Role of HCP5-miR-139-RUNX1 Feedback Loop in Regulating Malignant Behavior of Glioma Cells

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1 originl rtile The Amerin Soiety of Gene & Cell Therpy Role of HCP-miR-19-RUNX1 Feedk Loop in Regulting Mlignnt Behvior of Gliom Cells Ho Teng 1,2, Ping Wng, Yixue Xue, Xioi Liu 1,2, Jun M, Heng Ci 1,2, Zhuo Xi 1,2, Zhen Li 1,2 nd Yunhui Liu 1,2 1 Deprtment of Neurosurgery, Shengjing Hospitl of Chin Medil University, Shenyng, People s Repuli of Chin; 2 Lioning Reserh Center for Trnsltionl Mediine in Nervous System Disese, Shenyng, People s Repuli of Chin; Deprtment of Neuroiology, College of Bsi Mediine, Chin Medil University, Shenyng, People Repuli of Chin Aerrnt expression of long nonoding RNAs hs reently een reported in tumorigenesis nd plys pivotl role in regulting mlignnt ehvior of ners. In this study, we onfirmed tht the long nonoding RNAs humn histoomptiility leukoyte ntigen (HLA) omplex P (HCP) ws up-regulted in gliom tissues s well s in nd ells. Knokdown of HCP inhiited the mlignnt iologil ehvior of gliom ells y reduing prolifertion, migrtion nd invsion, nd induing poptosis. HCP regulted the mlignnt ehvior of gliom ells y inding to mirorna-19, whih funtions s tumor suppressor. Moreover, knokdown of HCP down-regulted Runt-relted trnsription ftor 1, diret nd funtionl downstrem trget of mirorna-19 tht is involved in mirorna- 19-medited tumor-suppressive effets in gliom ells. Runt-relted trnsription ftor 1 inresed promoter tivities nd upregulted expression of the onogeni gene stroyte elevted gene-1 (AEG-1). Runt-relted trnsription ftor 1 lso inresed the promoter tivities nd expression of HCP, whih showed positive feedk loop in regulting the mlignnt ehvior of gliom ells. In onlusion, this study demonstrted tht the HCP-miroRNA-19- Runt-relted trnsription ftor 1 feedk loop plys pivotl role in regulting the mlignnt ehvior of gliom ells, whih my provide potentil therpeuti strtegy for treting gliom. Reeived 1 Otoer 21; epted 1 My 21; dvne online pulition 19 July 21. doi:.8/mt.21. INTRODUCTION Gliolstom multiforme (GBM), whih ounts for 1% of ll intrrnil tumors, is the most ommon nd ggressive rin tumor in the entrl nervous system. 1 Due to the highly invsive nture nd insensitivity to rdiotherpy or hemotherpy, the prognosis for ptients with GBM is poor, with medin survivl of 1 months only. 2 Reent umulted evidene hs shown tht nonoding RNAs (nrnas) ply n importnt role in tumorigenesis nd progression,, whih my provide new strtegy for GBM tretment. Bsed on trnsript size, nrnas n e divided into two tegories: long nonoding RNAs (lnrnas) nd smll nrnas. LnRNAs re defined s RNA moleules longer thn 2 nuleotides tht re not trnslted into proteins. Aerrnt lnrna expression ws reently found to e ssoited with glioms. Severl lnrnas, suh s CASC2, TUG1, nd XIST, were found to e dysregulted in glioms nd re onsidered new therpeuti trgets. 8 LnRNA histoomptiility leukoyte ntigen (HLA) omplex P (HCP) is expressed primrily in immune system ells, suh s the spleen, lood, nd thymus, whih is onsistent with potentil role in utoimmunity. 9 A reent study reported tht HCP showed errnt expression in humn ners. HCP ws signifintly down-regulted in ptients with ovrin ner, ut ws onsidered suseptiility lous for HCV-ssoited heptoellulr rinom. 11 However, possile funtionl role of HCP in gliom remins elusive. MiroRNAs (mirnas) elong to lss of endogenous, smll nrnas tht regulte gene expression t post-trnsriptionl level. By inding to the -untrnslted region ( -UTR) of trget messenger RNAs, mirnas inhiit trnsltion or promote degrdtion of trget messenger RNAs. 12 MiRNA involvement in the mlignny proess hs een reported in vrious ners, 1 inluding glioms. 1 MiR-19, whih hs puttive inding sites with HCP s predited y Strse ( ws first studied in neurodegenertive diseses. 1 Reent reserh hs shown tht mir- 19 expression ws signifintly deresed in severl tumor types, inluding heptoellulr rinom, oloretl ner, nd rest ner, 1 18 nd funtions s tumor suppressor. However, little is known out the potentil role of mir-19 in humn glioms. Runt-relted trnsription ftor 1(RUNX1), lso known s ute myeloid leukemi 1 protein, is essentil for genertion of hemtopoieti stem ells nd is one of the most frequently mutted genes involved in humn leukemi. 19,2 Using the mirna trget predition softwre Trget Sn ( we identified RUNX1 s presumed trget of mir-19. Reent reserh identified RUNX1 s key regultor of tumorigenesis in vrious epithelil ners. 21,22 However, the role of RUNX1 in humn gliom ells remins unler. The mjor im of this study ws to investigte the expression of HCP, mir-19, nd RUNX1 in gliom tissues nd ell lines. Roles in regulting the mlignnt ehvior of gliom ells nd intertions mong HCP, mir-19, nd RUNX1 were lso explored. Correspondene: Yunhui Liu, Deprtment of Neurosurgery, Shengjing Hospitl of Chin Medil University, Shenyng 1, People s Repuli of Chin. E-mil: neurosurgery_sj@1.om 18 vol. 2 no., ot. 21

