CYP4Z1 30UTR represses migration of human breast cancer cells

CYP4Z130UTR represses migration of human breast cancer cells Bingyu Wang a,b,Lufeng Zheng a,b,Jinjiang Chou a,b,Cheng Li a,b,Yan Zhang a,b, Xia Meng a,b,Tao Xi a,b,*

a School of Life Science and Technology,China Pharmaceutical University,Nanjing210009,People's Republic of China

b Jiangsu Key Laboratory of Carcinogenesis and Intervention,China Pharmaceutical University,Nanjing210009,People's Republi

c of China

a r t i c l e i n f o

Article history:

Received13June2016 Accepted8August2016 Available online9August2016

Keywords:

CYP4Z1

Breast cancer

MicroRNA

Migration

EMT a b s t r a c t

To investigate the effects of CYP4Z130UTR in migration of breast cancer cells,a series of assays were used to con?rm that overexpression of CYP4Z130UTR could suppress the capacity of migration and adhesion of MCF-7and MDA-MB-231cells.EMT(Epithelial-mesenchymal transition)-related proteins were regulated by CYP4Z130UTR.Mesenchyma markers like Vimentin,MMP-2,and MMP-9were down-regulated,while the expression of E-cadherin was up-regulated with CYP4Z130UTR overexpression. Notably,luciferase reporter and qRT-PCR assays were applied to verify that CYP4Z130UTR was the po-tential target of miR-9.In addition,our results showed that CYP4Z130UTR repressed the expression of E-cadherin in a miRNA-dependent https://www.360docs.net/doc/e39752613.html,bining with our previous study,we have discovered the underlying link between CYP4Z1and E-cadherin.Therefore,those preliminary data suggest that CYP4Z1 30UTR could inhibit the migration and EMT of breast cancer cells via acting as a ceRNA for E-cadherin.

?2016Elsevier Inc.All rights reserved.

1.Introduction

The probability of developing breast cancer has been increasing since1970s in the world,and now the possibility is one woman in eight in the United States.Although mortality rates declined since 1990s,breast cancer is still the second leading cause of cancer death in women[1].However,breast carcinoma in situ is not a life-threatening cancer and most of the deaths were caused by metastasis.

Epithelial-mesenchymal transition(EMT)is de?nitely a way to enhance metastasis of breast cancer.EMT enables epithelial cells to become more like mesenchymal cells with increased motility and ability to invade.Markers of EMT are detected in many aggressive tumors[2,3].EMT-related markers such as E-cadherin could sup-press the process of EMT and metastasis of breast carcinoma. Down-regulation of E-cadherin would increase the possibility of metastasis and decrease the survival rate.

A regulatory mechanism,competitive endogenous RNA(ceRNA) hypothesis,was put forward recently.The ceRNA hypothesis is about how mRNAs affect the expression of each other through competitively binding for their shared microRNAs(miRNAs).This theory has revealed a reversed RNA/microRNA logic exists,which is different from the conventional microRNA/RNA function.All in all, ceRNA theory provides us a novel clue to understand the patho-logical mechanisms of human diseases and presents opportunities for new therapies with the progress of experimental tools.In hu-man genome,about20,000protein-encoding genes were found, and many of them contain microRNA response elements(MREs) [4].30UTRs are the main entities containing MREs as well as the essential building blocks of the ceRNA network.Thus changes in 30UTRs are thought to have profound effects on ceRNA network.For example,we have found that overexpression of FOXO130UTR could in?uence metastasis of breast cancer cells by acting as the ceRNA of E-cadherin[5].Our previous study has also shown that over-expression of CYP4Z2P30UTR exerts effects on angiogenesis in breast cancer by acting as the ceRNA of CYP4Z1[6].The cytochrome P450enzymes are a large family of constitutive and inducible mono-oxygenase enzymes[7].P450family4enzymes are generally distinguished by their capacity to active or degrade endogenous and xenobiotic substrates[8].CYP4Z1is a new member of the4Z. The gene for CYP4Z1localized on chromosome1p33,along with genes CYP4A11,CYP4A22,CYP4B1and CYP4Z2P.

