MicroRNA 17 5p induces drug resistance and invasion of ovarian carcinoma cells by targeting PTEN signaling

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1 DOI /s RESEARCH Open Access MicroRNA 17 5p induces drug resistnce nd invsion of ovrin crcinom cells y trgeting PTEN signling Ying Fng 1,2, Chngyn Xu 3 nd Yn Fu 1* Astrct Bckground: The ws overexpressed in ovrin cncer cells, nd those cells were treted with pclitxel. The prolifertion of ovrin cncer cells ws ssessed y MTT ssy. The Cspse-Glo3/7 nd TUNEL ssy were used to exmine the effect of on pclitxel-induced poptosis in ovrin cncer cells. The migrtion nd invsion of ovrin cncer cells were nlyzed y BD mtrigel ssys. Western lot ws performed to evlute the expression of poptotic proteins nd epithelil-mesenchyml trnsition mrkers in ovrin cncer cells. Results: The survivl rte of ovrin cncer cells ws incresed fter overexpression of. The poptosis decresed in overexpressed ovrin cncer cells. Altered expression ffected migrtion nd invsion of ovrin cncer cells. The overexpression of ltered the expression of EMT mrkers. ctivtes AKT y downregultion of PTEN in ovrin cncer cells. Conclusion: Our results indicte tht might serve s potentil moleculr therpeutic trget. Keywords: AKT, Apoptosis, PTEN, Metstsis, mirnas, mirna-17-5p, EMT Bckground Ovrin cncer ccounts for out 3 % of cncers mong women in USA. It is estimted tht out 21,29 women will receive new dignosis of ovrin cncer nd out 14,18 women will die from ovrin cncer in 215, even though the rte t which women re dignosed with ovrin cncer hs een slowly flling over the pst 2 yers. Only 2 % of ovrin cncers re found t n erly stge nd more thn 9 % of ptients live longer thn 5 yers if ovrin cncer cn e found t erly stge [1]. The most often used tests to screen for ovrin cncer re trnsvginl ultrsound nd the CA-125 lood test. However, the sensitivity nd specificity of trnsvginl ultrsound nd the CA-125 lood test re poor [2, 3]. Chemotherpy is the primry mode of tretment for ptients with ovrin cncer. However, the tretment filure is high due to resistnce [4]. Therefore, it is importnt to investigte the *Correspondence: yn.fu@ol.com 1 Deprtment of Gynecology, The First Hospitl of Jilin University, Chngchun, Jilin, People s Repulic of Chin Full list of uthor informtion is ville t the end of the rticle moleculr mechnisms nd identify vlule predictive mrkers in ovrin cncer. MicroRNAs (mirnas) re fmily of smll non-coding RNAs tht re 2 22 nucleotides in length. Studies hve demonstrted tht mirnas regulte the expression of trget genes t the post-trnscriptionl level nd ply importnt roles in the tissue-specific protein expression. An incresing numer of studies hve reported tht mirnas ply importnt roles in tumorigenesis, progression, dignosis nd prognosis of ovrin cncer [5]. The expression level of mirnas is different in ovrin cncer s demonstrted y mirna expression profiling studies [6, 7]. For exmple, mir-c nd mir-31 ply importnt roles in ovrin cncer metstsis [8]. Recent studies hve reported tht mirnas cn e used s prognostic iomrkers in ovrin cncer [9, 1]. Also, it hs een reported tht some serum mirnas could serve s iomrkers in ovrin cncer [11]. In the present study, we imed to exmine the role of in ovrin cncer. We found tht overexpression of induces drug resistnce, migrtion nd 215 Fng et l. This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1./) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Pge 2 of 1 invsion of ovrin cncer cells. Importntly, enhnced Epithelil-Mesenchyml Trnsition (EMT) of ovrin cncer cells y trgeting PTEN signling. Our findings indicte tht might serve s potentil iomrker to predict the tretment nd e trgeted for novel therpeutic strtegies. Results Overexpression of mir 17 5p induces drug resistnce of ovrin cncer cells To exmine the function of on prolifertion of ovrin cncer cells fter pclitxel