2 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells RESULTS Knokdown of HCP inhiited mlignnt ehviors of gliom ells Quntittive rel-time polymerse hin retion (qrt-pcr) ws performed to evlute HCP expression levels in norml rin tissues (NBTs), grde I II, grde III, nd grde IV gliom tissues, s well s in norml humn stroytes (NHAs) nd nd ell lines. HCP expression ws positively orrelted with the pthologil grde of gliom tissues (Figure 1) nd ws signifintly up-regulted in nd ell lines ompred with the NHA group (Figure 1). Stle HCP silened onstruts were used to ssess the funtionl role of HCP in nd ells. As shown in Figure 1, the Cell Counting Kit-8 (CCK8) ssy reveled tht prolifertion of nd ells ws deresed in the HCP ( ) group ompred with the negtive ontrol (NC) group. A similr trend ws oserved in trnswell ssy (Figure 1d), whih showed tht migrtion nd invsion of gliom ells were inhiited in the group ompred with the NC group. Figure 1e showed tht poptosis rtes were higher in the groups ompred with NC groups. These dt suggest tht knokdown of HCP exerts tumor-suppressive effets in humn gliom ells. HCP ound to mir-19 nd down-regulted its expression A possile mirna inding site for HCP ws predited y ioinformti dtses ( To verify whether mir-19 ws inding with nd regulted y HCP, mir-19 expression ws deteted in gliom nd ell lines with HCP knokdown. Figure 2 showed tht knokdown of HCP inresed mir-19 expression. We next loned reporter plsmids ontining the predited mir-19 inding site (HCP-Wt nd HCP-Mut) nd used dul-luiferse reporter ssy to investigte whether the mir-19 inding site within HCP ws funtionl. As shown in Figure 2, otrnsfetion of MiR-19-p gonist (gomir-19) nd HCP-Wt signifintly deresed luiferse tivity, while otrnsfetion of negtive ontrol of gomir (gomir-19nc) nd HCP-Wt did not hnge the luiferse tivity. Similrly, otrnsfetion of gomir-19 nd HCP-Mut did not hnge the luiferse tivity. These dt suggest tht the mir-19 inding site within HCP is funtionl. Moreover, we used iotin vidin pull-down system to investigte whether mir-19 ould pull down HCP. As shown in Figure 2, HCP ws pulled down y mir-19, ut the mir-19 muttion group ws unle to pull down HCP, mening tht reognition etween HCP nd mir-19 ws speifi. We lso used n inverse pull-down ssy to investigte whether HCP ould pull down mir-19 using iotin-leled speifi HCP proe (Figure 2d). The minimum free energy of the intertion etween HCP nd mir-19 ws lulted using n in silio method (see Supplementry Figure S1). In ddition, mir-19 expression ws deteted y PCR. The results indite tht mir-19 ws down-regulted in gliom tissues nd ell lines ompred with NBTs nd NHAs, respetively (Figure 2e,f). MiR-19 ted s tumor suppressor in gliom ell lines To further evlute the iologil role of mir-19 in glioms, gomir-19, nd mir-19-p ntgonist (ntgomir-19) were trnsfeted into humn gliom nd ells, respetively. Compred with the gomir-19 NC group, overexpression of mir- 19 resulted in signifint derese in ell prolifertion, migrtion, invsion, nd indued poptosis of nd ells, while mir- 19 inhiition promoted ell prolifertion, migrtion nd invsion, nd inhiited ell poptosis (Figure,,). These dt suggest tht mir-19 ts s tumor suppressor in gliom ell lines. MiR-19 inhiited RUNX1 expression y trgeting the -UTR Using Trget Sn ( to predit the onserved trgets of mir-19, we found tht the UTR of RUNX1 ontins puttive inding site (Tles 1 nd 2). We then ssessed whether mir-19 ould negtively regulte RUNX1 using Western lot. As shown in Figure, RUNX1 protein expression ws deresed in the gomir-19 group, ut ws inresed in the ntgomir-19 group. Susequently, we loned reporter plsmids ontining the widetype -UTR of RUNX1 (RUNX1- -UTR-Wt) to verify whether RUNX1 ws funtionl trget of mir-19. As shown in Figure, otrnsfetion of gomir-19 nd RUNX1- -UTR-Wt signifintly deresed luiferse tivity, while otrnsfetion of gomir-19 NC nd RUNX1- -UTR-Wt did not hve n effet. Cotrnsfetion of gomir-19 nd RUNX1- -UTR-Mut did not hnge the luiferse tivity. These findings indite tht mir-19 inds diretly to RUNX1 through its predited inding site nd down-regultes RUNX1 expression. We lso ssessed the messenger RNA expression of RUNX1 in gliom tissue nd ell lines. Compred with norml rin tissues nd NHA, signifint up-regultion of RUNX1 ws oserved in gliom tissues nd ell lines, respetively (Figure,d). Moreover, RUNX1 protein expression ws orrelted with the pthologil grde of gliom tissues (Figure e). RUNX1 ted s n onogene in gliom ell lines We explored the possile funtionl role of RUNX1 in gliom ells. As shown in Figure, the CCK8 ssy reveled tht prolifertion of nd ells ws inresed in the RUNX1(+) group ompred with the RUNX1(+)NC group, nd ws deresed in the RUNX1( ) group ompred with the RUNX1( ) NC group. As shown in Figure, the numer of ells involved in migrtion nd invsion ws signifintly inresed in the RUNX1(+) group ompred with the RUNX1(+)NC group. However, opposite effets were oserved in the RUNX1( ) group. As shown in Figure, up-regulted RUNX1 signifintly inhiited poptosis in oth nd ells ompred with the NC group. RUNX1 down-regultion led to opposite effets. These results indite tht RUNX1 funtions s n onogene in humn gliom ell lines. HCP knokdown suppressed RUNX1 expression y trgeting mir-19 The effets of otrnsfeted HCP nd mir-19 on nd ells were ssessed. HCP knokdown with mir-19 overexpression Moleulr Therpy vol. 2 no. ot

3 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells Reltive expression of HCP Ts NB d ~II ei d Gr II ei d Gr NC 1... Astroyte V ei d Gr Cell prolifertion Reltive expression of HCP NC 2 Migrtion Cell numer of migrtion 1 NC Invsion Cell numer of invsion 1 NC e NC 2 NC % of poptosis Figure 1 Expression of histoomptiility leukoyte ntigen (HLA) omplex P (HCP) in humn gliom tissues nd ell lines, nd the effets of HCP on gliom ells. () The expression HCP in norml rin tissues (NBTs), Grde I II gliom tissues, Grde III nd Grde IV gliom tissues. Error rs represent s the men ± SD (n =, eh group). P <.1, P <.1, P <.. () The expression of HCP in humn norml stroytes nd gliolstom multiforme (GBM) ell lines ( nd ). Error rs represent s the men ± SD (n =, eh group). P <.1. () Effet of HCP Knokdown on ell prolifertion of nd ells. (d) Effet of HCP Knokdown on ell migrtion nd invsion of nd ells. (e) Effet of HCP Knokdown on ell poptosis of nd ells. Error rs represent s the men ± SD (n =, eh group). P <.. Sle rs represent 2 μm. enhned the tumor suppressive effet used y HCP knokdown lone, while HCP knokdown with mir-19 inhiition prtilly resued HCP knokdown-medited redution of prolifertion, 188 migrtion nd invsion, nd deresed poptosis (Figure,,). These results indite tht the effets of HCP knokdown in gliom ells depend speifilly on mir-19 overexpression. vol. 2 no. ot. 21

