Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

997

Chen et al.:December MiR-144 Inhibits Cells Accepted: 16, 2014Proliferation in Lung Cancer 1421-9778/15/0353-0997$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Original Paper

MiR-144 Inhibits Proliferation and Induces Apoptosis and Autophagy in Lung Cancer Cells by Targeting TIGAR Shanshan Chena Ping Lia Juan Lia Yuanyuan Wangb Yuwen Dub Xiaonan Chenb :HQTLDR=DQJb+XDTL:DQJa+H\LQJ&KXa*XRTLDQJ=KDRb*XRMXQ=KDQJa 'HSDUWPHQWRI5HVSLUDWRU\0HGLFLQHWKH)LUVW$IÀOLDWHG+RVSLWDO=KHQJ]KRX8QLYHUVLW\=KHQJ]KRX China, b'HSDUWPHQWRI0LFURELRORJ\DQG,PPXQRORJ\&ROOHJHRI%DVLF0HGLFDO6FLHQFHV=KHQJ]KRX 8QLYHUVLW\=KHQJ]KRX&KLQD a

Key Words Lung cancer cell • MiR-144 • TIGAR • Proliferation • Apoptosis Abstract Background: MiRNAs are noncoding RNAs of 20–24 nucleotides that function as posttranscriptional negative regulators of gene expression. MiRNA genes are usually transcribed by RNA polymerase II in the nucleus. Their initial products are pre-miRNAs which have cap VHTXHQFHV DQG SRO\$ WDLOV 7KH SLQGXFHG JO\FRO\VLV DQG DSRSWRVLV UHJXODWRU 7,*$5  was discovered through microarray analysis of gene expression following activation of p53. +RZHYHUOLWWOHLVNQRZQDERXWWKHHIIHFWRIPL5RQFHOOSUROLIHUDWLRQDQGDSRSWRVLVDQG how it interacts with TIGAR. Methods: We performed real-time PCR, western blotting, CCK8, FRORQ\IRUPDWLRQWXPRUJURZWKÁRZF\WRPHWU\&DVSDVHDFWLYLW\+RHFKVWVWDLQLQJ 0'&VWDLQLQJRIDXWRSKDJLFFHOOVDQGOXFLIHUDVHUHSRUWHUDVVD\VWRGHWHFWWKHLQÁXHQFHRIPL5 144 to lung cancer cells. Results: miR-144 targeted TIGAR, inhibited proliferation, enhanced DSRSWRVLVDQGLQFUHDVHGDXWRSKDJ\LQ$DQG+FHOOVConclusions: Our study improves our understanding of the mechanisms underlying lung cancer pathogenesis and may promote the development of novel targeted therapies. Copyright © 2015 S. Karger AG, Basel

Introduction

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MiRNAs are noncoding RNAs of 20–24 nucleotides that function as post-transcriptional negative regulators of gene expression [1]. It is now well known that miRNAs regulate the ‡š’”‡••‹‘ ‘ˆ —Ž–‹’Ž‡ –ƒ”‰‡– ‰‡‡• ƒ† ƒˆˆ‡…– ƒ ˜ƒ”‹‡–› ‘ˆ …‡ŽŽ—Žƒ” ’ƒ–Š™ƒ›•ǡ •’‡…‹ϐ‹…ƒŽŽ› in cancer development and progression [2, 3]. MiR-144, sharing the same locus with miR-

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

998

Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

451[4], has been reported in many cancers. A report from Zhang et al. claimed that miR144 promoted proliferation, migration, and invasion of nasopharyngeal carcinoma through repression of phosphatase and tensin homolog (PTEN) [5]. Sureban et al. also showed that knockdown of doublecortin and CaM kinase-like-1 (DCAMKL-1) increased miR-144 expression, which in turn inhibited epithelial-mesenchymal transition (EMT) of pancreatic cancer [6]. These data impelled us to further study the relative mechanism of miR-144 acting on lung cancer behavior. Lung cancer is a major cause of cancer-related death worldwide. Although the diagnosis and therapy are in advances and the prognosis is encouraging, the number of cases and deaths related to lung cancer is still rising in many parts of the world. Lung cancer consisted of multiple sequential steps that are not completely understood to date, more investigation of this mechanism is urgently needed. Altered expression of miRNAs has been observed in lung cancer, suggesting that miRNA deregulation plays a role in lung carcinogenesis. For example, miR-145 inhibited lung cancer cell metastasis and EMT via targeting the Oct4 ‡†‹ƒ–‡† –ȀȾǦ…ƒ–‡‹ •‹‰ƒŽ‹‰ ’ƒ–Š™ƒ› ȏͶȐǤ ƒ ‡– ƒŽǤ ‡Ž—…‹†ƒ–‡† –Šƒ– ‹ǦͳͷƒȀͳ͸ …ƒ‡Šƒ…‡”ƒ†‹ƒ–‹‘•‡•‹–‹˜‹–›„›”‡‰—Žƒ–‹‰–Š‡ͳȀ ǦɈ•‹‰ƒŽ‹‰’ƒ–Š™ƒ›ƒ†ƒ…– as a potential therapeutic approach to overcome radioresistance for lung cancer treatment ȏ͹ȐǤƒ–ƒˆ”‘Šƒ‡–ƒŽǤŠƒ˜‡•Š‘™‡†‹ǦͳͶͶ‡š‡”–‡††‹”‡…–”‡‰—Žƒ–‘”›”‘Ž‡•‘‹…ϐ‹‰‡” X-chromosomal protein (ZFX) expression, which seems to be associated with tumorigenesis [8]. Our previously studies also found that the expression of miR-144 in these tissues was lower than in adjacent, paired non-tumor tissues by a miRNA chip-based expression analysis of lung cancer tissues. Relevant bioinfomatics were analyzed and we hypothesized that miR-144 interacted with the p53-induced glycolysis and apoptosis regulator (TIGAR). TIGAR was discovered through microarray analysis of gene expression following the activation of p53 [9]. It is a p53-induced gene that can reduce the level of fructose2, 6-bisphosphate in cells, resulting in inhibition of glycolysis, and plays a crucial role in apoptosis [10, 11]. TIGAR overexpression has been described in several types of tumor. Its expression is increased in primary colon cancer and associated metastases [12], as well as in invasive breast cancer when compared to normal tissue [13]. TIGAR is also overexpressed in glioblastoma [14]. The effect of miR-144 on lung cancer cells and how it interacts with TIGAR remain unknown. In the present study, we observed miR-144 expression levels in tissues from 67 lung cancer patients, and investigated the function of miR-144 in lung cancer cells. Materials and Methods Patients and tissue specimens A total of 67 cases of lung cancer cases were examined. All specimens were clinically and histologically †‹ƒ‰‘•‡†ƒ––Š‡ ‹”•–ˆϐ‹Ž‹ƒ–‡†‘•’‹–ƒŽ‘ˆŠ‡‰œŠ‘—‹˜‡”•‹–›„‡–™‡‡ʹͲͳʹƒ†ʹͲͳ͵ƒ†–Š‡›™‡”‡ “—‹…ˆ”‘œ‡‹Ž‹“—‹†‹–”‘‰‡ǤŠ‹••–—†›™ƒ•ƒ’’”‘˜‡†„›–Š‡–Š‹…•‘‹––‡‡‘ˆŠ‡‰œŠ‘—‹˜‡”•‹–› and written consent was obtained from all patients.

