithelial mesenchymal transition (EMT), PI3K/ $.7*6.DŽ 6PDG DQG -1. SDWKZD\V ZHUH GHWHFWHG E\ ZHVWHUQ EORW Results: MiR-196b H[SUHVVLRQZDVVLJQLÀFDQWO\GHFUHDVHGLQ$+DQG+FHOOOLQHVFRPSDUHGZLWKLQ WI-38 and HEL-1 cell lines. Overexpression of miR-196b suppressed cell viability, migration, LQYDVLRQDQGLQGXFHGDSRSWRVLVDVZHOODVLQKLELWHG7*)DŽLQGXFHG(07SURFHVVLQ$FHOOV In addition, Runx2 was a putative target of miR-196b, and Runx2 silence remarkably increased cell apoptosis and abolished the promotive effects of miR-196b suppression on cell viability, migration and invasion. Finally, miR-196b also mediated its action by inactivation of PI3K/ $.7*6.DŽ6PDGDQG-1.SDWKZD\VE\GRZQUHJXODWLRQRI5XQ[Conclusion: MiR-196b functions as a tumor suppressor that inhibited cell growth and metastasis of lung cancer cells E\WDUJHWLQJ5XQ[7KHVHÀQGLQJVSURYLGHGIXUWKHUHYLGHQFHVIRUWUHDWPHQWRIOXQJFDQFHU Introduction

© 2017 The Author(s) Published by S. Karger AG, Basel

Shucheng Hua

Department of Respiratory Medicine, The First Hospital of Jilin University, No. 71, Xinmin Street, Changchun 130021 (China) E-Mail [email protected]

Downloaded by: 154.16.58.156 - 11/28/2017 7:27:47 PM

Lung cancer is one of the most common malignancies and remains the leading cause of cancer-related deaths worldwide [1]. In the various histological subtypes, non-small cell

Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 www.karger.com/cpb and Biochemistry Published online: September 27, 2017

Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2

lung cancer (NSCLC) is the most prevalent subtype, which accounts for approximately 80% in all cases of diagnosed with lung cancer [1, 2]. NSCLC is characterized by a high mortality, ƒ† –Š‡ ‘˜‡”ƒŽŽ ϐ‹˜‡Ǧ›‡ƒ” •—”˜‹˜ƒŽ ”ƒ–‡ ”‡ƒ‹• Ž‡•• –Šƒ ͳͷΨ ȏʹȐǤ ‡”‹‘—•Ž›ǡ ‘•– ‘ˆ –Š‡ patients are diagnosed in the latest stage of lung cancer, especially in stage IV [3]. Despite the different therapeutic methods such as chemotherapy, radiotherapy, and surgery were utilized to improve the survival rate of patients with lung cancer, the inevitable metastasis ”‡ƒ‹ƒ‰”‡ƒ–…ŠƒŽŽ‡‰‡ˆ‘”–Š‡–”‡ƒ–‡–‘ˆŽ—‰…ƒ…‡”ȏͶǡͷȐǤ‡…‡ǡ–Š‡”‡‹•ƒ—”‰‡– need to introduce novel and effective therapies for treating lung cancer. MicroRNAs (miRNAs or miRs) are a small class of single-stranded and non-coding RNAs, which are participated in post-transcriptional regulation of more than 70% human genes ‡š’”‡••‹‘ȏͷǡ͸ȐǤ……——Žƒ–‹‰‡˜‹†‡…‡•‹†‹…ƒ–‡†–Šƒ–‹•’Žƒ›…”—…‹ƒŽ”‘Ž‡•‹ƒ™‹†‡ range of cellular biological processes, including cell division, proliferation, differentiation, metastasis and apoptosis of various cancers [7, 8]. In the studies of lung cancer, Yan et alǤ †‡‘•–”ƒ–‡† –Šƒ– ‹Ǧ͹͸ͳ ’”‘‘–‡† …‡ŽŽ ’”‘Ž‹ˆ‡”ƒ–‹‘ ƒ† ‡–ƒ•–ƒ•‹• ‘ˆ  „› targeting ING4 and TIMP2 [9]. In addition, Jiang et al. revealed that miR-21 suppressed the ’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‹‰”ƒ–‹‘‘ˆŽ—‰…ƒ…‡”ͷͶͻ…‡ŽŽ•ȏͳͲȐǤŽ–Š‘—‰Š‹•™‡”‡‹˜‘Ž˜‡† ‹–Š‡†‡˜‡Ž‘’‡–ǡ’”‘‰”‡••‹‘ƒ†‡–ƒ•–ƒ•‹•‘ˆŽ—‰…ƒ…‡”ǡ–Š‡‡ˆˆ‡…–‘ˆ‹Ǧͳͻ͸„‘ lung cancer was not straightforward [11]. ‹Ǧͳͻ͸„ ‹• ƒ ‡„‡” ‘ˆ ‹Ǧͳͻ͸ ˆƒ‹Ž›ǡ ™Š‹…Š Šƒ• „‡‡ ˜ƒŽ‹†ƒ–‡† ƒ• –—‘” suppressor in various cancers [12, 13]. As Rebucci et alǤ †‹•’Žƒ›‡† –Šƒ– ‹Ǧͳͻ͸„ ƒ• ƒ –—‘”•—’’”‡••‘”–Šƒ–•—’’”‡••‡†…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‹†—…‡†ƒ’‘’–‘•‹•‹‡’ ʹ…‡ŽŽ• [13]. Moreover, Bhatia et alǤƒŽ•‘ˆ‘—†–Šƒ– –Š‡”‘Ž‡‘ˆ‹Ǧͳͻ͸„ƒ•–—‘”•—’’”‡••‘”‹ „Ž‘‘†…ƒ…‡”•ȏͳͶǡͳͷȐǤ Ž—‰…ƒ…‡”ǡ”‡…‡–•–—†‹‡••Š‘™‡†–Šƒ–‹Ǧͳͻ͸„’”‘‘–‡†…‡ŽŽ invasion and induced epithelial mesenchymal transition (EMT) process in NSCLC cells [1, ͳ͸ȐǤ‹—Ž–ƒ‡‘—•Ž›ǡ‡ŽŽ‡œet alǤˆ‘—†–Šƒ–‹Ǧͳͻ͸„ƒ•ƒ–—‘”•—’’”‡••‘”™Š‘•‡•‹Ž‡…‹‰ early in lung carcinogenesis might provide a selective growth advantage to pre-malignant …‡ŽŽ•ȏͳ͹ȐǤ‘™‡˜‡”ǡ–Š‡†‡–ƒ‹Ž”‘Ž‡•‘ˆ‹Ǧͳͻ͸„‹Ž—‰…ƒ…‡”…‡ŽŽ•’”‘Ž‹ˆ‡”ƒ–‹‘ǡ‡–ƒ•–ƒ•‹• and apoptosis have not been investigated. Runt-related transcription factor 2 (Runx2) belongs to the Runx family, which is known to be an important regulator of organogenesis and cell differentiation [18]. Abnormal expressions of Runx genes are associated with variety of cancers, especially breast and osteosarcoma [18, 19]. Moreover, the effect of Runx2 on lung cancer has also been explored ‹•‡˜‡”ƒŽ•–—†‹‡•ȏʹͲǦʹʹȐǤ‘™‡˜‡”ǡ™Š‡–Š‡”—šʹƒ…–‡†ƒ•ƒ‡›”‡‰—Žƒ–‘”‹Ž—‰…ƒ…‡” cells proliferation, metastasis and apoptosis remain unclear.  –Š‹• •–—†›ǡ ™‡ ƒ‹‡† –‘ ‡š’Ž‘”‡ –Š‡ ‡ˆˆ‡…– ‘ˆ ‹Ǧͳͻ͸„ ‹ Ž—‰ …ƒ…‡”Ǥ ‹”•–Ž›ǡ ™‡ ‡šƒ‹‡†–Š‡‡š’”‡••‹‘•‘ˆ‹Ǧͳͻ͸„‹Ž—‰…ƒ…‡”…‡ŽŽŽ‹‡•ƒ†‘”ƒŽŽ—‰…‡ŽŽŽ‹‡•Ǥ Š‡ ™‡ —•‡† Ǧͺǡ ”ƒ•™‡ŽŽǡ ϐŽ‘™ …›–‘‡–”› ƒ••ƒ›• –‘ ƒƒŽ›œ‡ ͷͶͻ …‡ŽŽ• ˜‹ƒ„‹Ž‹–›ǡ migration, invasion and apoptosis. Lastly, the key factors expressions of apoptosis, EMT, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/ glycogen synthase kinase 3 „‡–ƒȋ ͵ȾȌǡƒ†ǡƒ† —Ǧ–‡”‹ƒŽ‹ƒ•‡•ȋ Ȍ’ƒ–Š™ƒ›•™‡”‡†‡–‡…–‡†„›™‡•–‡” blot. Our study will provide a new idea and theoretical basis for treatment of lung cancer. Materials and Methods Cell culture and treatment —ƒŽ—‰…ƒ…‡”…‡ŽŽŽ‹‡•ͷͶͻǡͳ͸ͷͲƒ†ͳʹͻͻ…‡ŽŽ•ƒ†Š—ƒŽ—‰…‡ŽŽŽ‹‡• Ǧ͵ͺƒ†Ǧ 1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). All these cells were ƒ‹–ƒ‹‡† ‹ ‘•™‡ŽŽ ƒ” ‡‘”‹ƒŽ •–‹–—–‡ ȋ Ȍ ͳ͸ͶͲ ‡†‹— •—’’Ž‡‡–‡† ™‹–Š ͳέ ƒ–‹„‹‘–‹…Ǧ ƒ–‹›…‘–‹… ‹š–—”‡ ȋ ‹„…‘ǡ ”ƒ† •Žƒ†ǡ Ȍ ƒ† ͳͲΨ ˆ‡–ƒŽ „‘˜‹‡ •‡”— ȋ ǡ ›…Ž‘‡ ƒ„‘”ƒ–‘”‹‡•ǡ ‘‰ƒǡȌƒ–͵͹鋐ƒƒ–‘•’Š‡”‡‘ˆͷΨ2ǦͻͷΨƒ‹”Ǥ”ƒ•ˆ‘”‹‰‰”‘™–Šˆƒ…–‘”Ǧ„‡–ƒͳȋ ǦȾͳǡͳͲ ng/ml) was used to induce EMT.