tretment, ws overexpressed in ovrin cncer cells with Lipofectmine nd the cell survivl rte ws mesured y MTT ssy. As shown in Fig. 1, d, the expression level ws significntly incresed in oth OVCAR-3 nd SKOV-3 cells fter trnsfection. The survivl rte of OVCAR-3 nd SKOV-3 cells ws incresed fter overexpression of when ovrin cncer cells were treted with pclitxel, compred to the negtive control group (Fig. 1, d, p =.25). The IC 5 ws 6.1 ± 1.1 µmol L 1 in control group, nd the IC 5 ws 8.2 ±.8 µmol L 1 in group. mir 17 5p decreses poptosis of ovrin cncer cells OVCAR-3 nd SKOV-3 cells were used to exmine the effect of on chemotherpy-induced poptosis y treting with different doses of pclitxel fter overexpression of. We found tht the reduced the sensitivity of ovrin cncer cells to the Reltive expression Dy 2 Dy 8 OD vlue OVCAR-3 Pclitxel p=.31. Dy 2 Dy 4 Dy 6 Dy 8 c Reltive expression 8 Dy Dy 8 d OD vlue SKOV-3 Pclitxel p=.25. Dy 2 Dy 4 Dy 6 Dy 8 Fig. 1 mir-17-5p decresed the chemo-sensitivity of ovrin cncer cells. The expression level ws exmined y qrt-pcr fter trnsfection in OVCAR-3 cells. The prolifertion of OVCAR-3 cells fter pclitxel tretment (6 µmol L 1 ) for 8 dys. c The expression level ws exmined y qrt-pcr fter trnsfection in SKOV-3 cells. d The prolifertion of SKOV-3 cells fter pclitxel tretment (6 µmol L 1 ) for 8 dys. All experiments were performed three times in triplicte

3 Pge 3 of 1 effects of pclitxel y inhiiting the cspse 3/7 ctivities (Fig. 2, ). The TUNEL ssy lso showed tht the numer of poptotic cells in overexpressed ovrin cncer cells ws less thn tht in control group (Fig. 2c, d). We further nlyzed the expression of poptotic relted proteins y western lot fter pclitxel tretment. As shown in Fig. 2e, f, the overexpression of inhiited the ility of chemotherpy to Fluorescence (RFU) p=.12 p=.91 p=.49 Fluorescence (RFU) 15 1 p=.15 p=.19 p= Pclitxel µmol L 1 Pclitxel µmol L 1 c OVCAR-3 SKOV-3 mir-17-5p mir-17-5p Hoechst TUNEL d 75 e f TUNEL-positive Cells/Totl cells (%) p=.11 OVCAR-3 p=.9 SKOV-3 BAX Bcl2 BAX Bcl2 Fig. 2 inhiits poptosis in ovrin cncer cells fter pclitxel tretment., The cspse 3/7 ctivity ws decresed in OVCAR-3 nd SKOV-3 cells trnsfected with fter pclitxel tretment. c The TUNEL positive cells fter OVCAR-3 nd SKOV-3 cells were treted with pclitxel. d The quntifiction of the percentge of TUNEL-positive cells. e The poptotic proteins level in OVCAR-3 cells trnsfected with mimic fter pclitxel tretment. f The poptotic proteins level in SKOV-3 cells trnsfected with fter pclitxel tretment. All experiments were performed three times in triplicte

4 Pge 4 of 1 increse BAX expression, while the Bcl-2 expression ws incresed in overexpressed ovrin cncer cells. mir 17 5p promotes migrtion nd invsion in ovrin cncer cells We further ssessed the effect of on migrtion nd invsion of ovrin cncer cells with BD trnswell migrtion nd mtrigel invsion ssys. We found tht migrtion nd invsion of OVCAR-3 cells (Fig. 3 d) nd SKOV-3 cells (Fig. 4 d) were enhnced fter trnsfection with. In contrst, the migrtion nd invsion of OVCAR-3 cells (Fig. 3e h) nd SKOV-3 cells (Fig. 4e h) were decresed with nti- inhiitor tretment. mir 17 5p ffects the expression of EMT mrkers nd ctivtes AKT in ovrin cncer cells The expression of EMT mrkers ws further exmined y western lot fter ovrin cncer cells were trnsfected with either or nti- inhiitor. We found tht the level of E-cdherin expression ws significntly decresed, nd the expression of N-cdherin, Snil nd Vimentin were incresed in oth OVCAR-3 nd SKOV-3 cells fter overexpression of mir- 17-5p (Fig. 5). In contrst, incresed E-cdherin expression nd decresed N-cdherin, Snil nd Vimentin were oserved in OVCAR-3 nd SKOV-3 cells trnsfected with nti- inhiitor (Fig. 5). Interestingly, we further found tht the expression of p-akt ws incresed, nd the PTEN expression level ws decresed in