‡ƒŽǦ–‹‡ϔŽ—‘”‡•…‡…‡“—ƒ–‹–ƒ–‹˜‡’‘Ž›‡”ƒ•‡…Šƒ‹”‡ƒ…–‹‘ƒƒŽ›•‹• —ƒ–‹–ƒ–‹˜‡”‡ƒŽǦ–‹‡™ƒ•’‡”ˆ‘”‡†–‘“—ƒ–‹–ƒ–‡‹ǦͳͶͶ‡š’”‡••‹‘ǡ—•‹‰”‡‹šš ƒ“ ȋŽ‹ƒ•‡Ž—•ȌȋƒƒƒǡƒŽ‹ƒǡŠ‹ƒȌ‹ƒ ͹ͷͲͲˆƒ•–•›•–‡ȋ’’Ž‹‡†‹‘•›•–‡•ǡƒ”Ž•„ƒ†ǡ

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RNA extraction For lung cancer tissues, if the proportion of lung cancer cells in a tissue section was >80% then the frozen block was subjected to RNA extraction. According to the manufacturer's protocol, total RNA was extracted from 67 pairs of snap-frozen cancer tissues and adjacent normal tissues using TRIzol reagent ȋ ˜‹–”‘‰‡ǡǡȌǤ ‘”Ž—‰…ƒ…‡”…‡ŽŽŽ‹‡•ǡ–‘–ƒŽ™ƒ•‡š–”ƒ…–‡†™‹–Šƒš–”ƒ…–‹‘‹–ȋ‹ƒ‰‡ȌǤ Š‡‹–‡‰”‹–›‘ˆ–Š‡™ƒ•‡˜ƒŽ—ƒ–‡†„›ƒƒ‘†”‘’ǦͳͲͲͲȋŠ‡”‘…‹‡–‹ϐ‹…ǡ‘”…‡•–‡”ǡȌǤŠ‡ value of OD260/OD280 is around 1.8 as a criterion of acceptable purity.

