New insights into the roles of the FOXO3 and P27Kip1 genes in signaling pathways ARTICLE New insights into the roles of the FOXO3 and P27Kip1 genes in signaling pathways Sabah Mayahia,b,c, Masood Golalipourc, Ahad Yamchid, Gagan Deep Jhingane and Majid Shahbazic,f aDepartment of Molecular Medicine, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran; bDepartment of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; cMedical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran; dDepartment of Biotechnology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran; eVProteomics, Ground Floor Green Park Main, New Delhi, India; fAryaTinaGene Biopharmaceutical Company, Gorgan, Iran ABSTRACT Background: The forkhead box O3 (FOXO3) and p27Kip1 are two important genes in breast cancer progression. In the present study we analyzed the effect of simultaneous FOXO3 silencing and p27Kip1 activation on breast cancer cell survival and the potential targets of these changes in cancer molecular pathways. Materials and methods: The present study involved the cloning of FOXO3a shRNA and p27Kip1 genes under the control of the bidirectional survivin promoter to down- and up-regulate FOXO3 and p27Kip1 genes, respectively. After transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by MTS and flow cytometry assays. Following the extraction of total mRNA and protein, the expression of target genes was evaluated by qPCR and Western blotting in both treated and untreated cell lines. Then, the downstream protein responses were examined by 2 D electrophoresis. The differentially expressed proteins were also identified by mass spectrometry. Results: Rates of cell proliferation were significantly inhibited in the transfected cell line 72 h post- transfection. Proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of FOXO3 and p27Kip1. The changes in expression of these markers suggested that certain signaling pathways contribute to the development of breast cancer. Conclusion: Simultaneous silencing of FOXO3 and activation of p27Kip1 in MDA-MB-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. It seems that the identified genes might serve as useful biomarkers for breast cancer. ARTICLE HISTORY Received 31 December 2018 Revised 17 May 2019 Accepted 21 May 2019 KEYWORDS Breast cancer; cell signaling; FOXO3a; gene therapy; P27Kip1; proteomics Introduction Breast cancer is an important global health concern and the second leading cause of cancer-related mortality among women (1). Omics studies have revealed several pathways and genes related to breast cancer (2), such as p27Kip1 and forkhead box O3 (FOXO3). The p27Kip1 protein interacts with cyclin E and cyclin-dependent kinase 2 (CDK2). In addition, the p27Kip1 gene arrests the progression of the cell cycle and leads to apoptosis, tumor suppression, and cell adhesion (3). Poor prognosis in patients with colorectal, gastric, pul- monary, and breast cancers is associated with a low expres- sion level of the p27Kip1 gene (4). The FOXO proteins, a subgroup of forkhead transcription factors, are involved in apoptosis, differentiation, stress response, cell cycle control, and cell metabolism (5). Overall, the FOXO3 gene is involved in many signaling pathways and makes major contributions to several processes, including cancer development (6). Therefore, the identification of FOXO3 inhibitors presents a promising strategy for future anticancer drugs (7). Proteomic analysis is generally applied in breast cancer serving two ends, namely the identification of novel molecular markers for breast tumor profiling and determination of the intracellular signaling pathways contributing to the growth of breast cancer cells (8). To our knowledge, there is no information on the effect of simultan- eously decreasing FOXO3 gene and increasing p27Kip1 gene expression, respectively, to inhibit breast cancer. With this background in mind, the present study aimed to analyze the effect of the FOXO3 and p27Kip1 genes on the control of the breast cancer