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Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 www.karger.com/cpb and Biochemistry Published online: September 27, 2017

Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2

Cell transfection ‹Ǧͳͻ͸„‹‹…ǡ‹Ǧͳͻ͸„‹Š‹„‹–‘”ƒ†…‘””‡•’‘†‹‰…‘–”‘Ž•™‡”‡•›–Š‡•‹œ‡†„› ‡‡Šƒ”ƒ Co. (Shanghai, China). In addition, Runx2 targeted small interfering RNA (si-Runx2) was also constructed by

‡‡Šƒ”ƒ‘ǤȋŠƒ‰Šƒ‹Ȍ–‘‹Š‹„‹–—šʹ‡š’”‡••‹‘Ǥ „”‹‡ˆǡͳέͳͲͷ…‡ŽŽ•™‡”‡…—Ž–—”‡†‹͸Ǧ™‡ŽŽ’Žƒ–‡ and all transfection cells were accomplished using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer’s protocol. Cell viability assay ‡ŽŽ• ™‡”‡ •‡‡†‡† ‹ ͻ͸Ǧ™‡ŽŽ ’Žƒ–‡ ™‹–Š ͷͲͲͲ …‡ŽŽ•Ȁ™‡ŽŽǡ ƒ† …‡ŽŽ ˜‹ƒ„‹Ž‹–› ™ƒ• ƒ••‡••‡† „› ƒ ‡ŽŽ ‘—–‹‰‹–ǦͺȋǦͺǡ‘Œ‹†‘‘Ž‡…—Žƒ”‡…Š‘Ž‘‰‹‡•ǡ ƒ‹–Š‡”•„—”‰ǡȌǤ”‹‡ϐŽ›ǡƒˆ–‡”–”ƒ•ˆ‡…–‹‘ˆ‘” ͶͺŠǡ–Š‡ͳͲɊŽǦͺ•‘Ž—–‹‘™ƒ•ƒ††‡†–‘–Š‡…—Ž–—”‡‡†‹—ǡƒ†–Š‡…—Ž–—”‡•™‡”‡‹…—„ƒ–‡†ˆ‘”ͳŠ ƒ–͵͹鋐Š—‹†‹ϐ‹‡†ͻͷΨƒ‹”ƒ†ͷΨ2ǤŠ‡ƒ„•‘”„ƒ…‡™ƒ•‡ƒ•—”‡†ƒ–ͶͷͲ—•‹‰ƒ‹…”‘’Žƒ–‡ ‡ƒ†‡”ȋ‹‘Ǧƒ†ǡ‡”…—Ž‡•ǡȌǤ Migration and invasion assay ‡ŽŽ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘ƒ••ƒ›•™‡”‡†‡–‡”‹‡†„›—•‹‰ƒ‘†‹ϐ‹‡†–™‘Ǧ…Šƒ„‡”…Šƒ„‡”•™‹–Š ƒ’‘”‡•‹œ‡‘ˆͺǤ ‘”‹‰”ƒ–‹‘ƒ••ƒ›ǡ…‡ŽŽ••—•’‡†‡†‹ʹͲͲρŽ‘ˆ•‡”—Ǧˆ”‡‡‡†‹—™‡”‡•‡‡†‡†‘ –Š‡—’’‡”…‘’ƒ”–‡–‘ˆʹͶǦ™‡ŽŽ”ƒ•™‡ŽŽ…—Ž–—”‡…Šƒ„‡”ǡƒ†͸ͲͲρŽ‘ˆ…‘’Ž‡–‡‡†‹—™ƒ•ƒ††‡† –‘–Š‡Ž‘™‡”…‘’ƒ”–‡–Ǥˆ–‡”‹…—„ƒ–‹‘ˆ‘”ͶͺŠƒ–͵͹ιǡ…‡ŽŽ•™‡”‡ϐ‹š‡†™‹–Š‡–Šƒ‘ŽǤ‘Ǧ–”ƒ˜‡”•‡† …‡ŽŽ•™‡”‡”‡‘˜‡†ˆ”‘–Š‡—’’‡”•—”ˆƒ…‡‘ˆ–Š‡ϐ‹Ž–‡”…ƒ”‡ˆ—ŽŽ›™‹–Šƒ…‘––‘•™ƒ„Ǥ”ƒ˜‡”•‡†…‡ŽŽ•‘–Š‡ Ž‘™‡”•‹†‡‘ˆ–Š‡ϐ‹Ž–‡”™‡”‡•–ƒ‹‡†™‹–ŠͲǤͳΨ…”›•–ƒŽ˜‹‘Ž‡–ȋ‡”…ǡƒ”•–ƒ†–ǡ ‡”ƒ›Ȍƒ†…‘—–‡†Ǥ ‘” invasion assay, the invasion capacity detection was conducted the same as migration assay, except utilizing ƒ–”‹‰‡Ž…‘ƒ–‡†‹•‡”–•™‹–Šͺ’‘”‡•‹œ‡ȋ…ƒ–ƒŽ‘‰‘Ǥ͵ͷͶͶͺͲǡ‹‘•…‹‡…‡•Ȍȏʹ͵ȐǤ Apoptosis assay ‡ŽŽ ƒ’‘’–‘•‹• ™ƒ• “—ƒ–‹ϐ‹‡† ™‹–Š ƒ ϐŽ‘™ …›–‘‡–”› —•‹‰ ’”‘’‹†‹— ‹‘†‹†‡ ȋ Ȍ ƒ† ϐŽ—‘”‡•…‡‹ ‹•‘–Š‹‘…›ƒ–‡ȋ ȌǦ…‘Œ—‰ƒ–‡†‡š‹•–ƒ‹‹‰ȋ‡›‘–‹‡‹‘–‡…Š‘Ž‘‰›ȌǤ”‹‡ϐŽ›ǡ…‡ŽŽ•™‡”‡™ƒ•Š‡† three times in phosphate buffered saline (PBS) and then were re-suspended in the staining buffer. —„•‡“—‡–Ž›ǡͳͲɊŽ Ǧ‡š‹ȋʹͲɊ‰ȀŽȌƒ†ͷɊŽ ȋͷͲɊ‰ȀŽȌ™‡”‡‹š‡†™‹–Š–Š‡•‡…‡ŽŽ•Ǥˆ–‡” ‹…—„ƒ–‹‘ˆ‘”ͳͲ‹‹ƒ†ƒ”ƒ–”‘‘–‡’‡”ƒ–—”‡ǡ–Š‡‹š–—”‡•™‡”‡‡šƒ‹‡†—•‹‰–Š‡ …ƒϐŽ‘™ …›–‘‡–”›•›•–‡ȋ‡…ƒ‘—Ž–‡”ǡ —ŽŽ‡”–‘ǡǡȌǤŠ‡†ƒ–ƒ™‡”‡ƒƒŽ›œ‡†„›—•‹‰ Ž‘™ ‘•‘ˆ–™ƒ”‡ (Tree Star, Inc., Ashland, OR, USA). qRT-PCR Total RNA was extracted from cells using Trizol reagent (Life Technologies Corporation, Carlsbad, CA, Ȍ ƒ……‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ…–—”‡”ǯ• ‹•–”—…–‹‘•Ǥ Š‡ –Š‡ ϐ‹”•–Ǧ•–”ƒ† …‘’Ž‡‡–ƒ”› • ȋ…•Ȍ ™‡”‡•›–Š‡•‹œ‡†—•‹‰ͳɊ‰•„›”‹‡…”‹’–ͳ•––”ƒ†…›–Š‡•‹•‹–ȋƒƒƒ‹‘ǡ‹‰ƒǡ ƒ’ƒȌǤ The One Step SYBR® PrimeScript®PLUS RT-RNA PCR Kit (TaKaRa) was used for the Real-Time PCR analysis –‘–‡•––Š‡‡š’”‡••‹‘Ž‡˜‡Ž•‘ˆ‹Ǧͳͻ͸„‹…‡ŽŽ•Ǥ͸™ƒ•—•‡†–‘‘”ƒŽ‹œ‡–Š‡‹Ǧͳͻ͸„Ž‡˜‡Ž•ƒ†