4 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells We next ssessed whether knokdown of HCP inhiits RUNX1 expression y up-regulting mir-19. As shown in Figure d, HCP knokdown signifintly down regulted RUNX1 protein expressions. Moreover, HCP knokdown with mir-19 overexpression enhned the RUNX1 suppression effet used y HCP knokdown lone, while HCP knokdown with mir-19 inhiition resued RUNX1 expression, suggesting tht HCP knokdown inhiits RUNX1 expression y up-regulting mir-19. RUNX1 promoted expression nd ound to AEG-1 promoters The stroyte elevted gene-1 (AEG-1) ws identified s diret downstrem trget of RUNX1 y ioinformti dtses (TFSEARCH). As shown in Figure 7, AEG-1 expression ws inresed in the RUNX1 (+) group, ut ws deresed in the RUNX1 ( ) group ompred with the NC group. To determine whether RUNX1 might e inding to the AEG-1 promoter, luiferse ssys were onduted. The trnsription Reltive expression of mir NC Reltive luiferse tivity gomir-19 NC gomir-19 Mut Reltive luiferse tivity gomir-19 NC gomir-19 HCP-Wt HCP-Mut HCP-Wt HCP-Mut d Fold enrihment of HCP 2 Bio-NC Bio-MiR-19-Wt Bio-MiR-19-Mut Fold enrihment of mir-19 1 Bio-NC proe Bio-HCP proe e Reltive expression of mir f Reltive expression of mir NBTs Grde I~II Grde III Grde IV. Astroyte Figure 2 MiroRNA-19 (mir-19) ws down-regulted y histoomptiility leukoyte ntigen (HLA) omplex P (HCP). () Reltive expression of mir-19 fter ells trnsfeted with the expression of HCP hnged. Error rs represent s the men ± SD (n =, eh group). P <.. () Reltive luiferse tivity ws performed y dul-luiferse reporter ssy. Error rs represent s the men ± SD (n =, eh group). P <.. () Quntittive rel-time polymerse hin retion (qrt-pcr) ws used to detet HCP in the smple pulled down y iotinylted mir-19. (n =, eh group). P <.1. (d) QRT-PCR ws used to detet mir-19 in the smple pulled down y iotinylted HCP proe. (n =, eh group). P <.1. (e) MiR-19 expression in norml rin tissues (NBTs), Grde I II gliom tissues, Grde III nd Grde IV gliom tissues. Error rs represent s the men ± SD (n =, eh group). P <.1, P <.1, P <.. (f) MiR-19 expression in humn norml stroytes nd GBM ell lines ( nd ). Error rs represent s the men ± SD (n =, eh group). P <.1. Moleulr Therpy vol. 2 no. ot

5 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells Cell prolifertion gomir-19 NC gomir ntgomir-19 NC ntgomir-19.. gomir-19 NC gomir-19 ntgomir-19 NC 2 ntgomir-19 Migrtion Cell numer of migrtion 1 Invsion Cell numer of invsion 1.. gomir-19.. ntgomir-19 NC ntgomir-19. ntgomir-19 NC ntgomir gomir-19 NC gomir-19 NC gomir % of poptosis 2 1 gomir-19 NC gomir-19 ntgomir-19 NC ntgomir-19 Figure The effets of mirorna-19 (mir-19) on gliom ell lines. () Effet of mir-19 on ell prolifertion of nd ells. () Effet of mir-19 on ell migrtion nd invsion of nd ells. () Effet of mir-19 on ell poptosis of nd ells. Error rs represent s the men ± SD (n =, eh group). P <., P <.. Sle rs represent 2 μm vol. 2 no. ot. 21

6 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells Tle 1 Trget sore etween mirorna-19 (mir-19) nd Runt-relted trnsription ftor 1 (RUNX1) Trget Representtive Conserved sites Poorly onserved sites Totl Aggregte gene trnsript Gene nme Totl 8mer 7mer-m8 7mer-1A Totl 8mer 7mer-m8 7mer-1A ontext+ sore PCR RUNX1 NM_17 Runt-relted trnsription ftor PCR, polymerse hin retion. Tle 2 Trget rnks of RUNX1 ompred with other mir-19 trgets Rnk Gene nme Gene/Isoform desription 1 NRA2 Nuler reeptor sufmily, group A, memer 2 2 PTAR1 Protein prenyltrnsferse lph suunit repet Trget Seed Conserved seed mthes Non-onserved seed mthes Refseq ID rnk sore mth totl 8mer M8 7mer A1 7mer mer 8mer M8 7mer A1 7mer mer NM_ NM_ ELTD1 EGF, ltrophilin nd seven trnsmemrne domin NM_ TMF1 TATA element modultory NM_ ftor 1 ADCY1 rin denylte ylse 1 NM_ TRIM7 Triprtite motifontining 7 protein NM_ DCBLD2 Disoidin, CUB nd LCCL NM_ domin ontining 2 8 PSD ADP-riosyltion ftor NM_ gunine nuleotide 9 DIS KIAA8 NM_ APLP2 Amyloid et (A) NM_ preursor-like protein 2 11 XPO Exportin NM_ GPMA Glyoprotein MA NM_ isoform 2 1 GRSF1 G-rih RNA sequene NM_ inding ftor 1 isoform 1 1 RNF2 Ring finger protein 2 NM_ TCF Trnsription ftor NM_ isoform 1 HNRPU Heterogeneous nuler NM_ rionuleoprotein U 17 SEPT11 Septin 11 NM_ FBN2 Firillin 2 preursor NM_ CDK Cylin-dependent kinse NM_ ANK2 Ankyrin 2 isoform 1 NM_ MGA MAX gene ssoited NM_ UBEA Uiquitin protein ligse NM_ EA isoform 2 RUNX1 Runt-relted trnsription NM_ ftor 1 isoform 2 SLC1A2 Exittory mino id NM_ trnsporter 2 2 SGK29 NKF kinse fmily NM_ memer 2 PRDM1 PR domin ontining 1 NM_ isoform 2 27 CUL ullin NM_ PHF PHD finger protein NM_ isoform 1 29 LAPTMB Lysosoml ssoited NM_ trnsmemrne protein HOXA Homeoox A isoform NM_ Moleulr Therpy vol. 2 no. ot