MDA-MB-231 cell line, using a bidirectional sur- vivin promoter. Moreover, the changes in the protein profile CONTACT Majid Shahbazi shahbazimajid@yahoo.co.uk Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, 4916668197, Iran Supplemental data for this article can be accessed here � 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UPSALA JOURNAL OF MEDICAL SCIENCES 2019, VOL. 124, NO. 3, 149–157 https://doi.org/10.1080/03009734.2019.1623351 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1623351&domain=pdf&date_stamp=2019-09-09 https://doi.org/10.1080/03009734.2019.1623351 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1623351 http://www.tandfonline.com of the transfected cell line were investigated in order to find cancer molecular markers. Materials and methods Cell line, plasmid constructs, and transfection The MDA-MB-231 cell line adopted in this study was supplied by the Pasteur Institute (Tehran, Iran). Cells were cultured in a high-glucose Dulbecco’s modified Eagle’s medium (ThermoFisher scientific, Waltham, MA, USA), supplemented with penicillin-streptomycin (100 units/mL) and fetal bovine serum (10% FBS; Gibco BI102-100) in a damp atmosphere (5% CO2) at 37 �C. The cells were transfected with a plasmid construct designed in a bidirectional promoter (dual target- ing of FOXO3a shRNA and simultaneous induction of p27Kip1 gene). In addition, an empty plasmid of pcDNA3.1þ expression vector with EGFP was used as the control (Figure 1). The vec- tors were purchased from BioMatic, Toronto, Ontario, Canada. To verify the identity of the inserted sequences, all plasmids were examined through direct sequence analysis. Twenty-four hours before transfecting DNAs into the cells, some of the cells were applied in the growth medium with- out antibiotics. Transfection was performed as recommended by the manufacturer using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). Cell viability assay The viability of the cells (2–5 � 103/well) was tested in a 96- well plate in a final volume of 200 lL/well, incubated in a CO2 incubator (5% CO2) at 37 �C overnight. Then, 20 lL/well of MTS reagent (Promega, Fitchburg, WI, USA) were added to each well and incubated for 4 h at 37 �C. The plate was shaken briefly, and then the absorbance of the treated and untreated cells was measured at 490 nm 24 h, 48 h, and 72 h after transfection, using a plate reader (BioTek, Winooski, VT, USA). Quantitative real-time polymerase chain reaction assay The extraction of total RNA was accomplished using TRIzol solution (Invitrogen). Total RNA (2 mg) was used for comple- mentary DNA synthesis as recommended by the manufac- turer (Thermo Scientific, Vilnius, Lithuania). Quantitative real- time reverse transcriptase polymerase chain reaction (qPCR) assay was performed using ABI 7300 RT-PCR systems (Applied Biosystems, Foster City, CA, USA) to determine the relative expression of the p27Kip1 and FOXO3a genes (Table 1). The threshold cycle (Ct) value was estimated, and the REST 2009 software was used to assess the amplification plots. In addition, the comparative Ct (2�DDCt) method was applied to analyze the data (8). Apoptosis assay To identify apoptotic cells, the FITC Annexin V Detection Kit with propidium iodide (PI) (BioLegend, San Diego, CA, USA) was exclusively developed. After seeding the cells (2 � 105 cells/mL) in tissue culture plates, they were incubated over- night, and then treated after 24 h, 48 h, and 72 h. In the next stage, the cells were washed with a cell-staining buffer, and then re-suspended in the annexin V binding buffer at a con- centration of 0.25–1.0 � 107 cells/mL. Subsequently, the PI solution (10 lL) and FITC annexin V (5 lL) were added. The cells were vortexed and incubated at 25 �C for 15 min in darkness. Then, the annexin V binding buffer (400 lL) was Figure 1. (A) Map of pcDNA3.1(þ) EGFP; (B) pSur (shFOXO3-p27Kip1) vector with a survivin promoter. 