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

999

Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

ǡȌǤ͸•ƒŽŽ—…Ž‡ƒ”ȋ•Ȍˆ‘”‹ǦͳͶͶ‘” ˆ‘” ™ƒ•—•‡†ƒ•ƒ‡†‘‰‡‘—• control. All samples were analyzed three times. The qRT-PCR results were expressed relative to miR-144 expression levels at the threshold cycle (Ct), which were then converted to fold changes (2Ȃȟȟ–). Cell lines and reagents ͷͶͻƒ†Ͷ͸ͲŠ—ƒŽ—‰…ƒ…‡”…‡ŽŽŽ‹‡•™‡”‡’—”…Šƒ•‡†ˆ”‘‡”‹…ƒ›’‡—Ž–—”‡‘ŽŽ‡…–‹‘ ȋƒƒ••ƒ•ǡǡȌǤ‘–Š…‡ŽŽŽ‹‡•™‡”‡…—Ž–—”‡†™‹–Š—Ž„‡……‘ǯ•‘†‹ϐ‹‡†ƒ‰Ž‡ǯ•‡†‹—ȋǢ ‹„…‘ǡ Ȍǡ ™Š‹…Š …‘–ƒ‹‡† ͳͲΨ ˆ‡–ƒŽ „‘˜‹‡ •‡”— ȋ Ǣ ‹„…‘ǡ Ȍǡ ͳͲͲ ȀŽ ’‡‹…‹ŽŽ‹ ƒ† ͳͲͲ ȀŽ streptomycin, and the cells were incubated at 37°Cunder 5% CO2 atmosphere. ”ƒ•ˆ‡…–‹‘™‹–Š‹• The miR-144 agomir (GMR-miRmicroRNA-144 agomir) and miR-144 scramble were synthesized „› Šƒ‰Šƒ‹ ‡‡Šƒ”ƒ ‘Ǥ –†Ǥ –”‡ƒ–‡†ͷͶͻƒ† Ͷ͸Ͳ…‡ŽŽ•ǡ ‰”‘™‹‰ ‡š’‘‡–‹ƒŽŽ›ǡ ™‡”‡ ’Žƒ–‡† ƒ– 2×107Ȁ™‡ŽŽ‹ʹǤͷŽ‡†‹—ˆ‘”ʹͶŠ‘•‹šǦ™‡ŽŽ’Žƒ–‡•Ǥ…‡…‡ŽŽ•”‡ƒ…Š‡†ƒ„‘—–ͷͲΨ…‘ϐŽ—‡…‡ǡ –”ƒ•ˆ‡…–‹‘™ƒ•…‘†—…–‡†ǤʹͲͲͳȋƒ–—”‡‰‡‡ǡ‡™ƒ˜‡ǡǡȌ™ƒ•—•‡†‹ƒŽŽ–”ƒ•ˆ‡…–‹‘ ’”‘…‡••‡•ƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•Ǥƒ…Š…‡ŽŽŽ‹‡™ƒ••‡’ƒ”ƒ–‡†‹–‘–Š”‡‡‰”‘—’•ǣ–Š‡ ‘Ǧ–”ƒ•ˆ‡…–‡†„Žƒ‰”‘—’ȋ„ŽƒȌǢ•…”ƒ„Ž‡†‹ǦͳͶͶ–”ƒ•ˆ‡…–‡†‡‰ƒ–‹˜‡…‘–”‘Ž‰”‘—’ȋ•…”ƒ„Ž‡ȌǢ and the miR-144 agomir transfected group (miR-144). ‡•–‡”„Ž‘–ƒƒŽ›•‹• The total protein content of cultured cells was extracted using RIPA buffer containing ’Š‡›Ž‡–Šƒ‡•—Žˆ‘›ŽϐŽ—‘”‹†‡Ǥ’”‘–‡‹ƒ••ƒ›‹–ȋ‡›‘–‹‡ǡƒ‹‡ǡŠ‹ƒȌ™ƒ•—•‡†–‘†‡–‡”‹‡–Š‡ ’”‘–‡‹…‘…‡–”ƒ–‹‘Ǥ”‘–‡‹•™‡”‡•—„Œ‡…–‡†–‘Ǧ ƒ†–”ƒ•ˆ‡””‡†‘–‘’‘Ž›˜‹›Ž‹†‡‡†‹ϐŽ—‘”‹†‡ ‡„”ƒ‡•Ǥˆ–‡”„Ž‘…‹‰™‹–ŠͷΨ•‹‹Žˆ‘”ͳŠƒ–͵͹Ǐǡ–Š‡‡„”ƒ‡™ƒ•‹…—„ƒ–‡†™‹–Š’”‹ƒ”› ƒ–‹„‘†‹‡•ǡ ’‘Ž›…Ž‘ƒŽ ”ƒ„„‹– ƒ–‹Ǧ  ȋͳǣ͵ͲͲȌ ȋƒ–ƒ ”—œ ‹‘–‡…Š‘Ž‘‰›ǡ ƒ–ƒ ”—œǡ ǡ Ȍ ƒ– 4°Covernight. To detect autophagy, the markers of autophagic vacuoles, microtubule-associated protein 1 Ž‹‰Š– …Šƒ‹ ͵ ȋ͵Ȍ ƒ† ‡…Ž‹ ͳ ™‡”‡ —•‡†ǡ ƒ† •‘ ’‘Ž›…Ž‘ƒŽ ”ƒ„„‹– ƒ–‹Ǧ͵  ƒ† ƒ–‹Ǧ͵  ȋͳǣʹͲͲȌǡ ’‘Ž›…Ž‘ƒŽ ”ƒ„„‹– ƒ† ’‘Ž›…Ž‘ƒŽ ”ƒ„„‹– ƒ–‹Ǧ ‡…Ž‹Ǧͳȋͳǣ͵ͲͲȌ ȋƒ–ƒ ”—œ ‹‘–‡…Š‘Ž‘‰›ǡ ƒ–ƒ ”—œǡ ǡ Ȍ™‡”‡ƒŽ•‘‹…—„ƒ–‡†ƒ–Ͷι‘˜‡”‹‰Š–ǤŠ‡‡„”ƒ‡•™‡”‡™ƒ•Š‡†–Š”‡‡–‹‡•™‹–Š”‹•Ǧ„—ˆˆ‡”‡† •ƒŽ‹‡ƒ†™‡‡ʹͲȋȌƒ†‹…—„ƒ–‡†™‹–Š†‹Ž—–‡†ȋͳǣ͵ͲͲͲȌŠ‘”•‡”ƒ†‹•Š-peroxidase -conjugated goat ƒ–‹Ǧ”ƒ„„‹– ‰ ȋƒ–ƒ”—œ‹‘–‡…Š‘Ž‘‰›Ȍˆ‘”ͳŠƒ–”‘‘–‡’‡”ƒ–—”‡Ǥˆ–‡”–Š”‡‡™ƒ•Š‡•‹ǡ–Š‡ ϐ‹Ž•‘ˆ‹—‘”‡ƒ…–‹˜‡’”‘†—…–•™‡”‡•…ƒ‡†—•‹‰–Š‡—’‡”‹‰ƒŽ—„•–”ƒ–‡‡•–‡”„Ž‘––‹‰†‡–‡…–‹‘ •›•–‡ȋ‹‡”…‡ǡ‘…ˆ‘”†ǡ ǡȌǤƒ–‹„‘†›ƒ‰ƒ‹•– ȋƒ–ƒ”—œ‹‘–‡…Š‘Ž‘‰›Ȍ•‡”˜‡†ƒ•ƒ endogenous reference. ‡ŽŽ‰”‘™–Šƒ••ƒ› ‡ŽŽ‘—–‹‰‹–ȋͺǢ‘Œ‹†‘ǡ ƒ’ƒȌ™ƒ•—•‡†–‘‡ƒ•—”‡…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘Ǥ‡ŽŽ•™‡”‡…—Ž–—”‡† ‹ ͻ͸Ǧ™‡ŽŽ ’Žƒ–‡• ȋͳͲͲɊŽȀ™‡ŽŽȌ‹ …‘’Ž‡–‡  ƒ† ™‡”‡ ƒ‹–ƒ‹‡† ‹ ƒ ‹…—„ƒ–‘” ƒ– ͵͹ι ƒ†ͷΨ CO2Ǥ•‘Ž—–‹‘ȋͳͲɊŽȌ™ƒ•ƒ††‡†–‘‡ƒ…Š™‡ŽŽǤ’–‹…ƒŽ†‡•‹–›ȋȌ˜ƒŽ—‡™ƒ•‡ƒ•—”‡††ƒ‹Ž›‘˜‡”ˆ‘—” consecutive days at 490 nm (OD490) to estimate viable cell numbers. The assay was repeated in three independent experiments.