Our group has found that30UTR of the pseudogene CYP4Z2P produced reactive vessel substances by acting as a ceRNA for CYP4Z1[6],but systematic researches are yet absent on CYP4Z1 30UTR to regulate migration of breast cancer.To investigate the

*Corresponding author.School of Life Science and Technology,China Pharma-ceutical University,Nanjing210009,People's Republic of China.

E-mail address:xitao18@https://www.360docs.net/doc/e39752613.html,(T.

Xi).Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage:www.elsev https://www.360docs.net/doc/e39752613.html,/locat e/ybb

rc

https://www.360docs.net/doc/e39752613.html,/10.1016/j.bbrc.2016.08.048

0006-291X/?2016Elsevier Inc.All rights reserved.

Biochemical and Biophysical Research Communications478(2016)900e907

effects of CYP4Z130UTR in migration of breast cancer cells,CYP4Z1 30UTR was overexpressed and knocked down in MCF-7and MDA-MB-231breast cancer cells.Particularly,we have used two pairs of siRNA to avoid the off-target effects of siRNA.Our research has proved that CYP4Z130UTR could suppress the migration and EMT of breast cancer via acting as a ceRNA for E-cadherin.

2.Materials and methods

2.1.Cell culture

Human breast cancer cells MCF-7,MDA-MB-231were obtained from the ATCC(Manassas,VA,USA)and cultured in Dulbecco's modi?ed Eagle's medium(DMEM,Gibco,Grand Island,NY)and L-15medium(KeyGEN BioTECH,Nanjing)with10%fetal bovine ser-um(FBS,Gibco,Grand Island,NY)at37 C in a humidi?ed atmo-sphere with5%CO2.

2.2.Plasmid construction

The pSilencer4.1-CMV-CYP4Z130UTR construct was made in our lab,and was denoted as CYP4Z130UTR.pSilencer4.1-CMV Vector (Ambion,Austin,USA)was preserved by our lab.

2.3.Transfection

Cells at a density of3?105e5?105/well were seeded in a6-well plate.Genetic material consisting of2.5m g of DNA or50nM of RNA(miR-9mimics;mimics NC;CYP4Z1siRNA;siRNA-2;Dicer siRNA;siRNA NC)(Biomics Biotechnology Inc.,China)was trans-fected into cells using the Lipofectamine2000Reagent(Invitrogen, Carlsbad,CA),following the manufacturer's protocol.

2.4.Real-time PCR

Total RNA was extracted using the Trizol reagent(Invitrogen, USA).Synthesis of the?rst strand cDNA was performed using M-MLV(Promega,USA)following the standard protocols.To deter-mine the mRNA expression levels,the EzOmics?SYBR qPCR kits (Biomics Biotechnology Inc.,China)and a qRT-PCR detection sys-tem(ABI,USA)were used.miR-9,CYP4Z1and E-cadherin relative expression level of each group were calculated using the2-△△Ct method.

2.5.Wound healing assay

MCF-7and MDA-MB-231cells were transiently transfected with CYP4Z130UTR,CYP4Z1siRNA and their corresponding negative control in a6-well plate.After24h,cells were allowed to grow to 80e90%-con?uent in complete medium.Cell monolayers were wounded with a sterile micropipette tip,and washed with phos-phate buffer solution(PBS)for three times to remove cell debris, and then serum-free medium was added for further incubating.As MDA-MB-231cells have the faster migrate rate than MCF-7cells, MDA-MB-231and MCF-7cells were measured every12h and every 24h.The distances between two edges were scaled for three po-sitions each time at over three time points.

2.6.Transwell assay

Transwell assays were performed using24-well MILLIcell Hanging Cell Culture inserts with8mm PET(MILLIPORE).1?105 transfected cells were added to the upper chamber after being trypsinized and suspended in serum-free medium.20%FBS was used as a chemo-attractant in the medium of the bottom chamber.MCF-7cells were stopped after48h,and MDA-MB-231cells were stopped after12h.A cotton swab was used to remove cells on the top surface of the membranes.Cells migrated to the underside were ?xed in methanol and stained with0.1%viola crystalline solution. Five random?elds from each of the triplicate migration assays were observed by phase contrast microscopy.Quanti?cation was measured by Microplate Reader(OD570nm)after being destained with glacial acetic acid.