150 S. MAYAHI ET AL. added to the tubes. Finally, a BD AccuriTM C6 cytometer (Seattle, WA, USA) was used to perform flow cytometry. Protein extraction and immunoblotting A protein lysis buffer (100 mM Tris-HCl, 150 mM NaCl, 5% SDS, 1 mM dithiothreitol, 5 mM EDTA, pH of 7.4, 5% sodium deoxycholate, and 10% glycerol) was used for lysing the har- vested cells collected via trypsinization. A protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO,USA) mixture was added to 1 mM of PMSF. The total protein in the resultant supernatant was used for Western blotting analysis. After separating the total protein using polyacrylamide gel electro- phoresis, it was transferred to a nitrocellulose membrane, which was blocked with bovine serum albumin (5% w/v bovine serum albumin; Sigma-Aldrich) in TBST. Furthermore, the primary antibody was used against FOXO3a and p27Kip1 antibodies (Santa Cruz, Santa Cruz, CA, USA). Visualization of the bands was accomplished by means of the Enhanced Chemiluminescence Detection Kit (Parstous, Tehran, Tehran, Iran) following the manufacturer’s protocol. In addition, the National Institutes of Health ImageJ program (Bethesda, MD, USA) was used to determine the band density. Two-dimensional gel electrophoresis Cell pellets were resuspended by adding an adequate amount of 2 D lysis buffer, containing 2 M thiourea, 1% dithiothreitol, 4% CHAPS, 7 M urea, 0.001% bromophenol blue, and 0.5% immobilized pH gradient (IPG) buffer (pH of 3–10; GE Healthcare, USA), followed by extracting the total protein. In addition, a 2 D Quant Kit (GE Healthcare) was used to measure the protein level with bovine serum albu- min as the standard. To this end, after rehydrating the IPG strips (7 cm 3–10 NL, Immobilone Dry Strip; GE Healthcare) at a pH gradient of 3–10, 300 mg of each protein sample was loaded on the IPG strip. The 2 D electrophoresis was carried out based on our previously published method (9). For stain- ing the gels, a colloidal Coomassie Brilliant Blue dye was used as described by Candiano et al. (10). A scanner (GE Healthcare Life Sciences, Chicago, IL, USA) was also employed to scan the stained gels. The analysis of the pro- tein spots in the gel images was performed by the ImageMaster 2 D Platinum 6.0 (GE Healthcare Life Sciences). Finally, the intensities of the treated and untreated gels were compared, and their ratios were determined. Protein identification via liquid chromatography-mass spectrometry After cutting the protein spots into 1-mm3 pieces, they were removed from the stained gel manually and added to tubes. The pieces were destained with an equal solution of ammo- nium bicarbonate and acetonitrile. Then, they were digested with trypsin (Promega, USA) at 37 �C for a minimum of 16 h. Trifluoroacetic acid (TFA) 10% was also added to prevent digestion. Extraction of the gel pieces was accomplished using an equal solution of 0.1% TFA and acetonitrile (50 lL). Supernatants from the pooled samples were dried, using Speed-Vac centrifugation (Thermo Scientific, Waltham, MA, USA). Then, 2% acetonitrile (10 lL) and 0.1% TFA were used for dissolving the samples. The Q-TRAP nLC-MS/MS system (Applied Biosystems) was used for all analyses, as described (11). Mass data were collected and analyzed using the Analyst software (Applied Biosystems). For finding the SwissProt-Trembl subdatabase, the MS/MS lists were searched, and Mascot 3 (version 2.2., Matrix Science, Boston, MA, USA) was used to blast the data lists against databases. Data analysis Data were analyzed in Prism, version 6.0 (GraphPad Software Inc, San Diego, CA, USA) using two-way ANOVA and Student’s t test. The data were also presented as mean- ± standard error of mean of at least three independent anal- yses. A p values less than 0.05 was considered statistically significant. Results Combinatorial effects of induced p27Kip1 and shFOXO3a on cell viability The p27Kip1 mRNA expression level was significantly higher, and FOXO3a mRNA expression significantly lower, in treated cells 72 h post-transfection in comparison with that found in controls (P < 0.05). Analysis of the qPCR results