MicroRNA 125b promotes tumor metastasis through targeting tumor protein 53 induced nuclear protein 1 in patients with non small cell lung cancer

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1 Li et l. Cner Cell Int (15) 15:8 DOI 1.118/s x PRIMARY RESEARCH MiroRNA 15 promotes tumor metstsis through trgeting tumor protein 53 indued nuler protein 1 in ptients with non smll ell lung ner Open Aess Qinhun Li 1, Yng Hn, Chunhong Wng 3, Shn Shn 3, Yunyun Wng 3, Jingng Zhng nd To Ren 3* Astrt Bkground: Lung ner, predominntly non-smll-ell lung ner (NSCLC), is the leding use of ner deths worldwide. There is gret need to identify ritil effetors involved in metstsis of NSCLC tht will filitte the development of new therpeuti strtegies. Here we evluted the potentil role of mir-15 in the metstsis of NSCLC ells. Methods: Humn NSCLC ells were isolted from surgil tissues with Cner Cell Isoltion Kit. Expressions of mir- 15 nd TP53INP1 were deteted with rel-time PCR nd western lot. Humn mir-15 mimis, mir-15 inhiitor, TP53INP1 expression plsmid nd TP53INP1 sirna were trnsfeted into NSCLC ells with nuleofetor trnsfetion kit. NSCLC metstsis ws determined with dhesion ssy, invsive ssy nd lung tumor metstsis model. Results: The expression of mir-15 ws signifintly higher in poorly differentited NSCLC ells tht re endowed with high metstti potentils. Up-regultion of mir-15 ould enhne the metstti potentil of NSCLC ells in vitro nd in vivo, while down-regultion of mir-15 resulted in deresed metstti potentils in vitro nd in vivo. Further, tumor protein 53-indued nuler protein 1 (TP53INP1) ws n importnt trget of mir-15 involved in metstsis of NSCLC ells. TP53INP1 served s negtive regultor of NSCLC metstsis. Deresed expression of TP53INP1 in tumor tissues ws inversely ssoited with their expression of mir-15, signifintly lower in poorly differentited tumors nd inversely orrelted with the linil stges in ptients with NSCLC. Conlusions: These findings demonstrted tht mir-15 promoted tumor metstsis vi trgeting TP53INP1 in humn NSCLC ells, whih unovered rel linil relevne of mirornas in tumor iology, nd provided novel potentil ndidtes for NSCLC linil prtie. Keywords: NSCLC, Metstsis, mir-15, TP53INP1 Bkground Lung ner, espeilly non-smll-ell lung ner (NSCLC), is the most ommon use of humn ner relted mortlity [1 ]. Although omined tretments *Correspondene: rentosh@1.om Qinhun Li, Yng Hn nd Chunhong Wng ontriute eqully to this work 3 Deprtment of Respirtory Mediine, Est Hospitl, Tongji University Shool of Mediine, 15 Jimo Rod, Pudong New Are, 1 Shnghi, Chin Full list of uthor informtion is ville t the end of the rtile inluding surgery, hemotherpy, rdiotherpy nd trgeted therpy were pplied in linil mngement, the urrent outome of NSCLC ptients is still fr from stisfied, whih is mostly euse of NSCLC metstsis [ 7]. Therefore, explortion of ritil effetors involved in NSCLC metstsis is urgently needed. MiroRNAs (mirnas) re non-oding RNAs nd regulte trget genes t post-trnsriptionl level [8, 9]. It is well knowledged tht deregultion of mirnas ws involved in tumor initition nd progression [1]. MiR-15, humn homologue of lin-, ws reported 15 Li et l. This rtile is distriuted under the terms of the Cretive Commons Attriution. Interntionl Liense ( retiveommons.org/lienses/y/./), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( zero/1./) pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Li et l. Cner Cell Int (15) 15:8 Pge of 1 to e involved in tumor progression [11 15]. MiR-15 ould regulte tumor ellulr poptosis nd prolifertion [1 1]. In NSCLC, serum mir-15 ws signifintly inresed nd ws positively ssoited with NSCLC stges nd poor ptient