Repression of miR-143 Mediates Cr (VI)–Induced Tumor Angiogenesis via IGF-IR/IRS1/ERK/IL-8 Pathway

JunHe 1 2 XuQian 0 1 RichardCarpenterQ 1 2 ingXu 0 1 LinWang 0 1 YantingQiZ 1 2 i-XuanWang 1 2 Ling-ZhiLiu 1 2 Bing-HuaJiang 0 1 2 0 State Key Laboratory of Reproductive Medicine, Department of Pathology, Nanjing Medical University , Nanjing 210029, P.R. China 1 The Author 2013. Published by Oxford University Press on behalf of the Society of Toxicology 2 Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University , Philadelphia, Pennsylvania 19107 D o w carcinogenesis are poorly defined partly due to the lack of suitalon Hexavalent chromium [Cr (VI)] is a well-known human carcin- ble experimental models that can closely mimic chronic Cr (VI)aed trohegmeenamianesscshouacninacitlseemdarwudnitudheertthloyeintihngecrtlheaaecskeCdorfr(isVsukIi)to-afibnlludenuegcxepcdaenrcicamerrce.innHtoaoglwemneveosedirs-, 2ex00p0o;suCroesitnaehtuaml.a,n20l1u0n;gLceveyllest(aAl.z,eat1ad9l8.,62).010; Balanskyetal., frhodm ilteonhlsnga.gtnI-ontmenrittRhmui-ms1e4osxt3rpuiogdesexyun,prirwceehetsosudimoCenvaren(lloeVlvupIene)l.dgsBeawypneiutrihteniellidviziaritnlaroBgmEmtahAtoiicsSda-eml2llByobdycreeeltl,plrsrwaetnehsssrffeoooduurmngihdn- seautnsaTdtlh.a,mei2ni0cep0rdo9ot;soWeaxntaitednilaagltleiittvaeeml.ie,sn2cts0hrte1aas1nsba,iis)lcm.ioBtsymesop(iHrdfoeomCselttrimshaeeecld.ssa,erD2cg0Nien0An8o;ogrtReeonopxeadisricriisgesufyfienesstccetlmsu,d, e .:tt/t/ifscpxoxo (CVrI)(-VinI)d-utrcaendscfoerllmmeadlicgenllasn. tTthraenrsefporremssaitoionnoafnmdiRan-1g4io3gelendestios vCiar carintuicmableervoefntstsufdoirecseslhlowmatlhiagtnaanbtertraranntsfgoernmeateixopnreinssdioucnesdabrye Cjrrdouor auwnpedrefogiununslaudtliitonhnarteocfineiptnetrsoulreluinsku-ilbniks-t8eraigstreot-h1wet(hmIRafSajoc1tr)orue-xp1prerrgeesucsleiapotnteo.dr Ma(nIoGgrieFoog-vIeRenr-), (roeVfgImu)l(icMartoeiRdoanNn.AetsTah(lme.,i2Ra0Nb1eA2r;rsRa)noptdrorivegixudpeersseenstsoaiolv.e,n2l0om0f9e)cm.hTiaRhneNisdAmissscoohvafesgryebneeen .l/rsaongb esis factor induced by Cr (VI) through activation of IGF-IR/IRS1 frequently observed in many human cancers including lung ygu faaxcitsofro-l1low/NeFd-byBascitgivnaatliionng opfadtohwwnasyt.reTahmesEeRfiKnd/hinypgsoxeiast-ainbdliushceda cancer (Liuetal., 2011). However, there is little information on tseo causal role and mechanism of miR-143 in regulating Cr (VI)- the involvement of miRNAs in environmental carcinogenesis.nO induced malignant transformation and tumor angiogenesis. It remains to be determined whether the loss or gain of certaintcbo - of Sciences, Shanghai, China) and maintained in pathogen-free conditionCs.olony isolation. BEAS-Cr cells were seeded in soft agar for at least 2 BEAS-2B cells, BEAS-Cr cells, BEAS-Cr cells stably expressing miR-14w3e,eks. A number of colonies were then picked up from the agarose with a or BEAS-Cr cells stably expressing miR control were injected sc into the pflipaentkte, seeded into a 6-cm dish with complete medium, and cultured to the of nude mice (2 106 cells in 150l). Bidimensional tumor volume measure-confluence. All surviving colonies (BEAS-Cr clones) were kept in complete ments were obtained with calipers three times a week. Tumor volumes wmereedium (DMEM + 10% FBS) and used for subsequent experiments. calculated according to the formula (2widltehngth)/2. The mice were euthanized after 28days, and tumors were weighed. HIF-1 forward: 5-TCCATGTGACCATGAGGAAA-3 HIF-1 reverse: 5-TATCCAGGCTGTGTCGACTG-3 IL-8 forward: 5-TAAATCTGGCAACCCTAGTC-3 IL-8 