7 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy RUNX1 RUNX1 gomir-19 NC gomir-19 ntgomir-19 NC ntgomir-19 2 kd 7 kd 2 kd 7 kd Reltive expression of RUNX gomir-19 NC gomir-19 ntgomir-19 NC ntgomir-19 Reltive expression of RUNX1 Reltive luiferse tivity 8 2 NBTs Grde I~II Grde III Grde IV gomir-19 NC gomir-19 RUNX1- UTR-Wt Reltive expression of RUNX1 Reltive luiferse tivity 1. d 1... RUNX1- UTR-Mut RUNX1- UTR-Wt RUNX1- UTR-Mut 8 2 Astroyte gomir-19 NC gomir-19 e RUNX1 RUNX1 NBTs Grde III Grde I~II Grde IV 2 kd 7 kd 2 kd 7 kd Reltive expression of RUNX NBTs Grde I~II Grde III Grde IV Figure MiroRNA-19 (mir-19) inhiited the expression of Runt-relted trnsription ftor 1 (RUNX1) y trgeting its -untrnslted region ( -UTR). () Western lot nlysis for RUNX1 in nd ells, fter ells trnsfeted with the expression of mir-19 hnged. Error rs represent s the men ± SD (n =, eh group). P <., P <.. () Reltive luiferse tivity ws performed y dul-luiferse reporter ssy. Dt represent men ± SD (n =, eh). P <.. () The expression of RUNX1 in norml rin tissues (NBTs), Grde I II gliom tissues, Grde III nd Grde IV gliom tissues. Error rs represent s the men ± SD (n =, eh group). P <.1, P <.1, P <.1, P <.1, P <.. (d) RUNX1 expression in humn norml stroytes nd gliolstom multiforme (GBM) ell lines ( nd ). Error rs represent s the men ± SD (n =, eh group). P <.1. (e) The protein expression of RUNX1 in humn gliom tissues. Error rs represent s the men ± SD (n =, eh group). P <.1, P <.1, P <.1, P <.1, P <.. strt site position of AEG-1 ws predited y DBTSS HOME. A puttive RUNX1 inding site t -829 in AEG-1 ws onfirmed. Wild-type (onstrut ), deletion onstrut (onstrut ) nd the puttive inding site were identified. As shown in Figure 7, deletion of the puttive RUNX1 inding site (-829 region) signifintly redued AEG-1 promoter tivity. These results indite tht the element neessry for high AEG-1 promoter tivity is likely to reside in the -829 region. To further determine whether RUNX1 ws diretly ssoited with AEG-1 promoters, Chromtin immunopreipittion (ChIP) vol. 2 no. ot. 21

8 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells 2. Cell prolifertion RUNX1(+)NC 1. RUNX1(+) RUNX1( )NC 1. RUNX1( ).. RUNX1(+) RUNX1( )NC RUNX1( ) Migrtion RUNX1(+)NC Cell numer of mgirtion Cell numer of invsion Invsion RUNX1(+)NC RUNX1(+) RUNX1( )NC RUNX1(+)NC RUNX1(+) % of poptosis 7.2 RUNX1( ) RUNX1( )NC RUNX1( ) RUNX1(+)NC RUNX1(+) RUNX1( )NC RUNX1( ) Figure The effets of Runt-relted trnsription ftor 1 (RUNX1) on gliom ell lines. () Effet of RUNX1 on ell prolifertion of nd ells. () Effet of RUNX1 on ell migrtion nd invsion of nd ells. () Effet of RUNX1 on ell poptosis of nd ells. Error rs represent s the men ± SD (n =, eh group). P <., P <.. Sle rs represent 2 μm. Moleulr Therpy vol. 2 no. ot

9 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy ssys were performed. As negtive ontrol, PCR ws onduted to mplify the 1, p upstrem region of the puttive RUNX1 inding site, whih ws not expeted to ssoite with RUNX1. There ws n intertion etween RUNX1 nd the AEG-1 puttive inding sites. There ws no intertion etween RUNX1 nd the ontrol region (Figure 7). RUNX1 medited the tumor-suppressive effets of mir-19 in gliom ell lines To verify whether RUNX1 is involved in regulting the influene of mir-19 on mlignnt ehvior of gliom ells, otrnsfetion of mir-19 nd RUNX1 ws onduted to ssess ell prolifertion, migrtion, invsion, nd poptosis. Compred with the ontrol group, prolifertion, migrtion nd invsion of gliom ells were deresed in the gomir-19&runx1( ) group, ut were inresed in the ntgomir-19&runx1(+) group. Prolifertion, migrtion nd invsion of gliom ells were deresed in the gomir-19&runx1( ) nd ntgomir-19&runx1( ) groups ompred with the gomir-19&runx1(+) nd ntgomir- 19&RUNX1(+) groups, respetively (Figure 8,). Moreover, ell poptosis ws inresed in the gomir-19&runx1( ) group ompred with the gomir-19&runx1(+) group, nd ws deresed in the ntgomir-19&runx1(+) group ompred with the ntgomir-19&runx1( ) group (Figure 8). These dt suggest tht mir-19 inhiits the mlignnt ehvior of gliom ells y down-regulting RUNX1. Western lot ws used to lrify whether mir-19 inhiited AEG-1 expression y down-regulting RUNX1. Compred with the NC group, AEG-1 expression ws deresed in the gomir-19 group, ut ws inresed in the ntgomir-19 group (Figure 8d). Compred with the gomir-19 group, AEG-1 expression ws signifintly inresed in the gomir-19&runx1(+) group. Compred with the ntgomir-19 group, AEG-1 expression ws signifintly deresed in the ntgomir-19&runx1( ) group. These results indite tht mir-19 my inhiit AEG-1 expression y down-regulting RUNX1. RUNX1 feedk promoted HCP expression y inding to HCP promoters We identified puttive RUNX1 inding site within the promoter region of HCP using ioinformti dtses (TFSEARCH). As shown in Figure 9, HCP expression ws inresed in the RUNX1(+) group ompred with the NC group, wheres expression ws deresed in the RUNX1( ) group. Luiferse ssys were onduted to ssess whether RUNX1 might e inding to the HCP promoter. A puttive RUNX1 inding site t the -97 position in HCP ws onfirmed. As shown in Cell prolifertion gomir-19 +ntgomir-19 Migrtion. + gomir-19 + ntgomir-19 Cell numer of migrtion Invsion Cell numer of invsion 2 1 +gomir-19 +ntgomir vol. 2 no. ot. 21

10 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells Figure 9, deletion of the region surrounding the -97 site signifintly redued HCP promoter tivity. Furthermore, ChIP ssys showed n intertion etween RUNX1 nd the HCP puttive inding sites, ut no intertion with the ontrol region (Figure 9).. HCP knokdown omined with mir-19 overexpression suppressed tumor growth in xenogrft mouse model To further onfirm the ove findings, n in vivo tumor model ws used. The results suggest tht the HCP ( ), mir-19 (+), nd +gomir-19 +ntgomir % of poptosis 2 +gomir-19 +ntgomir-19 d RUNX1 RUNX1 NC NC +gomir-19 NC +gomir-19 NC +ntgomir-19 NC +ntgomir-19 2 kd 7 kd 2 kd 7 kd Reltive expression of RUNX NC NC+gomir-19 NC +gomir-19 NC+ntgomir-19 NC +ntgomir-19 Figure MiroRNA-19 (mir-19) medited the tumor-suppressive effets of histoomptiility leukoyte ntigen (HLA) omplex P (HCP) knokdown on gliom ell lines. () Cell Counting Kit-8 (CCK8) ssy to evlute the effet of HCP nd mir-19 on ell prolifertion in nd ells. () Trnswell ssy to evlute the effet of HCP nd mir-19 on ell migrtion nd invsion of nd ells. () Flow ytometry nlysis to evlute the effet of HCP nd mir-19 on ell poptosis of nd ells. Error rs represent s the men ± SD (n =, eh group). P <., P <.1, P <.. Sle rs represent 2 μm. (d) Western lot nlysis for Runt-relted trnsription ftor 1 (RUNX1) in nd ells with the expression of HCP hnged. Error rs represent s the men ± SD (n =, eh group). P <., P <.. Moleulr Therpy vol. 2 no. ot