™ƒ•—•‡†–‘‘”ƒŽ‹œ‡‘–Š‡”•Ž‡˜‡Ž•Ǥƒ–ƒ™‡”‡ƒƒŽ›œ‡†™‹–Š–Š‡ʹǦȟȟ– method. Dual luciferase activity assay The 3’UTR target site was generated by PCR and the luciferase reporter constructs with the Runx2 ͵ǯ …ƒ””›‹‰ ƒ ’—–ƒ–‹˜‡ ‹Ǧͳͻ͸„Ǧ„‹†‹‰ •‹–‡ ‹–‘ ’‹Ǧ”‡’‘”– ˜‡…–‘” ȋ”‘‡‰ƒǡ ƒ†‹•‘ǡ  ǡ Ȍ ™‡”‡ƒ’Ž‹ϐ‹‡†„›Ǥ‡ŽŽ•™‡”‡…‘Ǧ–”ƒ•ˆ‡…–‡†™‹–Š–Š‡”‡’‘”–‡”…‘•–”—…–ǡ…‘–”‘Ž˜‡…–‘”ƒ†‹Ǧͳͻ͸„ mimic or mimic control using Lipofectamine 3000 (Life Technologies). Reporter assays were done using the dual-luciferase assay system (Promega) following to the manufacturer’s information. Western blot The protein used for western blot was extracted using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Basle, ™‹–œ‡”Žƒ†ȌǤ Š‡ …‘…‡–”ƒ–‹‘• ‘ˆ ’”‘–‡‹ ‡š–”ƒ…–• ™‡”‡ “—ƒ–‹ϐ‹‡† —•‹‰ –Š‡ ̻ ”‘–‡‹ ••ƒ› ‹– ȋ‹‡”…‡ǡ’’Ž‡–‘ǡ ǡȌǤŠ‡™‡•–‡”„Ž‘–•›•–‡™ƒ•‡•–ƒ„Ž‹•Š‡†—•‹‰ƒ‹‘Ǧƒ†‹•Ǧ”‹• ‡Ž•›•–‡ ƒ……‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ…–—”‡”ǯ• ‹•–”—…–‹‘•Ǥ ”‹ƒ”› ƒ–‹„‘†‹‡• ™‡”‡ ’”‡’ƒ”‡† ‹ ͷΨ „Ž‘…‹‰ „—ˆˆ‡”

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Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 www.karger.com/cpb and Biochemistry Published online: September 27, 2017

Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2

at a dilution of 1:1, 000 and were incubated with the membranes at 4°C overnight. Thereafter, the mem„”ƒ‡•™‡”‡™ƒ•Š‡†ƒ†‹…—„ƒ–‡†™‹–Š•‡…‘†ƒ”›ƒ–‹„‘†›ȋ†‹Ž—–‹‘‘ˆͳǣͷͲͲͲȌƒ”‡†„›Š‘”•‡”ƒ†‹•Š ’‡”‘š‹†ƒ•‡ ˆ‘” ͳ Š ƒ– ”‘‘ –‡’‡”ƒ–—”‡Ǥ ˆ–‡” ”‹•‹‰ǡ –Š‡ ’‘Ž›˜‹›Ž‹†‡‡ †‹ϐŽ—‘”‹†‡ ȋ Ȍ ‡„”ƒ‡• ȋ‹ŽŽ‹’‘”‡ǡ‡†ˆ‘”†ǡǡȌ…ƒ””‹‡†„Ž‘–•ƒ†ƒ–‹„‘†‹‡•™‡”‡–”ƒ•ˆ‡””‡†‹–‘–Š‡‹‘Ǧƒ†Š‡‹‘…̻ XRS system. The immunoreactive protein bands were visualized using chemiluminescence (PerkinElmer, ‡ŽŽ‡•Ž‡›ǡǡȌǤ Statistical analysis All experiments were repeated three times. The results of multiple experiments are presented as mean ± standard derivations (SD). Statistical analyses were performed using SPSS 19.0 statistical software (SPSS Inc., Chicago, IL). The P-values were calculated using one-way analysis of variance (ANOVA). A P-value of < ͲǤͲͷ™ƒ•…‘•‹†‡”‡†–‘‹†‹…ƒ–‡ƒ•–ƒ–‹•–‹…ƒŽŽ›•‹‰‹ϐ‹…ƒ–”‡•—Ž–Ǥ