OVCAR-3 (Fig. 6) nd SKOV-3 cells (Fig. 6) fter trnsfection with mimc. The trnsfection with nti- inhiitor decresed p-akt protein level nd incresed PTEN expression in OVCAR-3 (Fig. 6) nd SKOV-3 cells (Fig. 6). Discussion Growing evidence hs demonstrted tht mirna expression correltes with tissue type, differentition, ggression, response to therpy nd prognosis [12, 13]. Studies hve shown tht mirna ct s oncogenes or tumor suppressors in vriety types of tumors [14, 15]. Li et l. hs reported tht regultes cell cycle nd poptosis in ovrin cncer tissues nd serum of ovrin cncer ptients [16]. hs een found to e expressed differentilly in the serum of cncer tissue compred with tht of non-cncerous tissues [17]. Studies hve found tht is key regultor of the G1/S phse cell cycle trnsition [18]. In our study, we demonstrted tht the overexpression of promoted the prolifertion of ovrin cncer cells treted with pclitxel. These results indicte tht induces drug resistnce of ovrin cncer cells. By nlysis of cspse 3/7 ctivity nd TUNEL ssy, we found tht the overexpression of decreses the pclitxel-induced poptosis of ovrin cncer cells. Menwhile, the incresed Bcl-2 nd decresed BAX expression levels in overexpressed ovrin cncer cells confirm tht decreses the pclitxel-induced poptosis of ovrin cncer cells. These results indicte tht might ply n importnt role in conferring chemosensitivity to ovrin cncer cells. Chemotherpy drugs re most effective when given in comintion. Pclitxel is often comined with other chemotherpy drugs, such s cispltin. Therefore, further study is required to exmine the function of in comintion of chemotherpy tretment. Ovrin cncer hs higher incidence of distnt metstsis [19]. Once metstsis occurs, it ecomes n incurle disese with limited survivl time [2]. Metstsis is complex, multistep process y which tumor cells disseminte from their primry site nd form secondry tumors t distnt site [21]. Epithelil mesenchyml trnsition (EMT) hs een shown to ply criticl role in promoting metstsis. EMT is iologicl process tht llows epithelil cells to lose their epithelil chrcteristics nd cquire mesenchyml phenotype [22]. EMT plys criticl role in ovrin cncer metstsis. Mny mirnas, such s mir-7 [23], mirna-15 [24], mir-c [25], modulte the EMT nd metstsis of ovrin cncer cells. We showed tht incresed migrtion nd invsion in oth OVCAR-3 nd SKOV-3 cells fter forced expression of. In contrst, the migrtion nd invsion of ovrin cncer cells were decresed when ovrin cncer cells were treted with nti- inhiitor. Moreover, we found tht the expression level of EMT iomrkers ws chnged in ovrin cncer cells following errnt expression of. These results indicte tht plys importnt role in ovrin cncer progression. PTEN is tumor suppressor gene with decresed ctivity reported in mny humn cncers [26, 27]. The loss nd muttion of PTEN led to hyperctive PI3K signling, which is n importnt intrcellulr signling pthwy tht regulte mny cellulr processes, including cell survivl, cell prolifertion, nd cell growth. Studies hve found tht mir-26 cts s direct regultor of PTEN expression in high-grde gliom [28]. Some mirnas suppress PTEN expression y directly intercting with its 3 UTR in prostte epithelil nd cncer cells [29]. In the present study, we reported tht the expression of PTEN ws decresed nd the expression of pakt ws incresed in ovrin cncer cells. Our results indicte tht mir- 17-5p ffects drug-resistnce, poptosis nd invsion y regulting PTEN/Akt signling pthwy in ovrin

5 Pge 5 of 1 Invsion Invsion c Migr on cells p=.62 d Invsion cells p=.33 mimic mimic e f Anti- inhiitor Invsion Invsion g Migr on cells p=.22 inhiitor h Invsion cells p=.15 inhiitor Fig. 3 mir-17-5p increses migrtion nd invsion of ovrin cncer cells. d incresed migrtion nd invsion in OVCAR-3 cells fter trnsfection with. e h The migrtion nd invsion were decresed in OVCAR-3 cells fter trnsfection with nti- inhiitor