showed a 0.41-fold change in FOXO3a gene expression (down-regula- tion) and more than a three-fold change in p27Kip1 gene expression (up-regulation) 72 h after transfection (Figure 2). FOXO3 expression decreased (60%), while p27Kip1 expres- sion increased 72 h after transfection, compared to the con- trol expression (Figure 3). The expression of construct in MDA-MB-231 significantly reduced the growth of the cell line (P < 0.05) (Figure 4). The effect of the construct on the rate of cell proliferation was not statistically significant 24 h post-transfection. However, it was significantly inhibited at 72 h after treatment (P < 0.05; Figure 5). Flow cytometry Apoptotic cells included both early and late apoptosis (Annexin Vþ/PI- and Annexin Vþ/PIþ, respectively), while Annexin V and PI were negative for viable cells. In the con- trols, the majority of the cells (99.9%) were viable and non- apoptotic at baseline. However, early apoptotic cells showed a significant increase after treatment, compared to the untreated cells after 72 h (from 5.0% to 36.4%; Annexin Vþ/ PI–; Figures 6(A, B)). Effect of overexpression p27Kip1 and FOXO3a shRNA on downstream proteins To identify the downstream responsive proteins associated with the changes in p27Kip1 and shFOXO3a expression at UPSALA JOURNAL OF MEDICAL SCIENCES 151 72 h after transfection, the treated and untreated samples were compared by 2 D gel electrophoresis. There was differ- ential expression of 10 protein spots in the breast cancer cell line (Figure 7). Out of the 10 proteins, 3 and 7 proteins showed up-regulated and down-regulated expression, respectively (Figure 7). Table 2 presents the mass spectrom- etry (MS) information of the appeared spots. Succinate dehydrogenase (SDH; T1/C1), albumin (ALB; T3/C3), and ser- ine/threonine-protein kinase NLK (NLK; T4/C4) were signifi- cantly up-regulated, while tubulin alpha (TUBA1C; T2/C2), Figure 2. Relative expression of FOXO3 and p27Kip1 genes in the MDA-MB-231 cell line after transfection was significantly down-regulated and up-regulated, respectively, 72 h after transfection. Expression of the genes was analyzed by the REST program (P < 0.05). Figure 3. Relative band densitometry of Western blotting data, showing: (A) the incidence of the highest down-regulation of FOXO3a gene 72 h after transfection; (B) reduction of FOXO3a gene expression in the cells treated with the construct after 72 h, compared to the control; (C) the highest up-regulation of P27Kip1 gene in MDA-MB-231 cells occurring 72 h post-transfection; and (D) an increase in P27Kip1 expression in the cells treated with the construct 72 h after transfection, com- pared to the control. Implementation of quantification in three repeats (GAPDH was used as the control). Figure 4. Microscopic morphology of MDA-MB-231 cells under a fluorescent microscope after transfection for 24 h, 48 h, and 72 h with: (A) an empty vector; (B) a vector containing the genes after 24 h, (C) 48 h, (D) 72 h; and (E) negative signal of control cells without transfection. 152 S. MAYAHI ET AL. tubulin beta chain (TUBB; T10/C10), b-actin (ACTB) with two isoforms (T8/C8 and T9/C9), and vimentin (VIM) with three isoforms (T5/C5, T6/C6, and T7/C7) were significantly down- regulated (Figure 7). The theoretical and experimental masses and the isoelectric points of these differentially expressed proteins were also nearly the same, verifying the identified MS results (Table 2). Gene ontology (GO) analysis of 10 differentially expressed proteins consisted of protein– protein interactions using the GeneMANIA (https://genema- nia.org) and KEGG (https://www.genome.jp/kegg/kegg2.html) databases (Table 3). Based on the result of two-way ANOVA, after the transfection of genes in MDA-MB-231 cells for 72 h, FOXO3 gene expression showed the greatest reduction (P < 0.05), while P27Kip1 gene showed the highest increase, compared to the control (Figure 7). The 2 D gel Figure 5. Inhibition of MDA-MB-231 cell proliferation after 24 h, 48 h, and 72 h of treatment