survivl [9, 1]. Of note, mir- 15 expression in poorly differentited NSCLC ws signifintly higher thn those in well nd modertely differentited NSCLC [9, 1]. These findings indited n involvement of mir-15 in NSCLC metstsis, whih is lrgely undefined. In this study, we evluted the effet of mir-15 on metstsis of lung ner ells from NSCLC ptients. We oserved signifint higher expression level of mir- 15 in poorly differentited NSCLC ells. Of importnt, mngement of mir-15 expression ould modulte the NSCLC metstsis in vitro nd in vivo. Further, tumor protein 53-indued nuler protein 1 (TP53INP1) ws identified s ritil trget of mir-15 involved in NSCLC metstsis. Our findings provided new insights into the funtion of mir-15 during the metstsis of humn NSCLC nd were helpful for developing novel strtegy in tretment of NSCLC ptients. Results High expression of mir 15 in poorly differentited humn NSCLC Detetion of mir-15 expression in linil NSCLC ptients showed tht the expression level of mir-15 ws higher in lung ner tissue ompred with djent tissue (Fig. 1). We further showed tht the level of mir-15 ws signifintly higher in poorly differentited NSCLC thn those in well nd modertely differentited ners (Fig. 1). Besides, mir-15 expression ws positively orrelted with the linil stges of ptients with NSCLC (Fig. 1). These findings suggested tht mir-15 might e involved in NSCLC metstsis of linil ptients. Up regultion of mir 15 promoted humn NSCLC metstsis To determine the effet of mir-15 on metstti potentil of humn NSCLC, NSCLC ells were trnsfeted with mir-15 mimis nd then nlyzed for their metstti potentil. As shown in Fig., trnsfetion with mir- 15 mimis effetively enhned its expression level in NSCLC ells (p < 5). Given tht dhesion of tumor ells to extr-ellulr mtrix nd sement memrnes re onsidered to e the initil step in the invsive proess for metstti tumor ells, NSCLC ells trnsfeted with mir-15 mimis were exmined for their dhesion tivities to the sustrtes preoted with fironetin, whih is sement memer omponent. We found tht trnsfetion with mir-15 mimis signifintly elevted the Reltive expression of mir-15 Reltive mir-15 expression Reltive mir-15 expression 8 Adjent P<1 P<1 P<1 Tumor P=7 Well Modertely Poorly Differentition stte r=.733 P<1 1 3 TNM stge Fig. 1 Expression of mir-15 in humn NSCLC ells. Reltive expression of mir-15 ws determined y rel time PCR in NSCLC lung ner smples. The reltive expression of mir-15 in the well, modertely nd poorly differentited tumor tissues were shown. The orreltion etween the reltive mir-15 expression nd the TNM stge of NSCLC ptients ws nlyzed. Eh dot represented the results from one ptient

3 Li et l. Cner Cell Int (15) 15:8 Pge 3 of 1 Reltive mir-15 expression P<1 Control mimis MiR-15 mimis Reltive dhension ells P= Control mimis MiR-15 mimis 8 P=38 d.8 P=3 % Invsion Lung weight (g)... Control mimis MiR-15 mimis Control mimis MiR-15 mimis Fig. Up-regultion of mir-15 enhned NSCLC metstsis. NSCLC ells from different ptients were trnsfeted with mir-15 mimis for 1 h nd then ssyed for their expressions of mir-15. Eh dot represented the results from one ptient., NSCLC ells from different ptients were trnsfeted with mir-15 mimis or the ontrol, respetively, nd then ssyed for their dhesion tivity (n = ) nd invsion (n = 5). d Nude mie were hllenged LPS nd NSCLC ells tht were trnsfeted with mir-15 mimis or the ontrol. Lung tumor urden ws deteted y nlyzing lung weight. Dt were presented s mens (±SD) from five nude mie in eh group dhesion tivities of NSCLC ells (Fig. ). Then, we nlyzed the invsive potentil of NSCLC ells fter trnsfetion with mir-15 mimis, nd reveled tht trnsfetion with mir-15 mimis effetively