2.7.Adhesion assay

The96-well plate was coated with Matrix gel(BD Biosciences) which was diluted with serum-free medium to1:7.Put the96-well plate into incubator,37 C,4h.Then the wells were blocked for1h with1%BSA in PBS.1?105transfected cells were added to the wells after being trypsinized and suspended in serum-free me-dium.After1h,PBS was used to wash away cells which were not adherent.The colorimetric MTT-assay was used to measure the number of adherent cells.

Adhesion ratee%T?

A adhered cellàA control

A total cellàA control

?100%

2.8.Western blot

After being transfected with different DNA or RNA,cells were washed three times with ice-cold PBS and lysed in buffer RIPA (Beyotime,China),which contains1mM PMSF(Beyotime),for 30min on ice.They should be vortexed every5min and then centrifuged at4 C for15min(12000rpm).The concentration of the supernatant was determined by Bradford assay.30m g samples of protein extracts were separated using10%SDS-PAGE and trans-ferred onto polyvinylidinedi?uoride(PVDF)membranes(Millipore, USA).After blocked with5%non-fat milk for2h,the membranes were incubated with appropriate primary antibodies overnight at 4 C.After washed at room temperature3times and then incubated with horseradish peroxidase labeled secondary antibodies for 40min at37 C.The E-cadherin,MMP-2,MMP-9antibodies were purchased from Wanlei biotechnology,China.The Vimentin anti-body was purchased from Abcam,USA.The speci?c protein bands were detected using the High-sig ECL Western Blottin Substrate (Tanon,China).Images were taken by the Tanon5200imaging system.The protein expression level was normalized to b-actin.

2.9.Luciferase reporter assay

CYP4Z1wild-type and mutant-type fragments with binding sites for miR-9were synthesized by Sangon Biotech(Shanghai, China).The annealed oligonucleotides were inserted into pMiR-Report.

The fragments of pMiR-Report wild-type(WT)were as follows: forward,50-CTAGTGAATTAATGAGACAATTTTCCTACCAAAGGAA-GAACAAAAGGATAAATATAA-30and reverse,50-AGCTTTA-TATTTATCCTTTTGTTCTTCCTTTGGTAGGAAAATTGTCTCATTAATTCA-3’;the fragments of pMiR-Report mutant-type(MUT)were as follows:forward,50-CTAGTGAATTAATGAGA-CAATTTTCCTCATCGCTGAAGAACAAAAGGATAAATATAA-30and reverse,50-AGCTTTATATTTATCCTTTTGTTCTTCAGCGATGAGGAAAAT TGTCTCATTAATTCA-3’.MCF-7cells were seeded in a24-well plate (4?104cells/well)and co-transfected with WT and MUT se-quences,miR-9mimics or cel-67mimics and pMiR-Report b-gal control plasmid(Ambion)using Lipofectamine2000.After48h, cells were collected and luciferase activity was measured by a

B.Wang et al./Biochemical and Biophysical Research Communications478(2016)900e907901

Glomax 96luminometer (Promega).The transfection ef ?ciency was normalized to b -galactosidase activity.

2.10.Statistical analyses

All the data were shown as the mean ±SEM,n !3.Statistical analyses were analyzed using a two-sided Student's t -test,and P <0.05was considered to be signi ?cant.

3.Results

3.1.CYP4Z130UTR suppressed the migration of MCF-7and MDA-MB-231cells

In order to con ?rm the effects of CYP4Z130UTR on migration of MCF-7and MDA-MB-231cells,recombinant product CYP4Z130UTR was employed for up-regulating CYP4Z130UTR level,

while

Fig.1.(A e C)Transfection ef ?ciency of CYP4Z130UTR and two pairs of siRNA;(D e E)Effects of CYP4Z130UTR on cell migration and quanti ?cations of the wound healing assay;(F e I)Effects of siRNA and siRNA-2on cell migration and quanti ?cations of the wound healing assay.Data were presented as mean ±SEM,n ?3,*P <0.05,**p <0.01,***p <0.001vs.control group.