˜‹˜‘–—‘”‰”‘™–Šƒ••ƒ› Tumor heterotransplantation was performed to investigate tumorigenic ability of miR-144 ‹ ˜‹˜‘. A549 cells transfected with miR-144(miR-144 group) or with scrambled miRNA (scramble group) and ͷͶͻ…‡ŽŽ•ȋ„Žƒ‰”‘—’Ȍ™‡”‡•—„…—–ƒ‡‘—•Ž›‹‘…—Žƒ–‡†‹–‘–Š‡†‘”•ƒŽϐŽƒ‘ˆ͸Ǧ™‡‡Ǧ‘Ž†ˆ‡ƒŽ‡Ȁ… —†‡‹…‡–Šƒ–™‡”‡’—”…Šƒ•‡†ˆ”‘Š‡ƒ„‘”ƒ–‘”›‹ƒŽ‹–‘ˆ–Š‡‹˜‡”•‹–›‘ˆŠ‡‰œŠ‘—Ǥ‹ƒŽ handling and experimental procedures were approved by the Animal Experimental Ethics Committee of –Š‡‹˜‡”•‹–›‘ˆŠ‡‰œŠ‘—Ǥƒ…Š‰”‘—’…‘–ƒ‹‡†ϐ‹˜‡‹…‡Ǥ—‘”•‹œ‡™ƒ•‡ƒ•—”‡†‡˜‡”›™‡‡ˆ‘”ͳ month. The mice were killed and tumors were removed, photographed and weighed. Tumor volumes were

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‘Ž‘›ˆ‘”ƒ–‹‘ƒ••ƒ› Cells were trypsinized, counted and propagated after transfection, and then cultured in six-well plates at 100 cells/well. The number of colonies was counted at 12daysafter seeding. The colonies formed were stained with crystal violet, and the colonies with >50 cells were scored as surviving colonies. This experiment had three replicates.