in wells (in triplicate) using MTS assay. Data are expressed as the mean cell viability percentage versus that of the control. Figure 6. Apoptosis detection via flow cytometry. A: use of Annexin V-FITC staining in the treated and untreated MDA-MB-231 cells after 24 h, 48 h, and 72 h. The upper and lower right quadrants (Annexin Vþ/PI þ and Annexin Vþ/PI�, respectively) indicate late and early apoptosis, respectively (10.4% of cells died by lipo- fectamine, as shown in Supplementary Figure 1, available online). B: Apoptosis in the treated and control MDA-MB-231 cells 24 h, 48 h, and 72 h post-transfection. Data were presented as mean ± SD (P < 0.05). UPSALA JOURNAL OF MEDICAL SCIENCES 153 https://genemania.org https://genemania.org https://www.genome.jp/kegg/kegg2.html https://doi.org/10.1080/03009734.2019.1623351 electrophoresis of the plasmid construct and empty vector- transfected MDA-MB-231 cells indicated changes in the expression level. Out of the collected 25 spots, 10 spots (indicated by arrows, Figure 7) that had the highest scores were selected and confirmed to be involved in breast cancer. Discussion There are studies regarding the independent effect of P27Kip1 and FOXO3a genes on the inhibition of breast can- cer; however, they have reported a poor prognosis (12,13). The present study involved the design of a bidirectional expression vector suppressing FOXO3a and inducing p27Kip1 expression. Our results showed that the synergistic effects of p27Kip1 and shFOXO3a induced 36.4% apoptosis in cells. However, the overexpression of p27Kip1 alone has been reported to induce 12.73% apoptosis (12). Recently, prote- omic methods have been used as a strategy for the detec- tion of protein biomarkers (14). Expression of seven proteins changed after FOXO3a suppression and p27Kip1 induction (Figure 8). These proteins are all involved in apoptosis, metastasis, drug resistance, proliferation, and cell cycle control. Three up-regulated proteins included SDHA, albumin (with cell source), and NLK, while four down-regulated proteins included TUBA1C, TUBB, ACTB with two isoforms, and VIM with three isoforms. The SDH is identified as a mito- chondrial enzyme, involved in the Krebs cycle and electron transfer chain, and contributes to tumor suppression. This enzyme has four subunits, namely SDHA, SDHB, SDHC, and SDHD (15). Research over the past decades has shown an inverse relationship between albumin level and severity/risk of the progression of different cancers, including pulmonary, gastric, ovarian, and breast cancers. There are various mecha- nisms for the anticancer effects of albumin, such as DNA sta- bilization and duplication, calcium preservation and regulation, and antioxidant properties. In a study performed by Xiao-Jing Du et al., the growth of human cancer cells, such as breast cancer cells, was inhibited by high concentra- tions of albumin (16). The main cause of the reduction of the albumin level, especially in cancer patients, is the inhibition of albumin-regulating gene by tumor necrosis factor which results in mRNA expression reduction of approximately 90% in the liver (17). NLK, which belongs to the mitogen-acti- vated protein kinase family, is a vital regulator of various cancer types. In a study conducted by Lv et al., NLK inhibited non-small-cell lung cancer through the Wnt signaling path- way (18). In addition, Huang et al. showed that NLK inhibits cellular proliferation in breast cancer and induces apoptosis. Similar studies confirm that NLK is a tumor suppressor gene in ovarian cancer (19). The theoretical molecular weight of NLK is about 58 kDa, while its experimental molecular weight is about 35 kDa, which can be attributed to the degradation of this protein. Actin and its proteins have a major functional and structural role in cell motility, endocytosis, cell adhesion and division, and maintenance of cell morphology. The expression of ACTB increases in a metastatic state (20). Accordingly, Chunmei Guo et al. reported that ACTB Figure 