B.Wang et al./Biochemical and Biophysical Research Communications 478(2016)900e 907

902

synthesized CYP4Z1siRNA (siRNA)and CYP4Z1siRNA-2(siRNA-2)were used for down-regulating CYP4Z130UTR level.qRT-PCR was used to detect the transfection ef ?ciency (Fig.1A e C).Thus,CYP4Z130UTR recombinant products,siRNA and siRNA-2could be used to investigate the function of CYP4Z130UTR.The motility of MCF-7and MDA-MB-231cells was assessed by wound healing assay.Signi ?cant differences on cell migration were observed when compared with the control.Cell migration decreased by 8.18%(MCF-7)and 5.59%(MDA-MB-231)in CYP4Z130UTR trans-fected cells (Fig.1D e E).However,Cell migration increased by 12.96%(MCF-7)and 7.66%(MDA-MB-231)after knocking down with siRNA (Fig.1F e G).At the same time,a signi ?cant increase in

the siRNA-2group was observed as well (Fig.1H e I).As is shown in the transwell migration assay,CYP4Z130UTR overexpression signi ?cantly decreased the migration ability of these two cells (Fig.2A and 2D),while the migration ability was remarkably increased in siRNA (Fig.2B and E)and siRNA-2treated groups (Fig.2C and F).These results revealed that CYP4Z130UTR could suppress the migration of MCF-7and MDA-MB-231cells.3.2.CYP4Z130UTR suppressed the adhesion activities of MCF-7and MDA-MB-231cells

Tumor metastasis is a complex pathological and

physiological

Fig.2.(A e C)Effects of CYP4Z130UTR,siRNA and siRNA-2on migration of MCF-7and MDA-MB-231cells through the polycarbonate membrane stained with crystal violet (?200).(D e F)OD 570results of stain crystal violet for (A e C)respectively.(G e I)Effects of CYP4Z130UTR siRNA and siRNA-2on adhesion to matrix gel of MCF-7and MDA-MB-231cells.Data were presented as mean ±SEM,n ?3,*P <0.05,**p <0.01,***p <0.001vs.control group.(For interpretation of the references to colour in this ?gure legend,the reader is referred to the web version of this article.)

B.Wang et al./Biochemical and Biophysical Research Communications 478(2016)900e 907903

process,which relates to the interaction of tumor cells or mutual effects among cancer cells and host cells.During the process,cellular adhesion molecule (CAM)plays an essential role in the adhesion effects between cancer cells and other cells like vascular endothelial cells or lymphocyte.It's worth mentioning that CAM could mediate the adhesion of cancer cells and extracellular ma-trix [9,10].In our study,we have chosen matrix gel,which is used to simulate the blood-vessel wall,to investigate the in ?uences of

CYP4Z130UTR on the adhesion activities of those two cells.The adhesion activity decreased to 61.9%(MCF-7)and 80.0%(MDA-MB-231)(Fig.2G)after the transfection with CYP4Z130UTR,whereas this ability increased by 1.42-fold (MCF-7)and 1.23-fold (MDA-MB-231)(Fig.2H e I)in the siRNAs treated groups.In sum-mary,these results have showed that ectopic expression of CYP4Z130UTR could inhibit adhesion activity of breast cancer

cells.

Fig.3.Effects of CYP4Z130UTR (A,C,G)and two pairs of siRNA (B,D,E,F,H,I,J)on EMT-related proteins in MCF-7and MDA-MB-231cells.Data were presented as mean ±SEM,n ?3,*P <0.05,**p <0.01vs.b -actin.