11 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy &mir-19(+) groups hd smller tumors ompred with the NC+miR-19(+) NC group (Figure ). Tumors in the &mir-19(+) group hd the lowest volume nd weight ompred to the other groups (Figure,d). Moreover, mie in the &mir-19(+) group hd the longest survivl time (Figure e). DISCUSSION In this study, we demonstrted tht expression of the lnrna HCP ws inresed in gliom tissues nd ell lines. Knokdown of HCP inhiited prolifertion, ell migrtion nd invsion, nd promoted poptosis in gliom ells. HCP regulted the mlignnt ehvior of gliom ells y inding to mir-19, whih funtioned s tumor suppressor in gliom ells. HCP knokdown lso down-regulted the trnsription ftor RUNX1. We onfirmed tht RUNX1 is involved in mir-19-medited inhiition of mlignnt iologil properties in gliom ells. RUNX1 up-regulted the tivities of nd interted with AEG-1 promoters. In ddition, RUNX1 lso influened HCP expression, reting positivefeedk loop. In vivo studies showed tht HCP knokdown omined with mir-19 overexpression produed the smllest tumor nd resulted in the longest survivl time in nude mie. Reently umulted evidene hs shown tht lnrnas ply n importnt role in tumorigenesis, progression, nd metstsis. 2 2 Understnding the moleulr mehnisms of lnrnas in gliom ells my provide new therpeuti strtegies for treting GBM. The funtion of HCP, loted on hromosome p21., is not well understood in humns. HCP ws found to e frequently down-regulted in humn ovrin ner, while nother study indited tht HCP ws suseptiility lous for HCV-ssoited heptoellulr rinom. 11 Our results suggested tht HCP expression ws positively orrelted with histopthologil grde in humn gliom tissues, nd ws muh higher in nd ells thn in NHAs. Furthermore, knokdown of HCP inhiited ell prolifertion, migrtion nd invsion, nd promoted poptosis in nd ells. These AEG-1 AEG-1 Puttive RUNX1 inding site 829 RUNX1(+)NC RUNX1(+) RUNX1( )NC RUNX1( ) Lu Lu 7 kd 7 kd 7 kd 7 kd AEG-1 promoter tivity Reltive expression of AEG RUNX1(+)NC RUNX1(+) RUNX1( )NC RUNX1( ) pex expty vetor pex-runx1, 2, 1, Puttive RUNX1 inding site +1 AEG-1 TSS 829 PCR2 99 p PCR1 11 p 2 1 Input PCR1 PCR2 PCR1 PCR2 IgG RUNX1 Input IgG RUNX1 Input IgG RUNX1 Input IgG RUNX1 Figure 7 Runt-relted trnsription ftor 1 (RUNX1) promoted the expression of stroyte elevted gene-1 (AEG-1) nd ound to the promoters of AEG-1. () Western lot nlysis for AEG-1in nd ells with the expression of RUNX1 hnged. Error rs represent s the men ± SD (n =, eh group). P <., P <.. () RUNX1 on the promoter tivity of AEG-1 in nd ell lines. Error rs represent s the men ± SD (n =, eh group). P <.. () RUNX1 ound to the promoter of AEG-1 in nd ell lines. Shemti representtion of the humn AEG-1 promoter region, p upstrem of the trnsription strt site (trnsription strt site (TSS), designted s +1). Polymerse hin retion (PCR) ws onduted with the resulting preipitted DNA vol. 2 no. ot. 21

12 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells findings suggested tht HCP my funtion s n onogene in gliom ells. To explore the mehnisms of gliom suppression medited y HCP knokdown, we identified mir-19 s novel trget of HCP. MiR-19 is onsistently reported to e down-regulted in vrious humn ners nd plys ruil role in tumor suppression. MiR- 19 inhiits the prolifertion nd metstsis of lryngel squmous rinom ells y trgeting hemokine reeptor. 2 Restored expression of mir-19 signifintly promoted ell poptosis y reduing Jun expression in gstri ner ells. 27 Overexpression of mir-19 inhiits invsion nd metstsis of oloretl ner ells vi inding with type I insulin-like growth ftor reeptor. 28 This study indited tht mir-19 ws expressed t low levels in gliom tissues nd ell lines ompred with NBTs nd NHAs, respetively. Furthermore, mir-19 funtioned s tumor suppressor in gliom ells y inhiiting prolifertion, migrtion nd invsion, s well s promoting poptosis. Consistent with our results, reent report demonstrted tht mir-19 sensitized humn gliom ells to temozolomide-indued poptosis. 29 Another study showed tht mir-19 suppressed GBM nd U172 ell migrtion nd invsion prtly y trgeting ZEB1 nd ZEB2. Our results reveled the signifine of the intertion etween HCP nd mir-19 in tumorigenesis given tht HCP knokdown inhiited the mlignnt ehvior of gliom ells y up-regulting mir-19. Reent studies hve highlighted the importne of RUNX1 in solid tumors, with funtions s either tumor promoter or suppressor. RUNX1 lso ppers to hve growth-promoting roles in severl epithelil tumors, suh s squmous ell rinoms. 21 Knokdown of RUNX1 in epithelil ovrin ner ells led to shrp derese in ell prolifertion, migrtion nd invsion, nd indued G1 ell yle rrest. 22 RUNX1 is overexpressed in humn neurofirom initition ells nd ontriutes to neurofiromtosis type 1 formtion. 1 Therpies trgeting RUNX1 through modultion of its posttrnsltionl modifitions re onsidered potentil strtegies for ner tretment. 2 In this study, RUNX1 expression ws inresed in gliom tissues s well s in nd ell lines. Overexpression of RUNX1 promoted ell prolifertion, migrtion nd invsion, nd inhiited ell poptosis, while RUNX1 knokdown showed opposite effets, suggesting tht RUNX1 funtions s n onogene in gliom ells. Our results showed tht mir-19 overexpression inhiited RUNX1 protein expression. MiR-19 nd RUNX1 o-overexpression signifintly reversed the inhiition effet indued y up regulting mir-19 lone, reveling tht mir-19 inhiits the mlignnt ehvior of gliom ells y inhiiting RUNX1 expression. AEG-1 ws first identified nd loned s novel gene indued in primry humn fetl stroytes fter infetion with humn immunodefiieny virus-1. Numerous reent studies hve shown tht AEG-1 is up-regulted in vrious tumor types 8 nd is overexpressed in more thn 9% of humn glioms. 9 AEG-1 funtions s n onogene vi multiple signling pthwys, suh s Wnt/β-tenin, PIK/Akt, 1 nd NF-κB. 2 Overexpression Cell prolifertion 1.. Agomir-19+RUNX1(+) Agomir-19+RUNX1( ) Antgomir-19+RUNX1(+) Antgomir-19+RUNX1( ) Invsion Migrtion Agomir-19 +RUNX1(+). Agomir-19 +RUNX1( ) Antgomir-19 +RUNX1(+) Antgomir-19 +RUNX1( ) Cell numer of invsion Cell numer of migrtion Agomir-19+RUNX1(+) Agomir-19+RUNX1( ) Antgomir-19+RUNX1(+) Antgomir-19+RUNX1( ) Moleulr Therpy vol. 2 no. ot