Results

MiR-196b was low expressed in different lung cancer cells Š‡‡š’”‡••‹‘Ž‡˜‡Ž•‘ˆ‹Ǧͳͻ͸„™‡”‡ƒ••‡••‡†‹†‹ˆˆ‡”‡–…‡ŽŽŽ‹‡•‘ˆŽ—‰…ƒ…‡” …‡ŽŽŽ‹‡•ͷͶͻǡͳ͸ͷͲƒ†ͳʹͻͻƒ†Š—ƒ‘”ƒŽŽ—‰…‡ŽŽŽ‹‡• Ǧ͵ͺƒ†Ǧͳ„› “ǦǤ ŽŽ –Š‡ Ž—‰ …ƒ…‡” …‡ŽŽ Ž‹‡• ”‡˜‡ƒŽ‡† ”‡†—…‡† ‡š’”‡••‹‘ Ž‡˜‡Ž• ‘ˆ ‹Ǧͳͻ͸„ compared to human lung cell lines (P < 0.01 or P < 0.001, ‹‰Ǥͳ). The expression level was ƒš‹ƒŽŽ›”‡†—…‡†‹–Š‡ͷͶͻŽ—‰…ƒ…‡”…‡ŽŽŽ‹‡ǡŠ‡…‡ͷͶͻ…‡ŽŽŽ‹‡™ƒ•…Š‘•‡‹–Š‡ following experiments. MiR-196b suppressed cell viability, migration, invasion, and induced apoptosis ‘ ƒ••‡•• –Š‡ ”‘Ž‡ ‘ˆ ‹Ǧͳͻ͸„ ‹ …‡ŽŽ ˜‹ƒ„‹Ž‹–›ǡ ‹‰”ƒ–‹‘ǡ ‹˜ƒ•‹‘ǡ ƒ† ƒ’‘’–‘•‹•ǡ ͷͶͻ…‡ŽŽ•™‡”‡–”ƒ•ˆ‡…–‡†™‹–Š‹Ǧͳͻ͸„‹‹…ǡ‹Ǧͳͻ͸„‹Š‹„‹–‘”ƒ†…‘””‡•’‘†‹‰ …‘–”‘Ž•Ǥ‡•—Ž–••Š‘™‡†–Šƒ––Š‡‡š’”‡••‹‘Ž‡˜‡Ž‘ˆ‹Ǧͳͻ͸„™ƒ••‹‰‹ϐ‹…ƒ–Ž›—’Ǧ”‡‰—Žƒ–‡† „›‹Ǧͳͻ͸„‹‹…ƒ††‘™Ǧ”‡‰—Žƒ–‡†„›‹Ǧͳͻ͸„‹Š‹„‹–‘”ȋP < 0.01 or P < 0.001, ‹‰Ǥ 2AȌǤ –‡”•‘ˆ…‡ŽŽ˜‹ƒ„‹Ž‹–›ǡ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘ǡ‹Ǧͳͻ͸„‘˜‡”‡š’”‡••‹‘”‡ƒ”ƒ„Ž› †‡…”‡ƒ•‡†…‡ŽŽ•˜‹ƒ„‹Ž‹–›ǡ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘‹ͷͶͻ…‡ŽŽ•…‘’ƒ”‡†–‘‹–•…‘–”‘Ž‰”‘—’ (PδͲǤͲͷȌǤ–•ƒ‡–‹‡ǡ–Š‡•‡‡ˆˆ‡…–•™‡”‡”‡˜‡”•‡†„›‹Ǧͳͻ͸„•—’’”‡••‹‘ȋPδͲǤͲͷǡ ‹‰Ǥ 2B-2D).  –‡”•‘ˆ…‡ŽŽƒ’‘’–‘•‹•ǡ–Š‡’‡”…‡–ƒ‰‡‘ˆƒ’‘’–‘–‹……‡ŽŽ•™ƒ••‹‰‹ϐ‹…ƒ–Ž›‡Šƒ…‡† „›‹Ǧͳͻ͸„‘˜‡”‡š’”‡••‹‘…‘’ƒ”‡†–‘…‘–”‘ŽȋP < 0.001, ‹‰Ǥʹ). There was no obviously ‡ˆˆ‡…– ‘ˆ ‹Ǧͳͻ͸„ •—’’”‡••‹‘ ‘ …‡ŽŽ ƒ’‘’–‘•‹•Ǥ ‡ƒ™Š‹Ž‡ǡ –Š‡ ™‡•–‡” „Ž‘– ”‡•—Ž–• ”‡˜‡ƒŽ‡†–Šƒ–‹Ǧͳͻ͸„‘˜‡”‡š’”‡••‹‘†‘™Ǧ”‡‰—Žƒ–‡†…ŽǦʹ‡š’”‡••‹‘ǡ—’Ǧ”‡‰—Žƒ–‡†ƒš expression as well as activated cleaved-caspase-3 and cleaved-caspase-9 expressions. MiRͳͻ͸„•—’’”‡••‹‘•Š‘™‡†ƒ‘’’‘•‹–‡”‡•—Ž–•‘ˆ…ŽǦʹƒ†ƒš‡š’”‡••‹‘•ǤŠ‡”‡™‡”‡‘ obviously changes of pro-caspase-3 and pro-caspase-9 ( ‹‰Ǥʹ ). Taken together, these data ‹†‹…ƒ–‡†–Šƒ–‹Ǧͳͻ͸„‹Š‹„‹–‡†…‡ŽŽ‰”‘™–Šƒ†‡–ƒ•–ƒ•‹•‘ˆͷͶͻ…‡ŽŽ•Ǥ MiR-196b suppressed EMT process in A549 cells To explore the efˆ‡…– ‘ˆ ‹Ǧͳͻ͸„ ‘  ’”‘…‡••ǡ ͷͶͻ cells were transfected ™‹–Š‹Ǧͳͻ͸„‹‹…ǡ ‹Ǧͳͻ͸„ ‹Š‹„‹–‘” and corresponding controls. After inducing by 10 ng/ml of