B.Wang et al./Biochemical and Biophysical Research Communications 478(2016)900e 907

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3.3.CYP4Z130UTR suppressed EMT in MCF-7and MDA-MB-231

Western blot was applied to explore the effects of CYP4Z130UTR on protein level of EMT markers.The results indicates that over-expression of CYP4Z130UTR increased E-cadherin expression (Fig.3A)and decreased the expression of MMP-2,MMP-9(Fig.3C). However,the expression of E-cadherin in MCF-7cells was reduced (Fig.3B and E),and protein levels of MMP-2,MMP-9were up-regulated(Fig.3D and F)in siRNA and siRNA-2treated groups. The similar results were observed in the MDA-MB-231cells transfected with CYP4Z130UTR(Fig.3G),or siRNA(Fig.3H),or siRNA-2(Fig.3I e J).Therefore,over-expression of CYP4Z130UTR increased E-cadherin expression,and decreased the levels of mesenchymal markers in MCF-7and MDA-MB-231cells.3.4.Regulation of E-cadherin by CYP4Z130UTR was miRNA-dependent in MCF-7cells

In order to investigate mechanism through which CYP4Z1 30UTR regulated the migration of breast cancer cells,bioinfor-matics software was adopted to predict that CYP4Z130UTR was the potential target of miR-9.miR-9could promote the progres-sion of migration in breast cancer through regulating E-cadherin directly and promoting EMT[11].Our previous study has shown that miR-9could binding with E-cadherin30UTR[5].Therefore,we assumed that CYP4Z130UTR could inhibit EMT via acting as a ceRNA for E-cadherin.Ef?ciency of transfection of miR-9mimics in MCF-7cells was con?rmed by qRT-PCR(Fig.4A).After up-regulation of miR-9level,luciferase activity of wild-type

CYP4Z1

Fig.4.(A)Transfection ef?ciency of miRNA-9(miR-9)mimics in MCF-7cells;(B)Luciferase reporter assay for con?rming the binding relationship between miR-9and CYP4Z1 30UTR.Luciferase activity was measured and normalized to b-galactosidase activity;(C)CYP4Z1mRNA expression in MCF-7cells was reduced after up-regulating miR-9level;Data were presented as mean±SEM,n?3,*P<0.05,**p<0.01vs.control group.(D)E-cadherin mRNA expression in MCF-7cells was increased after CYP4Z130UTR overexpression;

(E e F)CYP4Z130UTR inhibited the expression of E-cadherin in a miRNA-dependent manner.Data were presented as mean±SEM,n?3,*P<0.05,**p<0.01vs.b-actin.

B.Wang et al./Biochemical and Biophysical Research Communications478(2016)900e907905

30UTR reporter containing miR-9binding site was attenuated, which was not observed in mutant-type CYP4Z130UTR reporter in MCF-7cells(Fig.4B).As is illustrated in Fig.4C,transfection with miR-9mimics signi?cantly reduced mRNA level of CYP4Z1than NC group in MCF-7cells.The data above proved that CYP4Z1 30UTR might be the potential target of miR-9.Besides,the expression level of E-cadherin was up-regulated after the over-expression of CYP4Z130UTR,which con?rmed the relationship between E-cadherin and CYP4Z1(Fig.4D).As Dicer is a critical enzyme involved in the processing of mature miRNAs,its corre-sponding siRNA could be used to evaluate miRNA-dependent ef-fects.Notably,down-regulation of E-cadherin expression caused by CYP4Z1siRNA was profoundly attenuated and even reversed in siDicer-treated cells compared with NC.(Fig.4E e F).In conclusion, CYP4Z130UTR regulates the expression of E-cadherin in a miRNA-dependent way in MCF-7cells.

4.Discussion

The previous studies by others and our group have proved that CYP4Z1mRNA is preferentially expressed in mammary tissue [1,6,12].Mei-Hui Hsu et al.detected the expression of CYP4Z1 mRNA in several tissue samples.CYP4Z1exhibited lower mRNA level in liver,kidney,and skeletal muscle[12],but higher levels were found in two out of?ve tested mammary samples.The results indicated that CYP4Z1expressed in mammary tissue particularly, which suggested the conditional regulation of the CYP4Z1gene in breast tissue[12].CYP4Z1mRNA appeared to be expressed at high levels in52%of cancerous breast samples relative to the corre-sponding normal tissue but levels were decreased in22%of the cancerous samples compared to normal breast[1].Despite the high levels of CYP4Z1mRNA in breast cancer tissue,the biological function of CYP4Z1is required to be explored.