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

1000

Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

…ƒŽ…—Žƒ–‡†—•‹‰–Š‡ˆ‘ŽŽ‘™‹‰‡“—ƒ–‹‘ǣȋέ†2)/2, where D means the longest diameter and d means the shortest diameter. Ž‘™…›–‘‡–”›ƒ••ƒ› Cell cycle was synchronized for 24 h before collection and 20,000 cells were detected per group. The cells were suspended in 500 μl binding buffer and 10 μl AnnexinV-ϐŽ—‘”‡•…‡‹‹•‘–Š‹‘…›ƒƒ–‡ȋ Ȍƒ†ͷρŽ propidium iodidewere added for 15 min. All the procedures were done according to the instructions of the ‡š‹’‘’–‘•‹•‡–‡…–‹‘‹– ȋ‡•–‹‘ǡŠƒ‰Šƒ‹ǡŠ‹ƒȌǤŠ‡…‡ŽŽ•™‡”‡ƒƒŽ›œ‡†™‹–Šƒ …ƒ ϐŽ‘™…›–‘‡–‡”ȋ‹‘•…‹‡…‡•Ȍ‡“—‹’’‡†™‹–Š‡ŽŽ—‡•–•‘ˆ–™ƒ”‡ȋ‹‘•…‹‡…‡•ȌǤƒ…Š–”‡ƒ–‡–™ƒ• carried out in triplicate. ƒ•’ƒ•‡͹Ȁͽƒ…–‹˜‹–›ƒ••ƒ› Cells from each treatment group were harvested at 48 h post-transfection and caspase activity was †‡–‡…–‡† „› ƒ•’ƒ•‡Ǧ Ž‘ ͵Ȁ͹ ••ƒ› ‹– ȋ”‘‡‰ƒǡ Šƒ‰Šƒ‹ǡ Š‹ƒȌǤ ͳͲͲɊŽ ƒ•’ƒ•‡Ǧ Ž‘ ͵Ȁ͹ ‡ƒ‰‡– ™ƒ• added to each well of the plates, followed by 1 min mixing using an IKA MTS4 plate shaker. Plates were incubated at room temperature for 2 h. Absorbance values were measured with a microplate reader at 405 ȋ ϐ‹‹–‡ʹͲͲǢ‡…ƒǡƒ‡†‘”ˆǡ™‹–œ‡”Žƒ†ȌǤ ‘‡…Š•–͹͹͹ͺ͸•–ƒ‹‹‰ƒ••ƒ› ‡ŽŽ•‹‡ƒ…Š‰”‘—’™‡”‡•‡‡†‡†‹–‘ƒͻ͸Ǧ™‡ŽŽ’Žƒ–‡ƒˆ–‡”–”ƒ•ˆ‡…–‹‘Ǥ‘‡…Š•–͵͵͵Ͷʹȋ‹‰ƒǡ–‘—‹•ǡ ǡȌ™ƒ•ƒ††‡†–‘–Š‡…—Ž–—”‡‡†‹—ƒ–ƒϐ‹ƒŽ…‘…‡–”ƒ–‹‘‘ˆͷρ‰ȀŽǤŠ‡‹ƒ‰‡•™‡”‡”‡…‘”†‡† on a computer with a digital camera attached to the microscope, and the images were merged by computer. ‘”“—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ‘‡…Š•–͵͵͵Ͷʹ•–ƒ‹‹‰ǡ–Š‡’‡”…‡–ƒ‰‡‘ˆ‘‡…Š•–Ǧ’‘•‹–‹˜‡—…Ž‡‹’‡”‘’–‹…ƒŽϐ‹‡Ž†ȋƒ– Ž‡ƒ•–ͷͲϐ‹‡Ž†•Ȍ™ƒ•…‘—–‡†Ǥ’‘’–‘–‹……‡ŽŽ••Š‘™‡†„”‹‰Š–„Ž—‡ϐŽ—‘”‡•…‡…‡Ǥ •–ƒ‹‹‰‘ˆƒ—–‘’Šƒ‰‹……‡ŽŽ• Each group of cells was plated on 24-well plates after transfection for 24 h. Exponentially growing cells were treated with 0.05 mM MDC (Sigma) in DMEM at 37°C for 10 min. After that, the cells were washed three –‹‡•™‹–ŠǤŠ‡‹ƒ‰‡•™‡”‡…ƒ’–—”‡†—•‹‰ƒϐŽ—‘”‡•…‡…‡‹…”‘•…‘’‡ȋƒ”Ž‡‹••ǡ ‡ƒǡ ‡”ƒ›Ȍƒ– an excitation wavelength of 380 nm and emission wavelength of 450 nm. ‹‘‹ˆ‘”ƒ–‹…•ƒƒŽ›•‹• miR-144 data of 67 pairs of primary tumors and normal lung tissues were downloaded from The Cancer Genome Atlas (TCGA) website () [15], and analyzed using Multi-Experiment Viewer (MeV) software (version 4.9, Š––’ǣȀȀ™™™Ǥ–ͶǤ‘”‰Ȁ). The fold differences in miRNA expression were compared by paired –Ǧ–‡•–Ǥ Š‡ ˆ‘ŽŽ‘™‹‰ ‘Ž‹‡ •‘ˆ–™ƒ”‡ ™ƒ• —•‡† –‘ †‡–‡”‹‡ ’—–ƒ–‹˜‡ ‹ǦͳͶͶ –ƒ”‰‡–•ǣ ƒ”‰‡– …ƒ ͸Ǥʹ ȋŠ––’ǣȀȀ–ƒ”‰‡–•…ƒǤ‘”‰Ȍƒ†‹ƒ†ƒȋŠ––’ǣȀȀ™™™Ǥ‹…”‘”ƒǤ‘”‰Ȁ‹…”‘”ƒȀ‰‡– ‡‡ ‘”. do) [16, 17]. The ˆ—…–‹‘‘ˆ ™ƒ•‹˜‡•–‹‰ƒ–‡†„› „‹‘‹ˆ‘”ƒ–‹…•ȋŠ––’ǣȀȀ†ƒ˜‹†Ǥ‹ƒ‹†Ǥ‹ŠǤ‰‘˜ȌǤ

–ƒ–‹•–‹…ƒŽƒƒŽ›•‹• All data are expressed as means ± standard deviation. Statistical testing was analyzed using SPSS version 17.0 statistical software. The t-test and one-way analysis of variance were used to analyze data. P< ͲǤͲͷ™ƒ•–Š‡…”‹–‡”‹‘ˆ‘”•–ƒ–‹•–‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡Ǥ

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—…‹ˆ‡”ƒ•‡”‡’‘”–‡”ƒ••ƒ› The human TIGAR 3c—–”ƒ•Žƒ–‡†”‡‰‹‘ȋȌˆ”ƒ‰‡–…‘–ƒ‹‹‰’—–ƒ–‹˜‡„‹†‹‰•‹–‡•ˆ‘”‹Ǧ ͳͶͶ™‡”‡ƒ’Ž‹ϐ‹‡†„›ˆ”‘Š—ƒ‰‡‘‹…ǤŠ‡—–ƒ– ͵c•™‡”‡‘„–ƒ‹‡†„›‘˜‡”Žƒ’ ‡š–‡•‹‘ǤŠ‡ˆ”ƒ‰‡–•™‡”‡…Ž‘‡†‹–‘ƒ’‹” ”‡’‘”–‡”˜‡…–‘”ȋ”‘‡‰ƒǡƒ†‹•‘ǡ ǡȌǡ †‘™•–”‡ƒ‘ˆ–Š‡Ž—…‹ˆ‡”ƒ•‡‰‡‡ǡ–‘‰‡‡”ƒ–‡–Š‡”‡…‘„‹ƒ–˜‡…–‘”•’‹” Ǧƒ†’‹” ǦǤ For the luciferase reporter assay, A539 cells were co-transfected with miRNA (miR-144 agomir or scrambled-miR-144 negative control) and reporter vectors (pmirGLO-WT reporter vectors or pmirGLO ”‡’‘”–‡” ˜‡…–‘”•Ȍǡ —•‹‰   ʹͲͲͳ ‡Ž‡…–”‘’‘”ƒ–‘”Ǥ —…‹ˆ‡”ƒ•‡ ƒ…–‹˜‹–‹‡• ™‡”‡ ‡ƒ•—”‡† ™‹–Š ƒ —ƒŽǦ—…‹ˆ‡”ƒ•‡ ƒ••ƒ› ‹– ȋ”‘‡‰ƒǡ ƒ†‹•‘ǡ  ǡ Ȍ ƒ……‘”†‹‰ –‘ ƒ—ˆƒ…–—”‡”ǯ • ‹•–”—…–‹‘• ƒ– ʹͶŠ post-transfection. Experiments were repeated three times in triplicate.