Fig. 1.‹Ǧͳͻ͸„™ƒ•Ž‘™‡š’”‡••‡† ‹ Ž—‰ …ƒ…‡” …‡ŽŽ Ž‹‡•Ǥ ‹Ǧͳͻ͸„ ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› †‘™Ǧ”‡‰—Žƒ–‡† ‹ Ž—‰…ƒ…‡”…‡ŽŽŽ‹‡•ȋͷͶͻǡǦͳ͸ͷͲ ƒ† ǦͳʹͻͻȌ …‘’ƒ”‡† –‘ ‘”ƒŽ Ž—‰ …‡ŽŽ Ž‹‡• ȋ Ǧ͵ͺǡ ǦͳȌǤ ‹Ǧ ͳͻ͸„ǣ ‹…”‘Ǧͳͻ͸„Ǣ ȗȗǡ δͲǤͲͳǢ ȗȗȗǡ δͲǤͲͲͳ ™Š‡ …‘’ƒ”‡† ™‹–Š  Ǧ͵ͺ …‡ŽŽ Ž‹‡Ǣ ͓͓ǡ δͲǤͲͳǢ ͓͓͓ǡ δͲǤͲͲͳ™Š‡…‘’ƒ”‡†™‹–ŠǦͳ cell line.

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Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 www.karger.com/cpb and Biochemistry Published online: September 27, 2017

Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2

Fig. 2. ‹Ǧͳͻ͸„ ‹Š‹„‹–‡† …‡ŽŽ ˜‹ƒ„‹Ž‹–›ǡ ‹‰”ƒ–‹‘ǡ ‹˜ƒ•‹‘ǡ ƒ† ’”‘‘–‡† ƒ’‘’–‘•‹•Ǥ ͷͶͻ …‡ŽŽ• ™‡”‡ –”ƒ•ˆ‡…–‡†™‹–Š‹Ǧͳͻ͸„‹‹…ǡ‹‹……‘–”‘Žǡ‹Ǧͳͻ͸„‹Š‹„‹–‘”ƒ†‹Š‹„‹–‘”…‘–”‘ŽǤǣ‹Ǧͳͻ͸„ ‡š’”‡••‹‘™ƒ•—’Ǧ”‡‰—Žƒ–‡†‹‹Ǧͳͻ͸„‹‹…–”ƒ•ˆ‡…–‡†…‡ŽŽ•„—–†‘™Ǧ”‡‰—Žƒ–‡†‹‹Ǧͳͻ͸„‹Š‹„‹–‘”–”ƒ•ˆ‡…–‡†…‡ŽŽ•ǢǦǣ˜‡”‡š’”‡••‹‘‘ˆ‹Ǧͳͻ͸„‹Š‹„‹–‡†…‡ŽŽ˜‹ƒ„‹Ž‹–›ǡ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘ǡ„—– •—’’”‡••‹‘‘ˆ‹Ǧͳͻ͸„’”‘‘–‡†…‡ŽŽ˜‹ƒ„‹Ž‹–›ǡ‹‰”ƒ–‹‘ƒ†‹˜ƒ•‹‘‹ͷͶͻ…‡ŽŽ•Ǣǣ˜‡”‡š’”‡••‹‘‘ˆ ‹Ǧͳͻ͸„’”‘‘–‡†…‡ŽŽƒ’‘’–‘•‹•‹ͷͶͻ…‡ŽŽ•Ǣ ǣ˜‡”‡š’”‡••‹‘‘ˆ‹Ǧͳͻ͸„’”‘‘–‡†ƒ’‘’–‘•‹•Ǧ”‡Žƒ–‡†’”‘–‡‹•‡š’”‡••‹‘•ǡƒ†•—’’”‡••‹‘‘ˆ‹Ǧͳͻ͸„‹Š‹„‹–‡†ƒ’‘’–‘•‹•Ǧ”‡Žƒ–‡†’”‘–‡‹•‡š’”‡••‹‘•‹ ͷͶͻ…‡ŽŽ•Ǥ‹Ǧͳͻ͸„ǣ‹…”‘Ǧͳͻ͸„ǢȗǡδͲǤͲͷǢȗȗǡδͲǤͲͳǢȗȗȗǡδͲǤͲͲͳǤ