In this study,we have proved that CYP4Z130UTR suppressed the migration of MCF-7and MDA-MB-231cells.As normal tissue comprise multiple cell types,but tumors are derived from a single cell[12],there has been no idea about which type of cells could express CYP4Z1within the mammary tissue yet.Therefore,the increased level of CYP4Z1mRNA in breast tumors may re?ect an enrichment of a cell type which expresses CYP4Z1.ceRNA hy-pothesis,which had been proved in recent studies,is a unifying hypothesis about how messenger RNAs transcribed from genes like pseudogenes,long non-coding RNAs(lncRNAs)regulate each other's expression via miRNAs[13e17].ceRNA theory indicates that 30UTRs can sponge endogenous miRNAs,and also have a wide-spread in?uence on the mRNA translation and stability.Therefore, aberrant gene expression caused by30UTR may play a vital role in tumor development.Meanwhile,based on ceRNA theory,non-coding function of mRNA might be different from the coding function,creating functional complexity and diversi?cation in physiological and pathological conditions.Accordingly,exploring non-coding function of mRNA is of great importance in develop-ment of cancers.

Our results con?rm the link between CYP4Z130UTR and E-cadherin via competitively binding with miR-9.CYP4Z130UTR regulated EMT by relieving the suppression of E-cadherin caused by miR-9and promoting the expression of E-cadherin in breast cancer cells.To the best of our knowledge,this is the?rst report about the regulation of CYP4Z130UTR on miRNAs activity.In the present study,mRNA level of CYP4Z1in MCF-7transfected with miR-9 mimics is lower than NC group,and the expression level of E-cadherin was down-regulated after the knockout of CYP4Z130UTR, which was attenuated or even partially reversed with siDicer treatment.Those results above con?rmed the ceRNA network be-tween E-cadherin and CYP4Z1.

The previous study demonstrated the induction of CYP4Z1 mRNA can be in?uenced by dexamethasone and progesterone[12], suggesting that the detection of CYP4Z1mRNA in some tumors could be a result of conditional regulation,such as treatment of donors with glucocorticoids[12].The regulatory mechanism of CYP4Z1in breast cancer development still required future inves-tigation despite the high level in breast tumors.In researches of Magdalena Cizkova,genes encoding metal ion-binding proteins like CYP4Z1,LTF were detected in breast tumors with PIK3CA mutations [18].LTF encodes lactoferrin,a protein involved in non speci?c immunity and that may inhibit carcinogenesis and tumor growth [19].Most importantly,PIK3CA mutations have also been associ-ated with favorable outcome of breast cancer patients[20e24]. Therefore,appearance of CYP4Z1might be a useful signal for favorable outcome.Also,Diane Downie detected expression of cy-tochrome P450in ovarian cancer,but CYP4Z1level showed no signi?cant differences in primary tumors and metastases[7].Thus the inhibiting effects CYP4Z130UTR showed on cell migration might be the leading function in certain conditions.Additionally,signal pathways of humans involve many respects[25,26],and cross in-?uences among them would make different effects[27e29],which indicates us that further studies should be continued to investigate how CYP4Z130UTR regulated in breast cancer.

In conclusion,the identi?cation of relevant P450s and deter-mination of their catalytic pro?les could aid in the design of bene?cial breast cancer treatments and provide useful tumor markers.The relationship between CYP4Z1and breast cancer would be clear with further studies.The networks related to CYP4Z1might provide new perspectives into the diagnosis and treatment of breast cancer.

Acknowledgments

We thanked Prof.Wu for critically reviewing this manuscript.

This work was supported by the National Major Special Program of New Drug Research and Development(No.2013ZX09301303-005),National Natural Science Foundation of China(No.81372331), the Fundamental Research Funds for the Central Universities(No. 2015ZD004)and the Priority Academic Program Development (PAPD)of Jiangsu Higher Education Institutions and Qing Lan Project,Specialized Research Fund for the Doctoral Program of Higher Education(SRFDP).