6 Pge 6 of 1 c Migr on cells p=.77 d Invsion cells p=.83 mimic mimic e f Anti- inhiitor g Migr on cells p=.12 h Invsion cells p=.35 inhiitor inhiitor Fig. 4 mir-17-5p increses migrtion nd invsion of ovrin cncer cells. d incresed migrtion nd invsion in OVCAR-3 cells fter trnsfection with. e h The migrtion nd invsion were decresed in SKOV-3 cells fter trnsfection with nti- inhiitor. All experiments were performed three times in triplicte

7 Pge 7 of 1 E-cdherin N-cdherin Snil PTEN AKT p-akt E-cdherin N-cdherin PTEN Snil AKT Fig. 5 regultes EMT mrkers expression in ovrin cncer cells. The EMT mrkers level in OVCAR-3 cell fter trnsfection with or nti- inhiitor. The EMT mrkers level in SKOV-3 cells fter trnsfection with or nti-mir- 17-5p inhiitor cncer.however, no trget site in PTEN ws identified. Amplified in rest cncer 1 (AIB1) mplifiction nd overexpression hve een seen in rest nd ovrin cncer cell lines. AIB1 is steroid receptor coctivtor tht medites the trnscriptionl ctivities of nucler receptors nd other trnscription fctors. Hossin et l. hs shown tht regultes rest cncer cell prolifertion y trgeting AIB1 [3]. PTEN cn suppress AIB1 through decresing protein stility [31]. In our study, ffects the PTEN expression in OVCAR-3 cells nd SKOV3 cells. Further study should e done to exmine the interction etween PTEN nd AIB1 in these ovrin cncer cell lines. p-akt Fig. 6 ctivtes AKT expression y regulting PTEN in ovrin cncer cells. The expression of AKT nd PTEN in OVCAR-3 cells fter trnsfection with or nti- inhiitor. The expression of PTEN nd AKT in SKOV-3 cells fter trnsfection with or nti- inhiitor Conclusion plys importnt role in the regultion of tumorigenesis nd mlignnt progression in ovrin cncer. could e potentil moleculr trget in ovrin cncer tretment in the future. Methods Cell lines nd cell culture The humn nsophryngel crcinom cell lines, OVCAR-3 nd SKOV-3, otined from Americn Type

8 Pge 8 of 1 Culture Collection were cultured in RPMI164 medium (Invitrogen, USA) supplemented with 1 % het-inctivted fetl ovine serum nd 1 U ml 1 of penicillin nd 1 μg ml 1 of streptomycin (Sigm, USA). The cells were cultured t 37 C in humidified incutor in n tmosphere of 5 % CO 2-95 % ir. All cells were pssged when they reched pproximtely 8 % confluency. Trnsfection s nd nti- inhiitor (single-strnded, modified RNA molecule) were purchsed from GenePhrm (Shnghi, Chin). When OVCAR-3 nd SKOV-3 cells reched 7 % confluency, they were trnsfected with or nti-mir- 17-5p inhiitor using Lipofectmine (Invitrogen, USA) ccording to the mnufcturer s instructions. The mirna-lipofectmine complex ws mde in serum-free OPTI-MEM medium. The scrmled oligonucleotide ws used s negtive control. Rel time RT PCR quntifiction of mir 17 5p To exmine the expression fter trnsfection, totl RNA ws extrcted with mirvnmirna Isoltion kit (Amion, USA) ccording to the mnufcturer s instruction. cdna ws synthesized from the isolted RNA with Tqmn MicroRNA Reverse Trnscription kit (Thermofisher Scientific, USA). The PCR condition used ws: 95 C for 6 min, followed y 35 cycles of 95 C for 35 s, 6 C for 3 s nd 72 C for 3 s, nd dissocition stge. PCR ws performed using the TqMn Fst Universl PCR Mster Mix (Thermofisher Scientific, USA) nd CFX Connect Rel-Time PCR Detection System (Bio-Rd, USA). The endogenous reference gene ws used for RNA quntifiction. The PCR primers sequences used were: 5 -GTCTCCTCTGACTTCAACA- GCG-3 nd 5 -ACCACCCTGTTGCTGTAGCCAA-3 (). The effect of mir 17 5p on cell prolifertion fter chemotherpy regent tretment To evlute the effect of on cell prolifertion fter chemotherpy regent tretment, the MTT (3-(4,5-Dimethylthizol-2-yl)-2,5-Diphenyltetrzolium Bromide) ssy ws performed s descried previously [32]. Briefly, OVCAR-3 nd SKOV-3 cells, trnsfected with either scrmled oligonucleotide or mimic, were seeded in triplicte to 96-well pltes t