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

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Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

Results

‹Ǧͷͺͺ‡š’”‡••‹‘‹Ž—‰…ƒ…‡” To calculate relative miR-144 concentrations by fold changes (2-ᇞᇞCtǡ ȟ– α– ‡†‹ƒ ‹ǦͳͶͶ Ǧ – ‡†‹ƒ Ǣ ȟȟ–αȟ– …ƒ…‡” Ǧȟ– ‘”ƒŽȌǡ ™‡ ˆ‘—† –Šƒ– ‹ǦͳͶͶ ‡š’”‡••‹‘ ™ƒ• •‹‰‹ϐ‹…ƒ–Ž› lower in lung cancer tissues than in paired adjacent normal tissues (*P < 0.05). The clinicopathological characteristics of the 67 lung cancer cases are presented in Table 1. Of the 67 lung cancer patients, miR-144 expression was related to tumor size (͵…ǣͲǤ͵ʹάͲǤͲ͹ʹǡȗP < 0.01 ,Table 1). And miR-144 expression level •Š‘™‡† •‹‰‹ϐ‹…ƒ– †‹ˆˆ‡”‡…‡• ‹ ˜ƒ”‹‘—• TNM (*P < 0.01, Table 1). MiR-144 expression was also lower in the lymph node metastasispositive group than the negative group (0.31±0.069 vs. 0.42±0.116, respectively, *P δ ͲǤͲͳǡ ƒ„Ž‡ ͳȌǤ Š‹Ž‡ ‘ •‹‰‹ϐ‹…ƒ– differences were detected with CLPTM1L mRNA expression (P > 0.05, Table 1, Fig. 1F,

ȌǤ ‘ •‹‰‹ϐ‹…ƒ– †‹ˆˆ‡”‡…‡• ™ƒ• ‘„•‡”˜‡† between miR-144 expression and gender, age, smoking, differentiation or histology (P > 0.05, Table 1).

Table 1. Clinicopathological characteristics and miR144 expression levels of 67 lung cancer patients *In†‹…ƒ–‡†•–ƒ–‹•–‹…ƒŽ•‹‰‹ϐ‹…ƒ…‡ȋδͲǤͲͷȌ

’”‡‰—Žƒ–‹‘‘ˆ‹Ǧͷͺͺ‹Š‹„‹–•’”‘Ž‹ˆ‡”ƒ–‹‘‘ˆͻͺͿƒ†ͺͼͶ…‡ŽŽ• š’”‡••‹‘‘ˆ‹ǦͳͶͶƒ† ƒˆ–‡”–”ƒ•ˆ‡…–‹‘‹ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•‹••Š‘™ ‹ ‹‰Ǥͳƒ†ͳǤ‘’ƒ”‡†–‘–Š‡•…”ƒ„Ž‡‰”‘—’ƒ†„Žƒ‰”‘—’ǡ‹ǦͳͶͶ‡š’”‡••‹‘Ž‡˜‡Ž• ‘ˆ‹ǦͳͶͶ‰”‘—’™‡”‡•‹‰‹ϐ‹…ƒ–Ž›Š‹‰Š‡”‹ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ǡƒ† ‡š’”‡••‹‘ Ž‡˜‡Ž• ™‡”‡ •‹‰‹ϐ‹…ƒ–Ž› Ž‘™‡” ‹ –Š‡ ‹ǦͳͶͶ ‰”‘—’ ȋȗP δ ͲǤͲͷǡ ‹‰Ǥͳǡ ͳȌǤ ‡ŽŽ ‰”‘™–Š …—”˜‡•ƒ”‡’”‡•‡–‡†‹ ‹‰ǤͳǤŠ‡”‡™‡”‡‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡•‹490 values between the blank and scramble groups (P > 0.05). Compared with the blank and scramble groups, the OD490˜ƒŽ—‡•ˆ‘”–Š‡‹ǦͳͶͶ‰”‘—’ƒ–Ͷͺǡ͹ʹƒ†ͻ͸Š™‡”‡ƒŽŽ•‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡† (*PδͲǤͲͷȌ‹ͷͶͻȋ ‹‰ǤͳȌƒ†Ͷ͸Ͳȋ ‹‰ǤͳȌ…‡ŽŽ•Ǥ‘Ž‘›ˆ‘”ƒ–‹‘‹–Š‡‹ǦͳͶͶ‰”‘—’ ™ƒ•Ž‘™‡”–Šƒ‹–Š‡•…”ƒ„Ž‡ƒ†„Žƒ‰”‘—’•ˆ‘”ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ȋȗP < 0.05, Fig. 1E, 1F). The result remains coordinate in tumor heterotransplantation experiment that the tumor volumes of miR-144 group cells were relatively lower than scramble group and blank group (*PδͲǤͲͷǡ ‹‰Ǥͳ ǡͳȌǤ

’”‡‰—Žƒ–‹‘‘ˆ‹Ǧͷͺͺ‡Šƒ…‡•ƒ—–‘’Šƒ‰›‘ˆͻͺͿƒ†ͺͼͶ…‡ŽŽ•  •–ƒ‹‹‰ ™ƒ• ˜‹•—ƒŽ‹œ‡† „› ϐŽ—‘”‡•…‡…‡ ‹…”‘•…‘’›Ǥ Š‡ —„‡” ‘ˆ autophagicvacuoles was increased in the miR-144 group compared to the scramble and