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[1]M.A.Rieger,R.Ebner,D.R.Bell,A.Kiessling,J.Rohayem,M.Schmitz,A.Temme,

E.P.Rieber, B.Weigle,Identi?cation of a novel mammary-restricted cyto-

chrome P450,CYP4Z1,with overexpression in breast carcinoma,Cancer Res.

64(2004)2357e2364.

[2]J.J.Hung,M.H.Yang,H.S.Hsu,W.H.Hsu,J.S.Liu,K.J.Wu,Prognostic signi?-

cance of hypoxia-inducible factor-1alpha,TWIST1and Snail expression in resectable non-small cell lung cancer,Thorax64(2009)1082e1089.

[3] D.S.Micalizzi,H.L.Ford,Epithelial-mesenchymal transition in development

and cancer,Future Oncol.5(2009)1129e1143.

[4]R.C.Friedman,K.K.Farh,C.B.Burge,D.P.Bartel,Most mammalian mRNAs are

conserved targets of microRNAs,Genome Res.19(2009)92e105.

[5]J.Yang,T.Li,C.Gao,X.Lv,K.Liu,H.Song,Y.Xing,T.Xi,FOXO13'UTR functions

as a ceRNA in repressing the metastases of breast cancer cells via regulating miRNA activity,FEBS Lett.588(2014)3218e3224.

[6]L.Zheng,X.Li,Y.Gu,X.Lv,T.Xi,The3'UTR of the pseudogene CYP4Z2P

promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1,Breast Cancer Res.Treat.150(2015)105e118.

[7] D.Downie,M.C.McFadyen,P.H.Rooney,M.E.Cruickshank, D.E.Parkin,

https://www.360docs.net/doc/e39752613.html,ler, C.Telfer,W.T.Melvin,G.I.Murray,Pro?ling cytochrome P450

B.Wang et al./Biochemical and Biophysical Research Communications478(2016)900e907 906

expression in ovarian cancer:identi?cation of prognostic markers,Clin.Can-cer Res.11(2005)7369e7375.

[8]R.T.Okita,J.R.Okita,Cytochrome P4504A fatty acid omega hydroxylases,Curr.

Drug Metab.2(2001)265e281.

[9]K.Y.Na,P.Bacchini,F.Bertoni,Y.W.Kim,Y.K.Park,Syndecan-4and?bronectin

in osteosarcoma,Pathology44(2012)325e330.

[10] E.S.Williams, E.Wilson,K.S.Ramos,NF-kappaB and matrix-dependent

regulation of osteopontin promoter activity in allylamine-activated vascular smooth muscle cells,Oxid.Med.Cell Longev.2012(2012)496540.

[11]L.Ma,J.Young,H.Prabhala,E.Pan,P.Mestdagh,D.Muth,J.Teruya-Feldstein,

F.Reinhardt,T.T.Onder,S.Valastyan, F.Westermann, F.Speleman,

J.Vandesompele,R.A.Weinberg,miR-9,a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis,Nat.Cell Biol.12(2010)247e256.

[12]U.Savas,M.H.Hsu,K.J.Grif?n,D.R.Bell,E.F.Johnson,Conditional regulation of

the human CYP4X1and CYP4Z1genes,Arch.Biochem.Biophys.436(2005) 377e385.

[13] C.R.Bohl,S.Harihar,W.L.Denning,R.Sharma,D.R.Welch,Metastasis sup-

pressors in breast cancers:mechanistic insights and clinical potential,J.Mol.

Med.Berl.92(2014)13e30.

[14]Q.Li,R.R.Mattingly,Restoration of E-cadherin cell-cell junctions requires both

expression of E-cadherin and suppression of ERK MAP kinase activation in Ras-transformed breast epithelial cells,Neoplasia10(2008)1444e1458. [15]P.Navarro,M.Gomez,A.Pizarro,C.Gamallo,M.Quintanilla,A.Cano,A role for

the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis,J.Cell Biol.115(1991)517e533.

[16]K.Vleminckx,L.Vakaet Jr.,M.Mareel,W.Fiers,F.van Roy,Genetic manipu-

lation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role,Cell66(1991)107e119.