the density of cells well 1. After overnight growth, culture medium contining pclitxel (6 µmol L 1 ) (Sigm, USA) ws dded. On every ech other dy, MTT solution (2 μl, 5 mg ml 1 ) ws dded to ech well, nd the pltes were incuted in the drk for 4 h t 37 C, followed y removl of the culture medium nd ddition of 1 μl of dimethyl sulphoxide (DMSO). The sornce ws mesured t 49 nm, with 65 nm s the reference wve length. All experiments were crried out in triplictes. Cspse 3/7 ctivity OVCAR-3 nd SKOV-3 cells, trnsfected with either scrmled oligonucleotide or, were seeded in 24-well pltes t density of cells well 1. After overnight incution in n tmosphere of 5 % CO 2-95 % ir, the superntnt ws replced with culture medium contining different concentrtions of pclitxel (3, 6 nd 12 µmol L 1 ). The cells were grown for 48 h, then Cspse-Glo regent (Promeg, USA) ws dded to ech well nd incuted t room temperture for 8 h with gentle shking. The cspse 3/7 ctivity ws mesured using 1 min lg time nd.5 s well 1 red time with luminometer (Thermofisher Scientific, USA). The experiments were performed in triplicte. TUNEL ssy After overexpression of, OVCAR-3 nd SKOV-3 cells were seeded in 96-well pltes t density of cells well 1. After overnight incution, the superntnt ws replced with culture medium contining pclitxel (12 µmol L 1 ). The cells were grown for nother 48 h, then the TUNEL ssy ws performed using Click-iT TUNEL Alex Fluor Imging Kit (Invitrogen, USA) in ccordnce with the mnufcturer s protocol. In rief, fter the cells were fixed with 4 % prformldehyde in PBS t room temperture for 2 min nd permeilized with Triton X-1 (.25 % in PBS) for nother 2 min, the cells were wshed twice nd incuted with terminl deoxynucleotidyltrnsferse rection uffer for 1 min t room temperture. The TUNEL rection mixture contining terminl deoxynucleotidyltrnsferse ws dded nd the smples were incuted in humidified chmer t 37 C for 6 min. Then, smples were wshed three times with 3 % BSA in PBS for 2 min ech nd then incuted with Click-iT rection mixture (contining Alex 488 zide) for 3 min t room temperture. After wshed with 3 % BSA in PBS, the cell nuclei were counter stined with Hoechst (Thermofisher Scientific, USA) for 15 min t room temperture. The TUNEL-positive cells were counted in eight different, rndom fields for ech well. Mtrigel invsion ssys The cell invsion ws exmined y Mtrigel invsion ssys ccording to the mnufcturer s instruction (Promeg, USA). Briefly, OVCAR-3 nd SKOV-3 cells t density of per well, trnsfected with either mir- 17-5p mimic or nti- inhiitor, were plced to the upper BD Biocot Mtrigel Invsion Chmer (BD Bioscience, US) in.5 ml DMEM with.1 % BSA.

9 Pge 9 of 1 The DMEM medium contining 5 % FBS ws dded to the lower chmer. The cells were incuted for 18 h, nd then the non-invded cells were removed with cotton sw. The invded cells were stined y Diff Quik stin (Thermofisher Scientific, USA) nd counted under microscopy. The percentge of invsion ws expressed s the rtio of invding cells over cell numer normlized on dy 2 of the growth curve. Western lot ssy The trnsfected OVCAR-3 nd SKOV-3 cells were lysed with ice-cold RIPA uffer (Beyotiem, Chin). Then the smples were mixed with 6 loding uffer, oiled t 1 C for 5 min, trnsferred on ice nd loded to n SDS-PAGE gel. Proteins were seprted y SDS-PAGE nd trnsferred to PVDF memrnes (Sigm, USA). Then the memrnes were incuted in 5 % non ft dry milk in Tris-uffered sline Tween-2 uffer (TBST: 1 mmol L 1 Tris-Bse, 15 mmol L 1 NCl,.5 % Tween-2; ph 7.4) for 1 h t room temperture to lock nonspecific ntiody inding sites. After wshing with TBST uffer, memrnes were incuted overnight t 4 C with primry ntiodies (E-cdherin, N-cdherin, Snil, Vimentin, Bcl-2, Bx, AKT nd PTEN: Cell Signling Technology, USA) in TBST with gentle gittion. After wshed with TBST t room temperture, the memrnes were incuted with the