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’”‡‰—Žƒ–‹‘‘ˆ‹Ǧͷͺͺ‡Šƒ…‡•ƒ’‘’–‘•‹•‘ˆͻͺͿƒ†ͺͼͶ…‡ŽŽ• Flow cytometry indicated that the apoptosis rate in the miR-144 group was higher than ‹ –Š‡ •…”ƒ„Ž‡ ƒ† „Žƒ ‰”‘—’• ˆ‘” „‘–Š ͷͶͻ ƒ† Ͷ͸Ͳ …‡ŽŽ• ȋȗP δ ͲǤͲͷǡ ‹‰Ǥ ʹǡ ʹȌǤ Caspase3/7 activity in miR-144 group was higher than in the scramble and blank groups ˆ‘” ͷͶͻ ƒ† Ͷ͸Ͳ …‡ŽŽ•ǡ ™Š‹…Š ƒŽ•‘ ‹†‹…ƒ–‡† –Šƒ– ƒ’‘’–‘•‹• ”ƒ–‡ ‹ –Š‡ ‹ǦͳͶͶ ‰”‘—’ was increased compared to the other groups (*PδͲǤͲͷǡ ‹‰ǤʹǡʹȌǤ –Š‡‘‡…Š•–͵͵͵Ͷʹ •–ƒ‹‹‰ƒ••ƒ›ǡ•‹‹Žƒ””‡•—Ž–•™‡”‡‘„•‡”˜‡†„›ϐŽ—‘”‡•…‡…‡‹…”‘•…‘’›ˆ‘”„‘–ŠͷͶͻƒ† Ͷ͸Ͳ…‡ŽŽ•ȋȗP < 0.05, Fig. 2E, 2F).

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

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Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

Fig. 1. ‹ǦͳͶͶ‹Š‹„‹–•–Š‡’”‘Ž‹ˆ‡”ƒ–‹‘Ž‡˜‡Ž‘ˆͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ǤȋǡȌ‘’ƒ”‡†–‘–Š‡•…”ƒ„Ž‡ ‰”‘—’ƒ†„Žƒ‰”‘—’‹„‘–ŠͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ǡ–Š‡‡š’”‡••‹‘Ž‡˜‡Ž‘ˆ‹ǦͳͶͶ™ƒ••‹‰‹ϐ‹…ƒ–Ž›Š‹‰Š‡”ƒ† ™ƒ••‹‰‹ϐ‹…ƒ–Ž›Ž‘™‡”‹‹ǦͳͶͶ‰”‘—’ȋȗP < 0.05). (C, D) The cell growth rate (OD490) of ‹ǦͳͶͶ‰”‘—’‰‘‡••Ž‘™‡”–Šƒ•…”ƒ„Ž‡‰”‘—’ƒ†„Žƒ‰”‘—’„‘–ŠͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•Ǥȋǡ Ȍˆ–‡” approximately 2 weeks of incubation, the colony formation number of miR-144 group cells came to lower …‘Ž‘›–Šƒ–Š‡•…”ƒ„Ž‡‰”‘—’ƒ†„Žƒ‰”‘—’‹„‘–ŠͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ȋȗPδͲǤͲͷȌǤȋ ǡȌ ǣŠ‡–—‘”–‹••—‡•™‡”‡…‘ŽŽ‡…–‡†ˆ”‘ƒ–Š›‹…—†‡‘—•‡ƒˆ–‡”ˆ‘—”™‡‡•ǤǣŠ‡–—‘”˜‘Ž—‡•™‡”‡‡ƒ•—red every week. The tumor volumes of miR group cell were relatively lower than scramble group and blank group (*P < 0.05).

 ‹•ƒ†‹”‡…––ƒ”‰‡–‘ˆ‹Ǧͷͺͺ ‹‘‹ˆ‘”ƒ–‹…• ƒƒŽ›•‹• ’”‡†‹…–‡† –Šƒ– –Š‡ ͵̵ • ‘ˆ   …‘–ƒ‹‡† „‹†‹‰ •‹–‡• ˆ‘”‹ǦͳͶͶȋ ‹‰ǤͶȌǤ‘–Š™‹Ž†Ǧ–›’‡ƒ†—–ƒ– ͵̵•™‡”‡•Š‘™‹ ‹‰ǤͶǤ ‘–”ƒ•ˆ‡…–‹‘ ™‹–Š ‹ǦͳͶͶ •‹‰‹ϐ‹…ƒ–Ž› •—’’”‡••‡† Ž—…‹ˆ‡”ƒ•‡ ƒ…–‹˜‹–› ‘ˆ –Š‡ ”‡’‘”–‡” …‘–ƒ‹‹‰ –Š‡ ™‹Ž†Ǧ–›’‡ ͵̵  ȋȗP < 0.05, Fig. 4C). Our results indicate that miR-144 negatively regulates TIGAR expression by directly binding to putative binding sites in the ͵̵Ǥ

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blank groups (*P δ ͲǤͲͷǡ ‹‰Ǥ ͵ȌǤ  ƒ††‹–‹‘ǡ ͵ ǡ ͵  ƒ† ‡…Ž‹ ͳǡ –Š‡ ƒ”‡”• ‘ˆ autophagic vacuoles, were detected by Western blotting. Compared to the scramble and „Žƒ ‰”‘—’•ǡ –Š‡ ”‡Žƒ–‹˜‡ ‡š’”‡••‹‘ Ž‡˜‡Ž• ‘ˆ ͵  ’”‘–‡‹ ‹ ͷͶͻ ƒ† Ͷ͸Ͳ …‡ŽŽ• ™ƒ• Ž‘™‡”‹–Š‡‹ǦͳͶͶ‰”‘—’…‡ŽŽ•ǡƒ†‡…Ž‹ͳƒ†͵ ’”‘–‡‹•™‡”‡Š‹‰Š‡”ȋȗP < 0.05, ‹‰Ǥ͵ǡ͵ȌǤŠ‡•‡”‡•—Ž–•‹†‹…ƒ–‡†–Šƒ–—’”‡‰—Žƒ–‹‘‘ˆ‹ǦͳͶͶ…ƒ‡Šƒ…‡ƒ—–‘’Šƒ‰›‘ˆ ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•Ǥ