7. Two-dimensional gel electrophoresis of MDA-MB-231 cell line in treated and untreated cells, showing the increased expression of tubulin beta chain, vimentin (three isoforms), and b-actin (two isoforms) and decreased expression of succinate dehydrogenase complex, albumin, and NLK. Protein spots 6 and 7 were absent in the treated cells. The experiment was performed in two biological repeats. A ¼ absence. Table 1. Primers used in this study. Primer Sequence (5’!3’) FOXO3a (R) GACTATGCAGTGACAGGTTGTG FOXO3a (F) TCTACGAGTGGATGGTGCGTT P27 (R) TCCATTCCATGAAGTCAGCG P27 (F) GAGCAGACGCCCAAGAAGC GAPDH (R) AGGCAGGGATGATGTGGAGAG GAPDH (F) ATCGTGGAACTCAGTACCACA 154 S. MAYAHI ET AL. Ta b le 2. Pr ot ei n sp ot s co lle ct ed fr om tw o- d im en si on al g el s in M D A -M B- 23 1 ce ll lin e tr an sf ec te d w it h th e p la sm id co n st ru ct in co m p ar is on w it h th e co n tr ol (e m p ty ve ct or ) af te r 72 h . Sp ot ID A cc es si on N am e of p ro te in G en e Th eo re ti ca l M W (k D a) /P I Ex p er im en ta l M W (k D a) /P I Ra ti o Pe p ti d e Sc or e 1 P3 10 40 Su cc in at e d eh yd ro g en as e SD H A 72 .6 /7 .3 9 72 .2 /7 .8 2. 1 (u p -r eg ul at ed ) V G SV LQ EG G K 2. 65 2 Q 9B Q E3 a -T ub ul in TU BA 1C 51 /6 .0 49 .9 /7 .1 0. 2 (d ow n - re g ul at ed ) A V FV D LE PT V ID EV R; EI ID LV LD R; ED A A N N YA R; D V N A A IA TI K; TI G G G D D SF N TF FS ET G A G K; LS V D YG KK ; LS V D YG K 12 .4 9 3 P0 27 68 A lb um in A LB 69 .3 /6 .2 8 65 .1 /6 .5 1. 2 (u p -r eg ul at ed ) KV PQ V ST PT LV EV SR ; A EF A EV SK ; LV TD LT K 2. 49 4 Q 9U BE 8 Se ri n e/ th re on in e- p ro te in ki n as e N LK N LK 8. 13 /5 8. 28 34 ..6 /8 .7 4. 5 (u p -r eg ul at ed ) TQ EV V TQ YY RA PE IL M G SR H YS N A ID IW SV G IF A EL LG R; PN V FQ N LV SC K; IC D FG LA RV EE LD ES R 2. 6 5 P0 86 70 V im en ti n V IM 53 .6 5/ 5. 12 49 .8 /5 .5 0. 3 (d ow n -r eg ul at ed ) Q D V D N A SL A R; Q V Q SL TE V D A LK ; V EL Q EL N D R; Q Q YE SV A A K; EE A EN TL Q SF R; ET N LD SL PL V D TH SK ; EY Q D LL N V K; LQ EE M LQ R; 19 .8 2 6 P0 86 70 V im en ti n V IM 53 .6 5/ 5. 12 52 .5 /5 .7 a/ p (d ow n -r eg ul at ed ) EE EN FA V EA A N YQ D TI G R; A LD IE IA TY R; EK LQ EE LQ R; Q V Q SL TE V D A LK ; Q D V D N A SL A R; V ES LQ EE IA FL K; V EL Q EL N D R; EE A EN TL Q SF R; ET N LD SL PL V D TH SK ; Q Q YE SV A A K; LQ EE M LQ R; LL EG EE SR ; D N LA ED IR ; Q V D Q LT N D K 35 .7 1 7 P0 86 70 V im en ti n V IM 53 .6 5/ 5. 12 50 .4 /5 .6 a/ p (d ow n -r eg ul at ed ) V EL Q EL N D R; FA D LS EA A N R; Q YE SV A A KL G D LY EE EM R; EA EN TL Q SF R; A LD IE IA TY R; LQ EE M LQ R; Q ES TE YR ; ET N LD SL PL V D TH SK ; Q V Q SL TE V D A LK ; LG D LY EE ER ; D N LA ED IR ; LL EG EE SR ; LQ D EI Q N KE EA R; FA N YI D K 27 .5 8 8 P6 07 09 b -A ct in A C TB 41 .7 /5 .4 8 40 .5 /6 .5 0. 2 (d ow n -r eg ul at ed ) Q EY D ES G PS IV H R; G YS FT TT A ER ; D LT D YL M K; A PE EH PV LL TE A PL N PK ; D SY V G D EA Q SK R; D SY V G D EA Q SK ; EI TA LA PS TK ; D LT D YL K; D LY A N TV LS G G TT YP G IA D R 17 .0 2 9 P6 07 09 b -A ct in A C TB 41 .7 /5 .4 8 40 .8 9/ 6. 7 0. 1 (d ow n -r eg ul at ed ) Q EY D ES G PS IV H R; G YS FT TT A ER ; D LT D YL M K; A PE EH PV LL TE A PL N PK ; D SY V G D EA Q SK R; D SY V G D EA Q SK ; EI TA LA PS TK ; D LT D YL K; D LY A N TV LS G G TT YP G IA D R 10 .7 2 10 P0 74 37 Tu b ul in b et a ch ai n TU BB 51 .2 /5 49 .6 /5 .8 0. 3 (d ow n -r eg ul at ed ) IN TF SV V PS PK ; TA V D IP PR ; IS V YY N EA TG G K; N A A D PR ; LA V N V PF PR 46 .3 8 a/ p : ab se n ce /p re se n ce ; M W : m ol ec ul ar w ei g h t. Ta b le 3. Pr ot ei n – p ro te in in te ra ct io n s of al l p ro te in s fo r co lle ct in g th e d at a. G en e V im A C TB TU BA 1C TU BB SD H A N LK A LB Ex p an d Ph ys ic al in te ra ct io n SE RP IN H , M A G O H , EI FA 3, SP RM 4, KR T1 8, U PP 1, IT G B4 , BA SP 1, PP L, U RG D P, X PN PE P3 C FL 1, C FL 2, PF N 1, C C T8 , TC P1 , C C T3 , C C T4 , RU V BL 1, M YH 11 TU BA 4B , TU BB 6, TU BB , TU BA 4A , TU BB 4A , TU BB 4B , TU BB 3, TU