enhned the invsion of NSCLC ells (Fig. ). To onfirm these results in vivo, nude mie were hllenged with NSCLC ells tht were trnsfeted with mir-15 mimis or ontrol, respetively. Over-expression of mir-15 signifintly enhned the lung tumor urdens of NSCLC ells (Fig. d). These results demonstrted tht up-regultion of mir-15 ould promote the metstti potentil of humn NSCLC. Down regultion of mir 15 redued humn NSCLC metstsis To further eluidte the effet of mir-15 on the metstti potentil of humn NSCLC, NSCLC ells were trnsfeted with mir-15 inhiitors nd then nlyzed for their metstti potentil. As shown in Fig. 3, trnsfetion with mir-15 inhiitors effetively deresed its expression level in NSCLC ells. Further, trnsfetion with mir-15 inhiitors signifintly inhiited the dhesion tivities of NSCLC ells (Fig. 3). Expetedly, trnsfetion with mir- 15 inhiitors lso effetively rogted the invsion of NSCLC ells (Fig. 3). To further onfirm this phenomenon in vivo, nude mie were hllenged with NSCLC ells tht were trnsfeted with mir-15 inhiitors or ontrol, respetively. Deresed expression of mir-15 onsiderly redued the lung tumor urdens of NSCLC ells (Fig. 3d). These results demonstrted tht down-regultion of mir-15 ould inhiit the metstti potentil of humn NSCLC. TP53INP1 ws the dominnt trget of mir 15 in regulting NSCLC metstsis To further understnd the effet of mir-15 on the metstsis of humn NSCLC, we predited the trgets of mir-15 y predition progrms inluding TrgetSn nd PiTr, nd seleted eight possile trget

4 Li et l. Cner Cell Int (15) 15:8 Pge of 1 Reltive mir-15 expression Control inhiitors P<1 MiR-15 inhiitors Reltive dhension ells Control inhiitors P<1 MiR-15 inhiitors P=3 d. P=8 % Invsion Lung weight (g).. Control inhiitors MiR-15 inhiitors Control inhiitors MiR-15 inhiitors Fig. 3 Down-regultion of mir-15 redued NSCLC metstsis. NSCLC ells from different ptients were trnsfeted with mir-15 inhiitors for 1 h nd then ssyed for their expression of mir-15. Eh dot represented the results from one ptient., NSCLC ells from different ptients were trnsfeted with mir-15 inhiitors or the ontrol, respetively, nd then ssyed for their dhesion tivity (n = ) nd invsion (n = 5). d Nude mie were hllenged LPS nd NSCLC ells tht were trnsfeted with mir-15 inhiitors or the ontrol. Lung tumor urden ws deteted y nlyzing lung weight. Dt were presented s mens (±SD) from five nude mie in eh group inluding STARD13, ZNF79, SH3TC, IRF, FUT, BAK1, ARID3B, nd TP53INP1 for rel time PCR nlysis. We found tht the expression of TP53INP1 exhiited drmtilly elevtion in NSCLC ells trnsfeted with mir-15 inhiitors (Fig. ). To onfirm this result, we performed western lot to detet the expression of TP53INP1 in NSCLC ells trnsfeted with mir-15 inhiitors. We found tht the protein level of TP53INP1 ws signifintly inresed y trnsfetion with mir-15 inhiitors in NSCLC ells (Fig. ). Given tht TP53INP1 hs een proved to e diret trget of mir-15 [7], we further evluted the possile role of TP53INP1 in tumor promoting tivity of mir-15. We found tht trnsfetion of mir-15 mimis filed to enhne the dhesion tivity nd invsion of NSCLC ells tht were o-trnsfeted with TP53INP1 expression vetor (Fig. e). To onfirm this phenomenon, NSCLC ells were o-trnsfeted with TP53INP1 sirna nd mir-15 inhiitors, nd then nlyzed for their metstti potentil. As shown in Fig. f h, trnsfetion with TP53INP1 sirna effetively deresed TP53INP1 expression in NSCLC ells nd rogted the effet of mir-15 inhiitors on dhesion tivity nd invsion of NSCLC ells. These results suggested tht TP53INP1 ws on fide trget of mir-15 in regulting the metstsis of humn NSCLC. TP53INP1 negtively regulted the metstsis of humn NSCLC We deteted the diret effet of TP53INP1 on the metstsis of NSCLC ells. NSCLC ells were trnsfeted with TP53INP1 expression vetor nd then nlyzed for their dhesion tivity nd invsion. We found tht trnsfetion with TP53INP1 expression vetor effetively inhiited the dhesion tivity nd invsion of NSCLC ells (Fig. 5, ). Trnsfetion with TP53INP1 sirna signifintly inresed the dhesion tivity nd invsive potentil of NSCLC ells (Fig. 5,