reverse: 5-GCGTTCTAACTCATTATTCCGT-3 GADPH forward: 5-CCACCCATGGCAAATTCCATGGCA-3 GADPH reverse: 5-TCTAGACGGCAGGTCAGGTCCACC-3 Primers for SYBR-Green RT-qPCR IL-8 forward: 5-CACCGGAAGGAACCATCTCA-3 IL-8 reverse: 5-AGAGCCACGGCCAGCTT-3 GAPDH forward: 5-ATGGGTGTGAACCATGA GAAGTATG-3 GADPH reverse: 5GGTGCAGGAGGCATTGCT-3 miRNA luciferase reporter constructs and luciferase activity assay. The 3-UTR-luciferase reporter constructs containing the 3-UTR of IGF-IR and IRS with wild-type and mutant-binding sites of miR-143 were amplified using PCR method. The PCR products were cloned into the pMiR-luc reporter vector (Ambion), immediately downstream of the luciferase gene. The mutant 3-UTR constructs were made by introducing four to six mismatch mutations into the putative seed regions of IGF-IR and IRS1. All the constructs containing 3UTR inserts were sequenced and verified. The luciferase activity assay was performed as previously described (He etal., 2012). Statistical analysis. All the results were obtained from at least three independent experiments. Most results were presented as mean SE from independent experiments and analyzed by Studet-nttesst and one-way ANOVA. All results were analyzed by SPSS for Windows, version 11.5. Differences were considered significant at a value of p < 0.05. FIG. 1. miR-143 is downregulated in Cr (VI)transformed BEAS-2B cells and human lung cancer tissues. (A) An equal number of BEAS-2B and BEAS-Cr cells (10,000 cells) were seeded in 12-well plates to analyze cell proliferation by counting with trypan blue exclushio.n(Bf)oCrelvlesryw2e4re seeded in 96-well plates and cultured in medium with different concentrations of serum. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide- assay was p formed at 72h to assess the cell proliferation rates. (C) An equal cell number of cells (5000) were seeded in a six-well plate and cultured in soft agar and incu at 37C for 2 weeks. Representative images for BEAS-2B and BEAS-Cr are presented (left panel). Number of colonies from soft agar assay was counted for each group shown in lower panel. (DF) miR-143 expression levels were determined by Taqman RT-qPCR in parental Cr (VI)transformed BEAS-2B cells (BEAS-Cr) and different BEAS-Cr clones. Relative miRNA expression levels were represented as R2QusCiTnmgethods. The values were normalized to the U6 expression level and that in BEAS-2B cells. Mean SE values were from three separate experiments. **Significantly different compared with control (p < 0.01). were much lower in lung cancer cells A549 and H2195 comEc-topic Expression of miR-143 Inhibits Cr (VI)Induced pared with BEAS-2B (Fig.1F). Tumor Angiogenesis TABl E1 Tumor Growth Assay in Nude Mice As miR-143 is downregulated in BEAS-Cr cells, they might have biological functions related to cell transformation a tumor growth. Angiogenesis is the essential process for tumor development (Folkmaent al., 1971). We evaluated the effect of miR-143 overexpression in tumor-induced angiogenesis both in vitro and in vivo. First, we performed tube formation assay in vitro (Fig. 2A). HUVECs maintained in EBM-2 basic medium were not capable of tube formation. However, it was strongly induced using conditioned medium prepared from BEAS-Cr cells. Further, the ectopic expression of miR143 in BEAS-Cr impaired the tube formation by 35%. Next, FIG. 2. Ectopic expression of miR-143 inhibits Cr (VI)induced tumor angiogenesis. (A) HUVECs were cultured in serum-free medium overnight and resuspended in EBM-2 basic medium. To perform th (...truncated)