13 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy of AEG-1 is losely relted to ner progression, inluding onogenesis, prolifertion, invsion, metstsis, nd hemoresistne. In this study, we found tht AEG-1 is involved in RUNX1-indued onogeni effet in gliom ells. Overexpression of RUNX1 ould inrese AEG-1 expression. Luiferse nd ChIP ssys showed tht RUNX1 inresed promoter tivity nd ound to the promoter region of AEG-1, inditing tht AEG1 ws involved in RUNX1 regultion in gliom ells. Reent work hs indited tht lnrnas my e regulted y severl key trnsription ftors. For exmple, MYC hs een implited in ner ell prolifertion nd tumorigenesis y trgeting the lnrnas CDKN1A, nd CDKN2B. The trnsription ftor -my promotes olon ner ell prolifertion nd invsion y tivting the lnrna CCAT1. 7 The lnrna ERIC is regulted y trnsription ftor E2F nd modultes ellulr responses to DNA dmge. 8 This study showed tht the trnsription ftor RUNX1 my regulte the expression of the lnrna HCP. Overexpression of RUNX1 signifintly inresed HCP expression, while knokdown of RUNX1 led to shrp derese of HCP. Luiferse nd ChIP ssys lso indited tht RUNX1 inds to the promoter region of HCP nd up-regultes promoter tivities. There is positive feedk loop etween RUNX1 nd HCP, in whih the HCP downregultion deresed the expression of trnsription ftor RUNX1 y trgeting mir-19, wheres up-regulted RUNX1 promoted HCP expression. Other reports hve shown similr mehnisms in whih the lnrna H19 up-regultes expression of the trnsription ftor Slug in the pnreti ner ell line Agomir-19 +RUNX1(+) 7.2 Agomir-19 +RUNX1( ) 7.2 Antgomir-19 +RUNX1(+) 7.2 Antgomir-19 +RUNX1( ) % of poptosis 2 Agomir-19+RUNX1(+) Agomir-19+RUNX1( ) Antgomir-19+RUNX1(+) Antgomir-19+RUNX1( ) d AEG-1 AEG-1 Agomir-19 NC Agomir-19 Agomir-19 NC +RUNX1(+)NC Agomir-19 +RUNX1(+) Antgomir-19 NC Antgomir-19 Antgomir-19 NC +RUNX1( )NC Antgomir-19 +RUNX1( ) 7 kd 7 kd 7 kd 7 kd. IDVs of AEG1..2. & & Agomir-19 NC Agomir-19 Agomir-19 NC+RUNX1(+)NC Agomir-19+RUNX1(+) Antgomir-19 NC Antgomir-19 Antgomir-19 NC+RUNX1( )NC Antgomir-19+RUNX1( ) Figure 8 Runt-relted trnsription ftor 1 (RUNX1) medited tumor-suppressive effets of mirorna-19(mir-19). () Cell Counting Kit-8 (CCK8) ssy to evlute the effet of mir-19 nd RUNX1 on ell prolifertion in nd ells. () Trnswell ssy to evlute the effet of mir-19 nd RUNX1 on ell migrtion nd invsion of nd ells. () Flow ytometry nlysis to evlute the effet of mir-19 nd RUNX1 on ell poptosis of nd ells. Error rs represent s the men ± SD (n =, eh group). P <., P <., P <.. Sle rs represent 2 μm. (d) Western lot nlysis for stroyte elevted gene-1 (AEG-1) in nd ells with the expression of mir-19 nd RUNX1 hnged. Error rs represent s the men ± SD (n =, eh group). P <., P <., P <., & P < vol. 2 no. ot. 21

14 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells Puttive RUNX1 inding site 97 Reltive expression of HCP , 2, 1, Lu Lu HCP promoter tivity 2 Puttive RUNX1 inding site 97 RUNX1(+)NC RUNX1(+) RUNX1( )NC RUNX1( ) +1 HCP TSS pex empty vetor pex-runx1 PCR2 9 p PCR1 128 p 2 1 Input PCR1 IgG RUNX1 PCR2 PCR1 PCR2 Input IgG RUNX1 Input IgG RUNX1 Input IgG RUNX1 Figure 9 Runt-relted trnsription ftor 1 (RUNX1) feedk promoted histoomptiility leukoyte ntigen (HLA) omplex P (HCP) expression y inding to the promoters of HCP. () Reltive expression of HCP in nd ells with the expression of RUNX1 hnged. Error rs represent s the men ± SD (n =, eh group). P <., P <.. () RUNX1 on the promoter tivity of HCP in nd ell lines. Error rs represent s the mens ± SD (n =, eh). P <.. () RUNX1 ound to the promoter of HCP in nd ell lines. Shemti representtion of the humn HCP promoter region, p upstrem of the trnsription strt site (trnsription strt site (TSS), designted s +1). Polymerse hin retion (PCR) ws onduted with the resulting preipitted DNA. Pn-1. Slug n lso tivte H19 promoter to up-regulte H19 expression. 9 The results of this study my support the existene of positive feedk loop etween lnrnas nd trnsription ftors, nd the loop my ply key role in regulting the mlignnt ehvior of gliom ells. The mehnism underlying tumor suppression of gliom ells y HCP knokdown is shemtilly presented in see Supplementry Figure S2. In onlusion, this study highlights the importne of intertions mong lnrna HCP, mirorna-19, nd trnsription ftor RUNX1 in regulting the mlignnt ehvior of gliom ells. HCP down-regulted mir-19 to up-regulte RUNX1. RUNX1 promoted AEG-1 expression, whih ws involved in series of onogeni effets in gliom ells. RUNX1 lso up-regulted HCP expression, whih formed positive feedk loop. Thus, HCP/ mir-19/runx1 my e used s potentil therpeuti trgets for treting GBM. MATERIALS AND METHODS Clinil speimens. Gliom smples nd humn rin tissues were otined from the Deprtment of Neurosurgery t Shengjing Hospitl of Chin Medil University. All ptients signed n informed onsent form efore surgery, nd pprovl of the Institutionl Reserh Ethis Committee ws otined. All speimens were immeditely frozen in liquid nitrogen fter surgil resetion. Aording to the WHO lssifition of tumors in the entrl nervous system (27), gliom tissues were divided into three groups: Grde I II (n = ), Grde III (n = ), nd Grde IV (n = ). Humn NBTs otined from epilepsy nd rin trum ses were used s negtive ontrols (n = ). Cell ulture. Humn gliom ell lines ( nd ) were otined from the Chinese Ademy of Medil Sienes (Beijing, Chin) nd Shnghi Institutes for Biologil Sienes Cell Resoure Center, respetively. ells were ultured in high gluose Duleo s modified egle medium (DMEM) supplemented with % fetl ovine serum (FBS; Life Tehnologies, Crlsd, CA). ells were ultured in Duleo s modified egle Moleulr Therpy vol. 2 no. ot