5 Li et l. Cner Cell Int (15) 15:8 Pge 5 of 1 d Reltive dhension ells Reltive expression Ctrl mimis Control inhiitors MiR-15 inhiitors STARD13 ZNF79 SH3TC IRF FUT BAK1 ARID3B TP53INP1 P<1 MiR-15 mimis MiR-15 mimi + Ctrl vetor P= MiR-15 mimi + TP53INP1 vetor e % Invsion TP53INP1 GAPDH 8 Ctrl mimis Untreted Control inhiitors MiR-15 inhiitors P=31 P=33 MiR-15 mimis MiR-15 mimis + Ctrl vetor MiR-15 mimis + TP53INP1 vetor f Reltive TP53INP1 expression Reltive TP53INP1 expression P=3 Control vetor TP53INP1 vetor P<1 Control sirna TP53INP1 sirna g Reltive dhension ells Ctrl inhiitors P=3 MiR-15 inhiitors MiR-15 inhiitors + Ctrl sirna P=8 MiR-15 inhiitors + TP53INP1 sirna h % Invsion Ctrl inhiitors P=17 MiR-15 inhiitors MiR-15 inhiitors + Ctrl sirna P=3 MiR-15 inhiitors + TP53INP1 sirna Fig. TP53INP1 ws funtionl trget of mir-15. NSCLC ells from different ptients (n = 3) were trnsfeted with mir-15 inhiitors for 1 h nd then ssyed for their expression of the indited genes using rel time PCR. NSCLC ells from one ptient were trnsfeted with mir- 15 inhiitors for 8 h nd then ssyed for their expression of TP53INP1 using western lot. NSCLC ells from different ptients were trnsfeted with TP53INP1 expression vetor for 1 h nd then ssyed for their expression of TP53INP1 mrna level. d, e NSCLC ells from different ptients were o-trnsfeted with mir-15 mimis nd TP53INP1 expression vetor, nd then ssyed for their dhesion tivity (n = ) nd invsion (n = 5). f NSCLC ells from different ptients were trnsfeted with TP53INP1 sirna or the ontrol for 1 h nd then ssyed for their expression of TP53INP1. g, h NSCLC ells from different ptients were o-trnsfeted with mir-15 inhiitors nd TP53INP1 sirna, nd then ssyed for their dhesion tivity (n = ) nd invsion (n = 5). Eh dot represented the results from one ptient

6 Li et l. Cner Cell Int (15) 15:8 Pge of 1 Reltive dhension ells Control vetor P<1 TP53INP1 vetor % Invsion Control vetor P=7 TP53INP1 vetor Reltive dhension ells Control sirna P=1 TP53INP1 sirna d % invsion 8 Control sirna P= TP53INP1 sirna e.5 P=9 f.8 P=35 Lung weight (g) Lung weight (g)... Control vetor TP53INP1 vetor Control sirna TP53INP1 sirna Fig. 5 TP53INP1 suppressed NSCLC metstsis., NSCLC ells from different ptients were trnsfeted with TP53INP1 expression vetor nd then ssyed for their dhesion tivity (n = ) nd invsion (n = 5)., d NSCLC ells from different ptients were trnsfeted with TP53INP1 sirna nd then ssyed for their dhesion tivity (n = ) nd invsion (n = ). e, f Nude mie were hllenged LPS nd NSCLC ells tht were trnsfeted with TP53INP1 expression vetor, TP53INP1 sirna or the ontrols. Lung tumor urden ws deteted y nlyzing lung weight. Dt were presented s mens (±SD) from five nude mie in eh group d). In onsistent, enfored TP53INP1 llevited the lung tumor urden of NSCLC ells, while deresed TP53INP1 enhned the lung tumor urden of NSCLC ells (Fig. 5e, f). These dt suggested tht TP53INP1 ws negtive regultor of humn NSCLC metstsis. TP53INP1 expression in tumor tissues ws orrelted with mir 15 expression nd linil prmeters in ptients with NSCLC To further investigte the linil relevne of our ove findings, we deteted the reltionship etween the