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

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Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

Fig. 2.‹ǦͳͶͶ‡Šƒ…‡•–Š‡ƒ’‘’–‘•‹•”ƒ–‡‘ˆͷͶͻ…‡ŽŽ•ƒ†Ͷ͸Ͳ…‡ŽŽ•ǤȋǡȌ’‘’–‘•‹•”ƒ–‡‹‹ǦͳͶͶ ‰”‘—’™ƒ•Š‹‰Š‡”–Šƒ•…”ƒ„Ž‡‰”‘—’ƒ†„Žƒ‰”‘—’‹ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ȋȗP < 0.05). (C, D) Caspa•‡͵Ȁ͹ƒ…–‹˜‹–›‘ˆ‹ǦͳͶͶ‰”‘—’™‡–Š‹‰Š‡”–Šƒ–Šƒ–‘ˆ•…”ƒ„Ž‡ƒ†„Žƒ‰”‘—’‹ͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ• (*P < 0.05). (E, F) The hoechst 33342 staining assay come out that the apoptosis rate was higher in miR-144 group than the scramble group and blank group (*P < 0.05).

—…–‹‘‘ˆ‹Ǧͷͺͺ‹ͻͺͿ…‡ŽŽ•’ƒ”–‹ƒŽŽ›ƒ––”‹„—–‡†–‘–ƒ”‰‡–‹‰  We constructed si-TIGAR vector and transfected it into A549 cells (si-TIGAR group). ›™‡•–‡”„Ž‘––‹‰ǡ™‡‘„•‡”˜‡†–Šƒ–‡š’”‡••‹‘‘ˆ ’”‘–‡‹‹–Š‡•‹Ǧ ‰”‘—’ ™ƒ•Ž‘™‡”–Šƒ‹–Š‡‹ǦͳͶͶƒ†„Žƒ‰”‘—’…‡ŽŽ•ǡƒ†‡…Ž‹ͳƒ†͵ ’”‘–‡‹•™‡”‡ higher in A549 cells (Fig. 5A).The OD490 values of the si-TIGAR groupat48, 72 and 96 h were •‹‰‹ϐ‹…ƒ–Ž›†‡…”‡ƒ•‡†…‘’ƒ”‡†™‹–Š–Š‡„Žƒ‰”‘—’…‡ŽŽ•ǡ„—–‹…”‡ƒ•‡†…‘’ƒ”‡†™‹–Š the miR-144 group cells (*PδͲǤͲͷǡ ‹‰ǤͷȌǤŠ‡—„‡”‘ˆ…‘Ž‘‹‡•‹–Š‡•‹Ǧ ‰”‘—’ ™ƒ••‹‰‹ϐ‹…ƒ–Ž›Ž‘™‡”–Šƒ‹–Š‡„Žƒ‰”‘—’ǡ„—–Š‹‰Š‡”–Šƒ‹–Š‡‹ǦͳͶͶ‰”‘—’ȋ ‹‰Ǥ

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Fig. 3.‹ǦͳͶͶ‹†—…‡•ƒ—–‘’Šƒ‰›‹„‘–ŠͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ǤȋȌ—–‘’Šƒ‰‹…˜ƒ…—‘Ž‡•‘ˆ‹ǦͳͶͶ‰”‘—’ are much more than the scramble and blank group (*PδͲǤͲͷȌǤȋǡȌŠ‡‡š’”‡••‹‘•‘ˆƒ—–‘’Šƒ‰›Ǧ”‡Žƒ–‡† ’”‘–‡‹•™‡”‡†‡–‡…–‡†„›‡•–‡”„Ž‘––‹‰Ǥ ™ƒ•—•‡†ƒ•…‘–”‘ŽǤ „‘–ŠͷͶͻƒ†Ͷ͸Ͳ…‡ŽŽ•ǡ–Š‡ ‡š’”‡••‹‘Ž‡˜‡Ž•‘ˆ͵Ǧ ™ƒ•Ž‘™‡”ǡƒ†‡…Ž‹ͳƒ†͵Ǧ …ƒ‡‘—–Š‹‰Š‡”‹‹ǦͳͶͶ‰”‘—’•ȋȗP < 0.05).

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:997-1007 DOI: 10.1159/000369755 Published online: February 02, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

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Chen et al.: MiR-144 Inhibits Proliferation in Lung Cancer Cells

Fig. 4. ‹ǦͳͶͶ†‹”‡…–Ž›–ƒ”‰‡–• ǤȋǡȌ‹Ž†ƒ†—–ƒ––›’‡•‘ˆ ͵ǯ••‡‰‡–•ƒ”‡•Š‘™‡†Ǥ ȋȌ‘Ǧ–”ƒ•ˆ‡…–‹‘™‹–Š‹ǦͳͶͶ•‹‰‹ϐ‹…ƒ–Ž›•—’’”‡••‡†–Š‡Ž—…‹ˆ‡”ƒ•‡ƒ…–‹˜‹–›‘ˆ–Š‡”‡’‘”–‡”…‘–ƒ‹‹‰ –Š‡™‹Ž†Ǧ–›’‡͵̵ȋȗP