[17]L.Xie,https://www.360docs.net/doc/e39752613.html,hmorov,F.Leithauser,H.Guan,C.Steidl,J.Farbinger,C.Pelzer,

M.J.Vogel,H.J.Maier,R.D.Gascoyne,P.Moller,T.Wirth,FOXO1is a tumor suppressor in classical Hodgkin lymphoma,Blood119(2012)3503e3511.

[18]M.Cizkova,G.Cizeron-Clairac,S.Vacher,A.Susini,C.Andrieu,R.Lidereau,

I.Bieche,Gene expression pro?ling reveals new aspects of PIK3CA mutation in

ERalpha-positive breast cancer:major implication of the Wnt signaling pathway,PLoS One5(2010)e15647.

[19]M.Benaissa,J.P.Peyrat,L.Hornez,C.Mariller,J.Mazurier,A.Pierce,Expression

and prognostic value of lactoferrin mRNA isoforms in human breast cancer, Int.J.Cancer114(2005)299e306.

[20] B.Karakas,K.E.Bachman,B.H.Park,Mutation of the PIK3CA oncogene in

human cancers,Br.J.Cancer94(2006)455e459.

[21]S.Loi, B.Haibe-Kains,S.Majjaj, https://www.360docs.net/doc/e39752613.html,llemand,V.Durbecq, https://www.360docs.net/doc/e39752613.html,rsimont,

A.M.Gonzalez-Angulo,L.Pusztai,W.F.Symmans,A.Bardelli,P.Ellis,A.N.Tutt,

C.E.Gillett,B.T.Hennessy,https://www.360docs.net/doc/e39752613.html,ls,W.A.Phillips,M.J.Piccart,T.P.Speed,

G.A.McArthur,C.Sotiriou,PIK3CA mutations associated with gene signature

of low mTORC1signaling and better outcomes in estrogen receptor-positive breast cancer,Proc.Natl.Acad.Sci.U.S.A.107(2010)10208e10213. [22]L.H.Saal,K.Holm,M.Maurer,L.Memeo,T.Su,X.Wang,J.S.Yu,

P.O.Malmstrom,M.Mansukhani,J.Enoksson,H.Hibshoosh, A.Borg, R.Parsons,PIK3CA mutations correlate with hormone receptors,node metastasis,and ERBB2,and are mutually exclusive with PTEN loss in human breast carcinoma,Cancer Res.65(2005)2554e2559.

[23]Y.Samuels,Z.Wang,A.Bardelli,N.Silliman,J.Ptak,S.Szabo,H.Yan,A.Gazdar,

S.M.Powell,G.J.Riggins,J.K.Willson,S.Markowitz,K.W.Kinzler,B.Vogelstein, V.E.Velculescu,High frequency of mutations of the PIK3CA gene in human cancers,Science304(2004)554.

[24]L.Zhao,P.K.Vogt,Class I PI3K in oncogenic cellular transformation,Oncogene

27(2008)5486e5496.

[25]G.Nteliopoulos,S.B.Marley,M.Y.Gordon,In?uence of PI-3K/Akt pathway on

Wnt signalling in regulating myeloid progenitor cell proliferation.Evidence for a role of autocrine/paracrine Wnt regulation,Br.J.Haematol.146(2009) 637e651.

[26]T.Reya,H.Clevers,Wnt signalling in stem cells and cancer,Nature434(2005)

843e850.

[27] C.E.Ford,E.J.Ekstrom,J.Howlin,T.Andersson,The WNT-5a derived peptide,

Foxy-5,possesses dual properties that impair progression of ERalpha negative breast cancer,Cell Cycle8(2009)1838e1842.

[28] A.C.Leris,T.R.Roberts,W.G.Jiang,R.F.Newbold,K.Mokbel,WNT5A expres-

sion in human breast cancer,Anticancer Res.25(2005)731e734.

[29]M.T.Veeman,J.D.Axelrod,R.T.Moon,A second canon.Functions and mech-

anisms of beta-catenin-independent Wnt signaling,Dev.Cell5(2003) 367e377.

B.Wang et al./Biochemical and Biophysical Research Communications478(2016)900e907907