7 Li et l. Cner Cell Int (15) 15:8 Pge 7 of 1 expression of mir-15 nd TP53INP1 in linil NSCLC ptients. As shown in Fig., the expression of TP53INP1 ws signifintly lower in tumor tissues ompred with djent tissues. We further reveled tht the expression of mir-15 ws inversely orrelted with the expression level of TP53INP1 in tumor tissues (Fig. ). The expression of TP53INP1 ws signifintly deresed in poorly differentited NSCLC thn those in well nd modertely differentited ners, nd ws inversely ssoited with the linil stges of NSCLC ptients (Fig., d). These findings ssigned TP53INP1 s n importnt trget for mir-15 in promoting the metstsis of humn NSCLC. Disussion Tumor metstsis is the most prominent prolem in linil tretment of ner, s most ner mortlity is ssoited with disseminted disese rther thn the primry tumor []. However, the underlying mehnisms involved in the metstsis of tumor still remin unler. MiRNAs re known to regulte the expression of genes involved in tumor initition, prolifertion, poptosis nd metstsis [, 3]. In present study, we reported the ritil role of mir-15 in regulting the metstsis of NSCLC. We oserved higher expression level of mir-15 in poorly differentited NSCLC ells. We vlidted the elevted expression of mir-15 in tumor tissues nd its positive ssoition with the linil stges. Of importnt, up-regultion of mir-15 expression enhned NSCLC metstti potentil in vitro nd in vivo, while down-regultion of mir-15 expression deresed their metstti potentils. Our findings were onsistent with reent study whih showed tht serum mir-15 ws signifintly inresed in NSCLC ptients, ws positively ssoited with NSCLC stges nd poor ptient survivl, nd ws signifintly higher in poorly differentited NSCLC [9, 1]. Our findings ould enlrge our understnding of the potentil role of mir-15 in NSCLC nd suggested Reltive expression of TP53INP Adjent P<1 Tumor Reltive TP53INP1 expression r=-.71 P<1 Reltive mir-15 expression Reltive TP53INP1 expression P= P=5 Well Modertely Poorly 1 3 Differentition stte TNM stge Fig. Expression of TP53INP1 in tumor tissues of NSCLC ptients. Reltive expression of TP53INP1 ws determined y rel time PCR in NSCLC lung ner smples. The orreltion etween the reltive mir-15 expression nd the reltive TP53INP1 expression in NSCLC ptients ws nlyzed. The reltive expression of TP53INP1 in the well, modertely nd poorly differentited tumor tissues were shown. d The orreltion etween the reltive TP53INP1 expression nd the TNM stge of NSCLC ptients ws nlyzed. Eh dot represented the results from one ptient d Reltive TP53INP1 expression r=-.9 P=

8 Li et l. Cner Cell Int (15) 15:8 Pge 8 of 1 tht mir-15 ws promising trget for tretment of NSCLC. TP53INP1 is widely reognized s tumor suppressor gene with nti-prolifertive nd pro-poptoti funtions [, 5]. The expression of TP53INP1 ws redued in vrious humn ners, e.g., rest, pnres nd gstri ners [ 8]. Of interest, redution of TP53INP1 expression in gstri ner ws losely orrelted with their ggressive phenotypes [8]. These findings suggested TP53INP1 s n importnt effetor in tumor suppression. In this study, we found tht TP53INP1 ws n importnt trget of mir-15 in regulting the metstsis of NSCLC. We found tht over-expression of TP53INP1 ould signifintly rogte the tumor promoting effet of mir-15. Further, up-regultion of TP53INP1 ould signifintly inhiit the metstsis of NSCLC ells. Deresed expression of TP53INP1 effetively promoted NSCLC metstsis. Finlly, we found tht the deresed expression of TP53INP1 ws inversely orrelted with the expression of mir-15 nd the linil stges, nd ws deresed further in poorly differentited tumors in ptients with NSCLC. In NSCLC ells without tretment, expression of mir-15 ws lso negtively ssoited with TP53INP1 expression (Additionl