 ǦȾͳǡ–Š‡’”‘–‡‹Ž‡˜‡Ž•‘ˆǦ”‡Žƒ–‡†ˆƒ…–‘”•™‡”‡†‡–‡…–‡†„›™‡•–‡”„Ž‘–Ǥ•”‡•—Ž–• revealed in ‹‰Ǥ͵ǡ‹Ǧͳͻ͸„‘˜‡”‡š’”‡••‹‘’”‘‘–‡†Ǧ…ƒ†Š‡”‹‡š’”‡••‹‘„—–‹Š‹„‹–‡† Ǧ…ƒ†Š‡”‹ǡ‹‡–‹ǡͳǡƒ†ƒ‹Ž‡š’”‡••‹‘•Ǥˆ…‘–”ƒ•–ǡ‹Ǧͳͻ͸„•—’’”‡••‹‘Ž‡† –‘‘’’‘•‹–‡”‡•—Ž–•ˆ‘”–Š‡•‡ϐ‹˜‡ˆƒ…–‘”•‡š’”‡••‹‘•ǤŠ‡•‡”‡•—Ž–•†‡‘•–”ƒ–‡†–Šƒ–‹Ǧ ͳͻ͸„…‘—Ž†•—’’”‡••’”‘…‡••Ǥ

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Physiol Biochem 2017;43:757-767 Cellular Physiology Cell © 2017 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000481559 www.karger.com/cpb and Biochemistry Published online: September 27, 2017

Bai et al.: Role of Mir-196b in Lung Cancer by Targeting Runx2

Fig. 3.‹Ǧͳͻ͸„‹Š‹„‹–‡†’”‘…‡••ǤͷͶͻ …‡ŽŽ• ™‡”‡ –”ƒ•ˆ‡…–‡† ™‹–Š ‹Ǧͳͻ͸„ ‹‹…ǡ ‹Ǧͳͻ͸„ ‹Š‹„‹–‘” ƒ† …‘””‡•’‘†‹‰ …‘–”‘Ž•ƒ†–Š‡‹†—…‡†„›ͳͲ‰ȀŽ‘ˆ ǦȾͳǤ ˜‡”‡š’”‡••‹‘‘ˆ‹Ǧͳͻ͸„’”‘‘–‡†Ǧ…ƒ†herin expression but inhibited N-cadherin, Vi‡–‹ǡͳƒ†ƒ‹Ž‡š’”‡••‹‘•Ǥ‹Ǧͳͻ͸„ǣ ‹…”‘Ǧͳͻ͸„Ǣ ǣ ‡’‹–Š‡Ž‹ƒŽǦ‡•‡…Š›ƒŽ –”ƒ•‹–‹‘Ǣ  ǦȾͳǣ –”ƒ•ˆ‘”‹‰ ‰”‘™–Š ˆƒ…–‘”Ǧ„‡–ƒͳǢͳǣœ‹…Ǧϐ‹‰‡”Ǧ„‘š„‹†‹‰ homeobox 1.

Fig. 4.—šʹ™ƒ•ƒ–ƒ”‰‡–‘ˆ‹Ǧͳͻ͸„Ǥǣ‹Ǧͳͻ͸„ negatively regulated the mRNA and protein expres•‹‘•‘ˆ—šʹ‹ͷͶͻ…‡ŽŽ•Ǣǣ—šʹ™ƒ•ƒ–ƒ”‰‡–‘ˆ ‹Ǧͳͻ͸„‹ͷͶͻ…‡ŽŽ•Ǥ—šʹǣ—–Ǧ”‡Žƒ–‡†–”ƒ•…”‹’–‹‘ˆƒ…–‘”ʹǢ‹Ǧͳͻ͸„ǣ‹…”‘Ǧͳͻ͸„ǢȗǡδͲǤͲͷǢȗȗǡ P