15 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy NC+ mir-19(+) + mir-19(+)nc mir-19(+) NC+ mir-19(+) + mir-19(+)nc mir-19(+) NC+ mir-19(+)nc mir-19(+) + mir-19(+) 1, NC+miR-19(+)NC Tumor volume (mm ) 8 mir-19(+) +mir-19(+) 2 NC+ mir-19(+) + mir-19(+)nc mir-19(+) 1, NC+miR-19(+)NC 8 mir-19(+) +mir-19(+) Tumor volume (mm ) 2 d Tumor weight (wg) Dys fter injetion NC +mir-19(+)nc mir-19(+) +mir-19(+) Tumor weight (wg) Dys fter injetion NC +mir-19(+)nc mir-19(+) +mir-19(+) e Perent survil 1 NC+miR-19(+)NC mir-19(+) +mir-19(+) Perent survil 1 NC+miR-19(+)NC mir-19(+) +mir-19(+) Dys fter injetion Dys fter injetion Figure In vivo tumor xenogrfts study. The stle expressing ells were used for the in vivo study. () The nude mie rrying tumors from respetive groups were shown. () The smple tumor from representtive groups ws shown. () Tumor growth urves in nude mie. Tumor volume ws lulted every five dys fter injetion. (d) Tumors were hrvested on dy nd weighed. (e) The survivl urves of nude mie injeted into the right stritum. Error rs represent s the mens ± SD (n =, eh group). P <., P <., P < vol. 2 no. ot. 21