file 1: Figure S1). Our findings were, to some extent, in line with reent study whih showed tht mir-15 ould promote prolifertion nd migrtion of type II endometril rinom ells through trgeting TP53INP1 [9]. However, the preise mehnisms for how TP53INP1 funtioned in metstsis of NSCLC undoutedly deserved suessive studies. Besides, the numer of ptients enrolled in this study ws reltively limited nd dditionl study on lrge size of linil smples might sustntite our findings. Conlusions Herein, we reported ritil role of mir-15 in NSCLC metstsis vi trgeting TP53INP1 in linil ptients. These findings were derived from linil smples nd thus losely refleted the rel linil relevne. MiR-15 nd TP53INP1 might e promising trgets for developing novel therpeutis for NSCLC linil prtie. Methods Ptients The humn study ws pproved y the Ethis Committee of Tongji University. Totlly 37 NSCLC ptients were enrolled in this study nd given written informed onsent efore olleting surgil tissues nd linil prmeters. Review of pthology reports onfirmed the dignosis. Sujets with utoimmune diseses or infetions were exluded. Informtion regrding linil pthologil hrteristis of ptients ws summrized in Tle 1. Regents nd ell ulture NSCLC ells were isolted from surgil tumor tissues using Cner Cell Isoltion Kit (Pnomis). Aording to the mnufture s instrutions, ells were ultured for dys t 37 C under 5 % CO in omplete RPMI 1 medium (GIBCO, ontining 1 % het-intivted fetl ovine serum supplemented with mm glutmine, 1 IU/ml peniillin nd 1 mg/ml streptomyin sulfte), nd used for experimentl reserh. MiR-15 mimis nd mir-15 inhiitor were from Rioio (Gungzhou, Chin). Humn TP53INP1 expression plsmid ws purhsed from Origen. Humn TP53INP1 sirna ws from Snt Cruz. The nuleofetor trnsfetion kit ws purhsed from Amx. Adhesion ssy Cell dhesion ssy ws ssyed s desried previously [, 3]. In rief, mirotiter wells were oted with fironetin (Sigm, St. Louis, MO, USA) overnight nd were loked for 3 min with.5 % BSA in PBS. NSCLC ells were suspended t finl onentrtion of ells/ml in serum-free medium for seeding. The MTTssy (Cymn) ws used to determine the numer of remining ells (dherent ells). Invsive ssy The BD Bioot Mtrigel Invsion Chmer ssy ws performed s desried y the mnufturers (8 μm, BD Biosiene). Briefly, the Mtrigel inserts were rehydrted nd 5 1 NSCLC ells were resuspended in.5 ml of serum-free medi nd then seeded onto the upper hmer of Mtrigel-oted filters. In the lower Tle 1 The linil pthologil hrters of the NSCLC ptients Clinil pthologil prmeter Lymph nodl metstsis is ording to pthologil dignosis nd linil plption. Clinil stge is ording to TNM stge Numer Sex Mle Femle 11 Age (yers) Tumor stge T1/ T3/ 31 Nodl sttus N/1 7 N/3 3 Histologil type Adenorinom 9 Others 8

9 Li et l. Cner Cell Int (15) 15:8 Pge 9 of 1 hmers,.75 ml of omplete medium ws dded s hemottrtnt. The whole hmer ws pled in one well of -well plte, nd ells were ultured in routine onditions. After h, the ells on the upper side of the hmer were srped, nd the ones on the lower side of the hmer were fixed y methnol, stined with hemtoxylin, nd invded ells were ounted under the mirosope. Five predetermined fields were ounted for eh memrne, nd the men vlues from three independent experiments in triplites were used. Dt re expressed s the perentge of invsion through the Mtrigel Mtrix nd memrne reltive to the migrtion through the ontrol memrne ording to the mnufturer s mnul. Rel time PCR Quntittive Rel-time RT-PCR ws performed s previously desried [31, 3]. All the primers nd proes were otined from Applied Biosystems. Totl RNA ws extrted using TRIzol regent. DNA ws synthesized with the PrimeSript RT regent Kit (TKR). Quntittive