horserdish peroxidse-conjugted secondry ntiody for 1 h t room temperture. The immune lot signls were visulized using the EsySeeWetern Blot Kit (Trnsgen, Chin). The protein nds were detected y densitometric scnning (Tnon- 16 gel imge system, Shnghi, Chin). Sttisticl nlysis All of results were shown s men ± SD. Sttisticl nlyses were performed y Student s t test. Briefly, the experimentl results from control groups nd experimentl groups were entered in SPSS progrm (version 11., IBM. USA), the p vlues were clculted. Differences re considered sttisticlly significnt t p <.5. Authors contriutions YF: crried out the genetic nlysis nd cell prolifertion experiments. CX prticipted in design nd drft the mnuscript. YF: crried out the western lot nd nlyzed the results. All uthors red nd pproved the finl mnuscript. Author detils 1 Deprtment of Gynecology, The First Hospitl of Jilin University, Chngchun, Jilin, People s Repulic of Chin. 2 Deprtment of Gynecology, No. 28 Hospitl of Chinese People s Liertion Army, Chngchun, Jilin, People s Repulic of Chin. 3 Deprtment of Medicl Administrtion, The First Hospitl of Jilin University, Chngchun, Jilin, People s Repulic of Chin. Acknowledgements The project is supported y the Young Scientists Awrd (JLP21315) in Jilin Province. Competing interests The uthors declre tht they hve no competing interests. Received: 18 August 215 Accepted: 12 Octoer 215 References 1. Mezznznic D. Ovrin cncer: moleculrly insidious disese. Chin J Cncer. 215;34: Gsiorowsk E, Michlk M, Wrchoł W, Lemńsk A, Jsiński P, Spczyński M, et l. Clinicl ppliction of HE4 nd CA125 in ovrin cncer type I nd type II detection nd differentil dignosis. 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10 Pge 1 of Klluri R, Weinerg RA. The sics of epithelil-mesenchyml trnsition. J Clin Invest. 9;119: Zhou X, Hu Y, Di L, Wng Y, Zhou J, Wng W, et l. MicroRNA-7 inhiits tumor metstsis nd reverses epithelil-mesenchyml trnsition through AKT/ERK1/2 inctivtion y trgeting EGFR in epithelil ovrin cncer. PLoS One. 214;9:e Jin M, Yng Z, Ye W, Xu H, Hu X. MicroRNA-15 predicts fvorle prognosis in ptients with epithelil ovrin cncer, nd inhiits cell invsion nd metstsis y suppressing trnscriptionl repressor ZEB1. PLoS One. 214;9:e Chen D, Zhng Y, Wng J, Chen J, Yng C, Ci K, et l. MicroRNA-c overexpression inhiits tumorigenicity nd metstsis of CD117 + CD44 + ovrin cncer stem cells y regulting epithelil-mesenchyml trnsition. J Ovrin Res. 213;6:5. doi:1.1186/ Andjelkovic T, Bnkovic J, Milosevic Z, Stojsic J, Milinkovic V, Pesic M, et l. Concurrent ltertion of p16 nd PTEN tumor suppressor genes could e considered s potentil moleculr mrker for specific sugroups of NSCLC ptients. Cncer Biomrk ;1: doi:1.3233/ CBM Stewrt DJ, Nunez MI, Jelinek J, Hong D, Gupt S, Aldz M, et l. Impct of decitine on immunohistochemistry expression of the puttive tumor suppressor genes FHIT, WWOX, FUS1 nd PTEN in clinicl tumor smples. Clin Epigenet. 214;6:13. doi:1.1186/ Huse JT, Brennn C, Hmrdzumyn D, Wee B, Pen J, Rouhnifrd SH, et l. The PTEN-regulting microrna mir-26 is mplified in high-grde gliom nd fcilittes gliomgenesis in vivo. Genes Dev. 9;23: Tin L, Fng YX, Xue JL, Chen JZ. Four micrornas promote prostte cell prolifertion with regultion of PTEN nd its downstrem signls in vitro. PLoS One. 213;8:e doi:1.1371/journl.pone Hossin A, Kuo MT, Sunders GF. Mir-17-5p regultes rest cncer cell prolifertion y inhiiting trnsltion of AIB1 mrna. Mol Cell Biol. 6;26: Yng C, Li S, Wng M, Chng AK, Liu Y, Zho F, et l. PTEN suppresses the oncogenic function of AIB1 through decresing its protein stility vi mechnism involving Fw7 lph. Mol Cncer. 213;12: Mioli E, Torricelli C, Fortino V, Crlucci F, Tommssini V, Pcini A. Criticl pprisl of the MTT ssy in the presence of rottlerin nd uncouplers. Biol Proced Online. 9;11: Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t