16 The Amerin Soiety of Gene & Cell Therpy HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells medium/f12 mixed medium supplemented with % FBS. NHAs were purhsed from Sienell Reserh Lortories (Crlsd, CA) nd ultured ording to the instrutions provided y the mnufturer. All ells were inuted t 7 C in humidified tmosphere ontining % CO 2. RNA extrtion nd quntittive RT-PCR. Totl RNA ws extrted from tissues nd ells using Trizol regent (Life Tehnologies). RNA onentrtions were deteted y 2/28 nm sorne using Nno-drop spetrophotometer (ND-, Thermo, Wlthm, MA). HCP, mir-19, nd RUNX1 expression levels were deteted y RT-PCR nlysis through 7, Fst RT-PCR System. High Cpity DNA Reverse Trnsription kit (Applied Biosystems, Foster City, CA) ws used to detet HCP nd RUNX1 expression. TqMn MiroRNA Reverse Trnsription kit nd Tqmn Universl Mster Mix II (Applied Biosystems) were used to detet mir-19 expression. Glyerldehyde -phosphte dehydrogense () nd U were used s endogenous ontrols for HCP, RUNX1, nd mir-19 expression. For HCP nd RUNX1, PCR yling onditions were s follows: minutes t 2 C, seonds t 9 C, yles of seonds t 9 C, nd seonds t C. For mir-19, reverse trnsription ws set s follows: minutes t 1 C, minutes t 2 C, nd minutes t 8 C. PCR onditions were set s follows: 2 minutes t C, minutes t 9 C, yles of 1 seonds t 9 C nd 1 minutes t C. Fold hnge in gene expression ws lulted y reltive quntifition s 2 CT. Cell trnsfetion. The short hirpin RNA ginst humn HCP gene ws reonstruted in pgpu/neo vetor (HCP ( ); GenePhrm, Shnghi, Chin), nd its empty vetor ws used s negtive ontrol (NC) ( NC). Humn full-length RUNX1 gene ws ligted into pgcmv/mcs/ Neo vetor (RUNX1(+)), nd its empty vetor ws used s negtive ontrol (RUNX1(+)NC). The short hirpin RNA of RUNX1 ws onstruted into pgpu/neo vetor (RUNX1( ); GenePhrm), nd NC ws synthesized (RUNX1( )NC; GenePhrm). Gliom ells were seeded in 2-well pltes nd trnsfeted using Lipo, regent nd Opti-MEM I (Life Tehnologies) when the onfluene rehed t ~7%. Vetors were used t onentrtion of ng/µl ording to the mnufturer s instrutions. Genetiin (G18, Invitrogen, Crlsd, CA) ws used to onstrut stle ell lines, nd trnsfetion effiy ws evluted y qrt-pcr. Agomir-19, gomir-19nc, ntgomir-19 nd negtive ontrol of ntgomir (ntgomir-19nc;genephrm) were trnsiently trnsfeted into nd ells, whih stly trnsfeted HCP or RUNX1 ording to the mnufturer s instrutions, t onentrtion of ng/µl. Cells were hrvested 8 hours fter trnsfetion. Cell prolifertion ssy. nd ells were seeded in 9-well pltes t density of 2, ells per well nd ssyed 8 hours fter trnsfetion. A CCK8 (Beyotime Institute of Biotehnology, Jingsu, Chin) solution ws dded into eh well (2 μl), nd five replite wells were used for eh group. All ells were inuted for nother 2 hours t 7 C. Asorne ws mesured t nm. Cell migrtion nd invsion ssy. For ell migrtion, nd ells were resuspended in μl serum-free medium t density of 2 ells/ml nd ssyed in 2-well Trnswell hmers with n 8 μm pore size polyronte memrne (Corning, NY). After inution t 7 C for 2 hours, ells on the upper surfe of the memrne were mehnilly removed. The ell invsion ssy proedure ws similr to the migrtion ssy. The upper hmer ws preoted with ng/µl mtrigel solution (BD Biosienes, Frnklin Lkes, NJ). Cells tht migrted or invded on the lower surfe of the memrne were fixed with methnol nd glil eti id t rtio of :1 nd stined with 2% Giems. Stined ells were rndomly ounted in five rndom fields of eh hmer. Quntifition of poptosis y flow ytometry. Apoptosis ws deteted y stining with Annexin V-APC/7-AAD (BD Biosienes) ording to the mnufturer s instrutions. Cells were wshed twie with old phosphte-uffered sline nd resuspended in inding uffer t onentrtion of 1 ells/ml. A totl of μl llophyoynin (APC) nd μl 7-minotinomyin D (AAD) were dded to the ell suspension nd inuted for nother 1 minutes t room temperture in drk room, followed y ddition of μl inding uffer. Cell smples were nlyzed y flow ytometry (FACSn, BD Biosienes). Western lot nlysis. nd ells were lysed with old RIPA (Beyotime Institute of Biotehnology) uffer supplemented with 1% phenylmethyl sulfonyl fluoride nd entrifuged t 17, g for minutes t C. BCA protein ssy kit (Beyotime Institute of Biotehnology) ws used to determine smple protein onentrtions. Equl mounts of totl protein were nlyzed y sodium dodeyl sulfte-polyrylmide gel eletrophoresis (SDS-PAGE) nd eletrilly trnsferred onto polyvinylidenedifluoride memrne (Millipore, Shnghi, Chin). Memrnes were loked in Tween- Tris-uffered sline ontining % nonft milk for 2 hours t room temperture. Memrnes were inuted overnight t C with primry ntiodies (rit polylonl nti-runx1 ntiody, 1:,, Am, Cmridge, MA; rit polylonl nti-aeg-1 1:1,, Proteinteh, Rosemont, IL; mouse monolonl nti-, 1:,, Am). Memrnes were then wshed three times with Tween-Tris-uffered sline nd inuted with horserdish peroxidse onjugted seondry ntiody t room temperture for 2 hours. Blots were visulized y n enhned hemiluminesene (ECL, Snt Cruz Biotehnology, Dlls, TX) kit nd deteted y ECL Detetion Systems (Thermo Sientifi, Beijing, Chin). FLuor Chem2. softwre (AlphInnoteh, Sn Lendro, CA) ws used to lulte the reltive integrted density vlues. Bioinformtis predition nd luiferse reporter ssy. Potentil HCP mir-19 inding sites predited y omputer-ided lgorithms were otined from the ioinformtis tool Strse ( n/). Trget Sn ( used to predit ommon trgets of mir-19. Briefly, the inding site ws mplified y PCR, synthesized nd loned into the pmirglo dul-luiferse vetor (Promeg, Mdison, WI). Vetors were trnsfeted into nd ells seeded in 9-well pltes when onfluene rehed 7%. Reltive luiferse tivities were mesured nd expressed s the rtio of firefly luiferse tivity to renill luiferse tivity. Promoter tivities were mesured to determine responsive RUNX1-inding sites in humn AEG-1 or HCP promoters using dul-luiferse reporter ssy system. Humn AEG-1 or HCP promoter nd its frgments were mplified from humn genomi DNA, nd then suloned into pgl-bsi vetor (Promeg). Humn full-length RUNX1 ws onstruted into pex vetor (GenePhrm). Firefly luiferse tivity ws normlized to renill luiferse tivity for eh individul nlysis. Pull-down ssy with iotinylted mir-19. Biotinylted mir-19, mir- 19-Mut, nd negtive ontrol of mir-19(genephrm) were trnsfeted into nd ells. At 8 hours fter trnsfetion, ells were hrvested nd lyzed. Smples ( μl) were liquoted for input. Dyneds M-28 Streptvidin (Invitrogen) ws used to inute the remining smples ording to the mnufturer s protool. Beds were wshed nd treted in RNse-free solutions, then inuted with equl volumes of iotinylted mir-19 for 1 minutes t room temperture using gentle rottion. Beds with the immoilized mir-19 frgment were inuted in mmol/l ethylenediminetetrette, ph 8.2 with 9% formmide for minutes t C. RNAs were purified nd ssyed y qrt-pcr. Chromtin immunopreipittion ssy. The ChIP ssy ws performed using the Simple Chip Enzymti Chromtin IP kit (Cell Signling Tehnology, Dnvers, MA) ording to the mnufturer s instrutions. Cells were ross-linked with 1% formldehyde for minutes nd quenhed y dding glyine. Cells were then lysed with old uffer ontining phenylmethyl sulfonylfluoride nd resuspended with old phosphteuffered sline. Chromtin ws digested y mirool nulese. Lystes (2%) were used s n input referene. Other immunopreipittion smples were inuted with norml rit IgG or nti-runx1 ntiodies t C with rottion. Protein G grose eds were used to pture the hromtinimmune omplex, nd eds were wshed with low slt hip uffer nd Moleulr Therpy vol. 2 no. ot

17 HCP-miR-19-RUNX1 Feedk Loop in Gliom Cells The Amerin Soiety of Gene & Cell Therpy high slt hip uffer. DNA rosslinks were reversed y mol/l NCl nd proteinse K t C for 2 hours. DNA ws purified nd mplified y PCR. Tumor xenogrfts in nude mie. Stle, expressing ells were used for the in vivo study. An mir-19 overexpression plsmid nd pegfp-mir- 19-mimi sponge ws trnsfeted into stle trnsfeted ells using Lipo, nd seleted y ulture medium ontining mg/ ml G18. Experiments were divided into five groups: ontrol, NC+miR-19(+)NC,, mir-19(+), nd +mir-19(+). Four-week-old nude BALB/ thymi mie were purhsed from the Cner Institute of the Chinese Ademy of Medil Siene. All proedures were performed in ordne with the Cre nd Use of Lortory Animls, nd pprovls from the Animl Cre Committee of Shengjing Hospitl were otined. Eh nude mouse ws injeted with ells in the right flnk re. Tumor volume ws mesured every dys. The following formul ws used to lulted tumor volume: volume (mm ) = length width 2 /2. All tumors were hrvested nd weighed on dy. Orthotopitumor inoultions were performed y injeting ells into the right stritum of nude mie. The numer of surviving mie ws reorded every dys, nd survivl nlysis ws performed using Kpln Meier survivl urve. Sttistil nlysis. GrphPd Prism v.1 (GrphPd Softwre, L Joll, CA) softwre ws used for sttistil nlysis. All dt re presented s the men ± SD from t lest three independent replites. Sttistil nlysis of dt ws performed using the Student s t-test. Differenes were onsidered to e sttistilly signifint when P <.. SUPPLEMENTARY MATERIAL Figure S1. The minimum free energy of the intertion etween HCP nd mir-19 ws lulted in silio method. Figure S2. The shemti rtoon of the mehnism of HCP-miR- 19-RUNX1 feedk loop in nd ells. ACKNOWLEDGMENTS This work is supported y grnts from the Nturl Siene Foundtion of Chin (81272, nd 817), Lioning Siene nd Tehnology Pln Projet (No ), Shenyng Siene nd Tehnology Pln Projets (Nos. 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