RT-PCR (qrt-pcr) nlyses were rried out to detet mrna expression using SYBR Premix Ex Tq (TKR), nd β-tin ws used s n internl ontrol. TqMn miro-rna ssys (Applied Biosystems) were used to quntittive the expression levels of mture mir- 15, nd U smll nuler RNA ws used s n internl ontrol. Western lotting Cells were lysed with M-PER protein extrtion regent (Piere) supplemented with protese inhiitor oktil. Cytoplsmi nd nuler extrts were prepred using NE-PER nuler nd ytoplsmi extrtion regents (Piere). After entrifugtion t 13,g under C for 15 min, the superntnts were olleted, nd the protein onentrtion of the extrts ws mesured y BCA Protein Assy (Piere) ording to mnufturer s instrutions. Twenty mirogrms of the protein were loded onto 1 % SDS polyrylmide gels nd trnsferred for 9 min t 1 V onto polyvinylidene fluoride memrnes using wet trnsfer system. The memrnes were wshed in 5 % skim milk in phosphte uffered sline plus 5 % Tween (PBST) for h in order to lok nonspeifi protein inding sites on the memrne. Immunolotting ws performed using monolonl ntiodies to TP53INP1 nd GAPDH (Sigm) t dilution of 1:1 in nonft milk Tris uffer. The memrne ws then wshed in PBST, proed with seondry nti-rit ntiody onjugted to horserdish peroxidse (Amershm Life Sienes) t dilution of 1:5, developed using n ECL Western Blotting KIT (Piere), nd exposed to X-ry film (Kodk). Lung tumor metstsis model BALB/ nude mie were purhsed from niml enter of Tongji University nd housed under speifi pthogen-free onditions. Lung tumor metstsis model were performed s previously desried [33, 3]. Briefly, nude mie (n = 5 per group) were intrtrhelly hllenged with LPS (1 μg/mouse), nd injeted with 1 5 NSCLC ells vi til vein h lter. Two weeks lter, nude mie were deteted for their lung tumor metstses/urdens tht were refleted y totl lung weights. The mie experiments were pproved y Ethis Committee of Tongji University. Sttistil nlyses T tests nd Person orreltion were used for sttistil nlyses using the progrm PRISM. (GrphPd Softwre In., Sn Diego, CA, USA). A vlue of P < 5 ws onsidered sttistilly signifint. Additionl file Additionl file 1: Figure S1. Expressions of mir-15 nd TP53INP1 in isolted NSCLC ells were determined y qpcr nd nlyzed for their negtive orreltion. Authors ontriutions QL nd YH rried out the experimentl studies. CW prtiipted in the study design nd drfted the mnusript. SS, YW nd JZ performed the sttistil nlysis, prtiipted in study design nd experimentl studies. TR oneived of the study, prtiipted in its design nd oordintion, nd helped to drft the mnusript. All uthors red nd pproved the finl mnusript. Author detils 1 Deprtment of Crdiothori Surgery, Est Hospitl, Tongji University Shool of Mediine, Shnghi, Chin. Deprtment of Pthology, Est Hospitl, Tongji University Shool of Mediine, Shnghi, Chin. 3 Deprtment of Respirtory Mediine, Est Hospitl, Tongji University Shool of Mediine, 15 Jimo Rod, Pudong New Are, 1 Shnghi, Chin. Servie Center for Fmily plnning, Mternl nd Child Helth Cre, Lnshn, Linyi, Shndong, Chin. Aknowledgements This work ws supported y Innovtion Fund of Siene nd Tehnology Deprtment of Pudong New Are (PKJ11-Y33), Ntionl Nturl Siene Foundtion of Chin (813799, 81555), Key Speilty in Shnghi City Helth Bureu (ZK1A8), nd Medil Guided Projet supported y Shnghi Committee of Siene nd Tehnology (111979). Compline with ethil guidelines Competing interests The uthors delre tht they hve no ompeting interests. Reeived: Ferury 15 Aepted: August 15 Referenes 1. Yu SL, Chen HY, Chng GC, Chen CY, Chen HW, Singh S et l (8) Miro- RNA signture predits survivl nd relpse in lung ner. Cner Cell 13:8 57

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