BB 2A , TU BB 6, TU BB 2B , G A PD H , TH BS 1, C LI P1 , TC P1 1L 1, TU BB 2A , YB X 1, TU BA 1A , TU BA 1B , TU BA 1C , TU BA 1A , TU BA 4B , PN KD , TN PO 2, D N A JA 4 SL C 2A 14 , C PN E1 , RA B1 4, LR RC 8A , H IG D 1A , SD H A F2 , SD H A F2 , SD H C , SD H B, U BC M YB , RA N G A P1 , ZH X 3, M A P3 K7 , TA B1 , H IP K2 , LE F1 , TP 53 , TC F7 L2 F2 , H P, IT IH 1, A M BP , A H SG , A PO A 2, RB P4 , FC G RT C o- ex p re ss io n SE RP IN H , TT N C FL 1, PF N 1 TU BB 6, TU BB , TU BA 4A , TU BB 4B , TU BB 3, TU BB 2A , TU BB 6, TU BB 2B , G A PD H , TU BA 1A , TU BA 1B , TB C B, PF D N 1, ST M N 1 TU BA B, TU BB 2A , TU BG 2, YB X 1, TU BA 1B , TU BA 1C , TU BA 4A , TU BB 3, TU BB 4B , TU BG 1 RA B1 4, H IG D 1A , SD H C , SD H B, N D U FV 1, U BC , A LA D , U Q C RC 1 — F2 , H P, IT H 1, G C , A M BP , A FM , A H SG , A FP , A PO 2, RB P4 , SL C O 1B 1, SL C O 1B 3 C o- lo ca liz at io n SE RP IN H I — ST M N 1, TB C B, TU BA 1B , TU BA 1A , TU BB 2A — N D U FV 1, U Q C RC 1, N D U FS 1 RA N G A P1 , M A P3 K7 , TA B1 F2 , H P, IT H 1, G C , A M BP , A H SG , A PO 2, RB P4 , SL C O 1B 1, SL C O 1B 3 G en e in te ra ct io n — — — — — TP 53 — Ro le Pr ol ife ra ti on , ti ss ue in va si on , an d m et as ta si s C el l m ob ili ty , en d oc yt os is , ce ll ad h es io n , d iv is io n , d ru g re si st an ce , an d m et as ta si s C el lu la r m ob ili ty C el lu la r m ob ili ty Tu m or su p p re ss or In h ib it ce llu la r p ro lif er at io n , in d uc e ap op to si s St ab ili zi n g D N A , re g ul at io n ca lc iu m , an ti ox id an t p ro p er ti es UPSALA JOURNAL OF MEDICAL SCIENCES 155 increased in many cancers of the liver, kidneys, colon, stom- ach, pancreas, esophagus, lung, breast, prostate, and ovaries (21). These tumors are associated with increased drug resist- ance and metastasis, especially in MDA-MB-231 cells (21). Tubulins consist of microtubules, with two alpha and beta subunits (approximate weight of 50 kDa). They play a large role in cellular activities, such as cellular mobility. Dysfunction of the microtubule network causes disturbance in apoptosis and differentiation (20). In addition, VIM is one of the most filamentous proteins, expressed in various types of cancers, such as prostate, gastrointestinal, central nervous system, pulmonary, and breast cancers. In a study carried out by Satelli, it was shown that the increased invasive/migratory potential of cancer cells is related to VIM overexpression (22). The VIM is generally considered as an indicator of poor prog- nosis (22). In conclusion, the simultaneous silencing of FOXO3 and activation of p27Kip1 in MDA-MB-231 cells caused alterations in the expression level of several genes which are involved in cell apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. Therefore, it seems that these genes may serve as useful biomarkers for breast cancer. Acknowledgements We thank the Golestan University of Medical Sciences and Arya Tina Gene (ATG) Biopharmaceutical Company, Gorgan, Iran. Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by the Golestan University of Medical Sciences and Health Services (Grant number: 940714170 ) and AryaTinagene bio- pharmaceutical company. Notes on contributors Sabah Mayahi is a Ph.D. student of Molecular Medicine at Golestan University of Medical Sciences, Gorgan, Iran. 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UPSALA JOURNAL OF MEDICAL SCIENCES 157 Abstract Introduction Materials and methods Cell line, plasmid constructs, and transfection Cell viability assay Quantitative real-time polymerase chain reaction assay Apoptosis assay Protein extraction and immunoblotting Two-dimensional gel electrophoresis Protein identification via liquid chromatography-mass spectrometry Data analysis Results Combinatorial effects of induced p27Kip1 and shFOXO3a on cell viability Flow cytometry Effect of overexpression p27Kip1 and FOXO3a shRNA on downstream proteins Discussion Acknowledgements Disclosure statement Funding Notes on contributors References